1P PL1 PL2 PL3 PL4 Plenary Lectures

Transcription

London_Physiology_14-2 6/4/14 4:15 PM Page 1
Plenary Lectures
PL1
Preparing students for the world of work
D. Lewis
University of Leeds, Leeds, UK
In the current economic climate, students are unlikely to be
successful at interview unless they can provide evidence that
they possess key employability skills and have relevant work
experience. Given that the majority of physiology and other
life sciences graduates do not go onto careers in scientific
research, it is essential that universities provide opportunities
for their students to develop key skills and gain work experience more suited to the majority of our graduates final career
destinations. This lecture will review initiatives, both within
and outside of the taught curriculum, where this education,
training and experience is provided, for example “alternative”
final year research projects, educational research internships,
ethics and ethical awareness training and student-led public
engagement activities . It will not discuss employability modules per-se. The impact of these interventions and the benefits to students, in the development of graduate attributes,
Staff and Institutions will also be discussed.
Where applicable, the authors confirm that the experiments
described here conform with The Physiological Society ethical
requirements.
PL2
Intervening in ageing to prevent the diseases of ageing
L. Partridge
University College London, London, UK
We aim to discover genes and mechanisms that maintain
health during ageing and that are conserved in evolution, and
hence are of relevance to humans. In particular, we focus on
nutrition and on the signaling pathways that
sense nutrients: the insulin/IGF/TOR pathway. We investigate
the signaling mechanisms at work, the biochemical processes
that are altered to slow ageing, and the mechanisms by which
ageing acts as a risk factor for ageing-related neurodegeneration.
in The Journal of Physiology. Over the intervening decades, much
has been learned about molecular mechanisms of E-C and ES coupling. Excitation-transcription (E-T) coupling, the topic of
this lecture, is arguably a more general event in neurons and
other excitable cells. However, in contrast to E-C and E-S coupling, E-T coupling is much less understood at a mechanistic
level. It is generally accepted that one particular class of Ca2+
channels, CaV1 (also known as L-type channels) has a privileged
role in excitation-transcription coupling to nuclear CREB, a transcription factor critical in learning and memory. However, the
mechanism of even the earliest step in this signaling pathway
is not well understood: local Ca2+ elevations in the nanodomain
of CaV1 channels are thought to be the main trigger in the
signaling cascade, but CaV1 channels could also convey a voltage-dependent conformational signal (VCS) to nearby signaling intermediates, analogous to the conformational signal in
E-C coupling. We have devised an approach where conformational changes required to open the CaV pore are experimentally decoupled from Ca2+ influx into the channel nanodomain.
This molecular dissection uncovered a remarkable and unexpected requirement for the CaV1 VCS in excitation-transcription coupling. CaV1 signaling to CREB behaves as a coincidence
detector, where both Ca2+ and voltage-dependent movements
are necessary.
Another puzzle is how local signaling at CaV1 channels is
relayed onward to the nucleus. We have discovered a novel
mechanism that mediates long-distance communication
within cells: a shuttle that transports Ca2+ /calmodulin from
the surface membrane to the nucleus. We find that the shuttle protein is γCaMKII, that its phosphorylation at Thr287 by
βCaMKII protects the Ca2+ /CaM signal, and that calcineurin
(CaN) triggers its nuclear translocation. Both βCaMKII and CaN
act in close proximity to CaV1 channels, supporting their dominance, while γCaMKII operates as a carrier, not as a kinase.
Upon arrival within the nucleus, Ca2+ /CaM activates CaMKK
and its substrate CaMKIV, the CREB kinase. This mechanism
resolves longstanding puzzles about CaM/CaMK-dependent
signaling to the nucleus. The significance of the mechanism
is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII and
CaN in multiple neuropsychiatric disorders.
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PL4
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A historical perspective on reflex cardiovascular control in
man
P. Sleight
PL3
Excitation-transcription coupling: novel mechanisms and
implications for brain disease
R.W. Tsien
NYU Neuroscience Institute, New York, NY, USA
The primary language of excitable cells (action potential firing) is converted into the primary language of intracellular
activity (biochemical signaling) by voltage-gated Ca2+ channels (CaVs) – a key part of “excitation-response coupling”. This
kind of signaling is vividly exemplified by excitation-contraction (E-C) coupling and excitation-secretion (E-S) coupling,
processes respectively described by Hodgkin, Huxley and their
colleagues, and by Katz and Miledi, Llinas, and Douglas, mostly
University of Oxford, Oxford, UK
This presentation will supplement earlier Paton lectures concerning central nervous organisation of cardiovascular control
by concentrating on subcortical & peripheral pathways which
subserve circulatory homeostasis.
Baroreflex control of long term blood pressure - It had earlier
been proposed (by AC Guyton & others) that arterial baroreflex mechanisms were mainly involved in short term beat to
beat blood pressure (BP) control, but not longer-term levels of
pressure. This view arose from BP recordings made in isolated
conscious dogs that had earlier undergone carotid & cardiac
denervation. Their BP was raised by only a few mmHg, which
was interpreted then as relatively insignificant. At that time it
was not known that the arterial baroreflexes had effects on
sodium & water elimination via renal nerves, as well as the
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Plenary Lectures
more easily demonstrated effects on heart rate, cardiac contractility, & peripheral vascular tone. 40 years or so later T.
Thrasher showed that baroreceptor unloading caused nervously mediated renal sodium retention & thus longer term BP
elevation.
Interventions to control blood pressure - Earlier attempts to
lower malignant hypertension in man by splanchnic denervation (Smithwick) were successful but accompanied by severe
postural hypotension. Another surgical approach was to stimulate the carotid sinus nerves (CSN) by an implantable device.
Although this was initially successful longer-term results were
marred by damage to the nerve or its blood supply.
New methods of CSN stimulation in man are now successful
long term, by avoidance of dissection of the CSN using stimulation of the receptors by electrodes applied to the adventitia of the sinus. The implanted generator is now similar in size
to a cardiac pacemaker.
Non-drug therapy for hypertension has recently used renal
denervation by electrical damage applied by a catheter introduced percutaneously from the groin into each renal artery.
Earlier trials (Esler & colleagues in Melbourne) showed impressive long lasting BP reduction in patients with drug resistant
hypertension. Since this technique did not involve surgery or
device implantation it rapidly became the treatment of choice,
compared with baroreceptor stimulation, & is now in clinical
use. However the US FDA required a properly blinded controlled randomised trial before implementation in the US. In
January 2014 it was announced that this trial (Symplicity-3)
was negative! So far no further details are available. Preliminary results will be given at the meeting of the American College of Cardiology at the end of March 2014.
Methods to assess autonomic tone in man - I will now discuss
the use of methods to assess autonomic tone in man. Historically these have evolved from measurements of BP or pulse
rate to obtain a measure of arousal, or urine/blood catecholamines, which of course are very indirect assessments
(which do not take account of turnover or of tissue sensitiv-
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ity). Ambulatory BP (ABP) or home BP measures go some way
to avoid the arousal caused by medical/nurse observers, but
have other problems. Another more direct method is to measure sweating & skin conductivity. All these techniques are
imperfect & none (except heart rate) gives any measure of
parasympathetic tone. For this reason there has been considerable interest in Power Spectral Analysis (PSA) of heart rate
& BP variability which can give an indirect measure of sympatho-vagal balance. It is not possible to completely validate this
method, but responses to actions such as postural change from
lying to standing, or changes in arousal from sleep to wakefulness, alter PSA measures which demonstrate parallel
changes in sympathetic or in vagal tone.
Music therapy - The use of music is not only specific to man,
but dates back thousands of years. The appreciation of music
is confined to man, despite the so called Mozart effect of music
on milk yield in cows. The published data on the latter is sparse
& poorly controlled.
Music can entrain inherent autonomic rhythms in man. Specifically it can entrain the 10 sec Mayer waves in BP & can increase
measures of vagal tone. But claims that music therapy can be
tailored to individual people are poorly controlled. In experiments on young medical students we found that autonomic
responses to 6 differing styles of music (presented in random
order) were the same – whatever the individual’s pre-stated
musical preference. However the differing responses of the
students who were musically trained were greater than the
responses of the others.
Conclusion - Hypertension treatments have multiplied over
the last 40-50 years. But many patients fail to continue treatment long-term, due to side effects or cost. There are now
practical physiological alternatives to drug treatment of human
hypertension.
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Research Symposia
SA001
A digital reconstruction of the neocortical microcircuit
H. Markram
EPFL- BLUE BRAIN PROJECT, EPFL, CH-1015 Lausanne, Switzerland
A digital reconstruction of a prototypical neocortical microcircuit is presented. A novel strategy enabled a first draft of a
full reconstruction from partial anatomical and physiological
data on the cellular and synaptic organization of the
somatosensory cortex of a two-week old rat. This first draft
cellular-level map of the microcircuit predicts the number of
neurones of each morphological and electrical type per layer
(the neurome), all intrinsic and extrinsic synaptic connections
involved (the connectome), and complete maps of intrinsic
synapses for each neurones (the synaptomes). In silico simulations revealed a robust activity spectrum, ranging from synchrony to asynchrony (SA spectrum). Modulating synapses,
neurones and layers shift the network along the spectrum and
changes network properties. The digital reconstruction of the
neocortical microcircuit is being made freely available for independent investigation and to facilitate collaborative refinement of the predictions.
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SA002
A nanoscale view into cells through super-resolution
microscopy
R. Henriques
DNA, RNA and protein, part of the central molecules of biology, typically exist at dimensions of a few nanometers, well
beyond the resolving power of conventional fluorescent
microscopy (~300 nm). Super-resolution microscopy techniques hold to date the record in resolving power for light
microscopy. These have both the capacity to differentiate and
localize individual molecules at scales experimentally demonstrated of few nanometers (1-30 nm). In this family of methods, photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) as well as
their variants embrace the potential to fully resolve complete
cellular structures at the nanoscale, accurately localizing thousands to millions of individual fluorophores within a labeled
cell. In this seminar I will introduce several of the aspects in
super-resolution methods we have developed, ranging from
analytical methods to novel super-resolution probes. Some of
its applications will then be demonstrated, for example by
super-resolving host-pathogen in HIV infection.
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SA003
A tool-kit for dissecting mechanisms of auditory circuit
assembly
L. Goodrich
Department of Neurobiology, Harvard Medical School, Boston,
MA, USA
The precisely organized circuits of the auditory system allow
animals to detect, recognize, and locate sounds in the environment. The frequency, intensity, and timing of each sound
stimulus is encoded in the activity of spiral ganglion neurons,
which receive input from mechanosensitive hair cells in the
cochlea and transmit this acoustic information to a variety of
target neurons in the auditory brainstem. Frequency information is captured in the spatial pattern of activation, with
hair cells and spiral ganglion neurons organized from low frequencies in the apex to high frequencies in the base of the
cochlea. In addition, spiral ganglion neurons make specialized
synaptic contacts that enable unusually accurate preservation
of the timing of the original sound stimulus as electrical signals pass from hair cells to the spiral ganglion neurons and
from the spiral ganglion neurons to the cochlear nuclei. The
goal of our work is to understand how developing spiral ganglion neurons establish the correct pattern and types of connections necessary for the perception of sound.
Despite the fundamental importance of the sense of hearing
for animal survival and reproduction, the development and
function of this key sensory system have been challenging to
unravel. One obstacle has been the small size of the cochlea,
which houses too few hair cells and spiral ganglion neurons
for standard molecular approaches. In addition, the cochlea
is difficult to access experimentally, as it is surrounded by bone
and embedded in the skull. Unfortunately, few in vitro assays
are available, making it difficult to perform high throughput
unbiased screens to identify new molecular players or to define
mechanisms. My laboratory has been applying newly developed approaches to circumvent the difficulties of manipulating and labeling spiral ganglion neurons in mice. Using genetic
labeling techniques, we mapped the behavior of individual
developing spiral ganglion neurons from the initial extension
of peripheral and central processes towards their targets to
the formation of specialized synapses and the onset of hearing. We are now able to visualize these same events in real
time and in situ by time lapse confocal imaging of fluorescently
labeled neurons in embryonic cochlear explants. In parallel,
we performed a large scale analysis of gene expression to identify new markers and candidate genes, focusing on those genes
that are uniquely enriched in auditory neurons vs. the closely
related neurons of the vestibular system. These studies indicated a particularly prominent role for the transcription factor Gata3 during spiral ganglion neuron development and differentiation. Based on these findings, we have begun to
develop new high throughput methods for generating and
analyzing spiral ganglion neurons in vitro. With the introduction of progressively more sophisticated tools and technologies, the future holds the exciting opportunity to make important new discoveries into the development and function of the
auditory system.
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SA004
Nutrigenomics versus nutrigenetics for personalised
nutrition: implications for cardiovascular function
T.A. Sanders
Diabetes and Nutritional Sciences, Kings College London, London,
London, UK
Nutrigenetics are the fixed genetic variation (genotype) in
response to diet whereas nutrigenomics describes the influence of specific nutrients on gene expression. The unravelling of the human genome had led to hopes that individual
susceptibility to major risk factors (blood pressure, lipids,
inflammatory and haemostatic factors) might lead to the personalised nutritional advice to avoid cardiovascular disease
(CVD). The major problem is that dietary effects are difficult
to detect in the first place and diet x genotype interactions are
even more so. While relatively common mutations in the low
density lipoprotein (LDL) receptor increase by 25 fold the risk
of premature CVD, common polymorphisms have much milder
effects. Generally, the environmental effects (smoking, physical activity, diet and obesity) overwhelm genetic effects with
regard to risk of CVD. Polymorphisms that raise LDL cholesterol are associated with increased risk of CVD. The polymorphism in the apolipoprotein E phenotype is a good example
of how individuals differ in their LDL cholesterol response to
dietary cholesterol. Polymorphism in the angiotensinogen
gene AGT may explain some of the variability in response to
diet and blood pressure. Dietary folate and vitamin B12 intake
can influence DNA methylation and are potential candidates
for explaining the nutrigenomic effects of how maternal diet
can affect the outcome of the offspring. The potential nutrigenomic mechanisms by which early diet and development might
affect cardiovascular function, particularly blood pressure and
risk of type 2 diabetes which both increase risk of CVD in later
life, will be discussed.
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out life. Male offspring from both groups were weaned onto
chow and ex vivo cardiac function was studied in one per litter at 12 weeks of age by isolated Langendorff heart perfusion. Baseline measurements included left ventricular developed pressure, left ventricular end diastolic pressure and the
minimum and maximum velocity of contraction. Both
chronotropic and inotropic effects to muscarinic agonist carbachol and beta-adrenergic agonist isoprenaline were also
assessed.
Results: Cardiac hypertrophy was observed in offspring of
obese dams from 3 to 8 weeks of age and was accompanied
by an increase in cardiomyocyte cell area and re-expression of
pathological cardiac fetal genes. At any time, there was no difference in offspring body weight between groups. At 12 weeks
of age, offspring from obese dams had impaired systolic and
diastolic function (Figure 1A-D) and cardiac sympathetic dominance (p<0.001).
Conclusion: Maternal diet-induced obesity programmes cardiac dysfunction in the absence of any change in body weight
and despite the fact that the offspring themselves had been
eating a healthy low fat diet. These findings help to provide a
causal link to explain and further support recent human observations linking maternal obesity to premature death from CVD.
Figure 1: Left ventricular developed pressure (A). Myocardial contractility
(B). Left ventricular end diastolic pressure (C) Myocardial relaxability (D).
Sample number is represented inside the histogram. *p<0.05, **p<0.01.
This work was funded by the British Heart Foundation
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SA006
SA005
Programming of cardiac function by maternal diet-induced
obesity
H.L. Blackmore1, D. Fernandez-Twinn1, Y. Niu2, D.A. Giussani2
and S.E. Ozanne1
1Metabolic
Research Laboratories and MRC Metabolic Diseases
Unit, Wellcome Trust MRC-Institute of Metabolic Science, University
of Cambridge, Cambridge, UK and 2Physiology, Development and
Neuroscience, University of Cambridge, Cambridge, UK
Introduction: Obese pregnancies are not only associated with
adverse consequences for the mother but also the long-term
health of her child. Recent evidence from human studies has
shown that individuals born to obese mothers are at increased
risk of premature death from cardiovascular disease (CVD) in
adulthood. Whilst important, such epidemiological studies fail
to directly address causality. Our study aimed to address causality using a mouse model of maternal diet-induced obesity.
Methods: Obesity was induced in female C57BL/6 mice using
a diet high in simple sugars and saturated fat for 6 weeks prior
to mating, throughout pregnancy and lactation. Control
females were fed a standard laboratory chow diet through-
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Nutrigenomics of the nitric oxide pathway: Current
evidence and future perspectives
M. Siervo
Institute for Ageing and Health, Newcastle University, Newcastle
upon Tyne, UK
Endothelial function and cardiovascular risk: The preservation
of the structural and functional integrity of the endothelium
plays a critical role in the prevention of atherosclerotic damage and cardiovascular risk. Endothelial dysfunction has been
identified as a surrogate physiological biomarker for cardiovascular risk prediction as well as a target of nutritional and
pharmacological interventions (1). This prominent role is mechanistically explained by the selective activity of endothelial
cells in regulating cellular and molecular trafficking between
circulating fluids and interstitial space and several physiological functions via the secretion of molecules with paracrine
and endocrine signalling properties. One of the most important secretory endothelial products is nitric oxide (NO), a gasotransmitter with paracrine and endocrine actions involved in
the regulation of vascular resistance, immunity, coagulation,
memory formation and mitochondrial efficiency (1).
Research Symposia
Nitric Oxide Pathway: NO can be synthesised in humans
through an enzymatic and non-enzymatic pathway. The
endothelial isoform of Nitric Oxide Synthase (eNOS) uses arginine and molecular oxygen as precursors to tonically release
NO. The efficiency of the enzymatic pathway is dependent on
the availability of several cofactors including flavin adenine
dinucleotide (FAD), flavin mononucleotide (FMN), heme,
tetrahydrobiopterin (BH4) and calmodulin. The non-enzymatic
pathway is based on the progressive reduction of inorganic
nitrate into nitrite (NO2-) and NO in a complex system involving the recovery of circulating plasma nitrate through the salivary glands (entero-salivary circulation), reductase activity of
saprophytic oral bacteria and reductase-specific activity of
enzymes in conditions of low pH and oxygen tension (2, 3).
Nutrition and the NO pathway: Nutritional strategies to
increase NO production can target the different elements of
the NO pathway by increasing the bioavailability of the NO precursor (arginine) and co-factors (antioxidants, folic acid) and/or
raise the non-enzymatic generation of NO (inorganic nitrate).
L-Arginine appears to dilute the intracellular levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of
eNOS. Levels of ADMA are determined by the balance between
synthesis and degradation regulated by two enzymes, i.e., protein arginine methyltransferases (PRMT) and dimethylarginine
dimethylaminohydrolases (DDAH), respectively. The effects of
inorganic nitrate derive from increased non-enzymatic and
enzymatic generation of NO in environments with higher acidity and lower oxygen tension and specific host enzymes with
NO2- reductase activity (i.e., xanthine oxido-reductase (XOR),
deoxy-Hb) (2,3).
Nutrigenomics of the NO pathway: The close mechanistic links
between nutritional factors, enzymatic synthesis and degradation of NO represent an ideal feature for a multi-candidate
gene-nutrient approach based on the following characteristics: 1) genes involved in NO metabolism are responsive to
nutritional interventions; 2) genes are characterised by important functional variations; 3) genes can influence the synthesis and function of NO and have downstream, multi-systemic
effects and 4) the prevalence of functional gene polymorphisms in the general population is meaningful and 5) the function of the genes can be directly assessed by measuring gene
expression or direct (systemic NO production) and indirect
biomarkers (nitrate, nitrite, cGMP, ADMA, flow mediated dilation) of NO production (4).
The nutrigenomic approach to the NO pathway is gaining
momentum and recent data from epidemiological and clinical studies have investigated the association of several single
nucleotide polymorphisms (SnPs) in the genes involved in the
regulation of the NO pathway including eNOS, arginase, DDAH,
PMRT and XOR with health outcomes (4,5,6). For example,
eNOS (G894T (Glu298Asp, rs1799983) in exon 7 and T786C
(rs2070744) in the promoter region) and XOR polymorphisms
(– 337GA, 565+64CT) have been associated with raised blood
pressure (5,6). In addition, eNOS Glu298Asp genotype
appeared to differentially affect vasodilation and ex vivo
lipoprotein oxidation after consumption of fruit and vegetables in humans (7).
Future Directions: The effects of the individual allelic variants
for each SnPs in the various genes involved in the regulation
of the NO pathway are far from being elucidated. In addition,
there is currently limited evidence on the effects of nutritional
factors acting on the two synthetic mechanisms of NO production (enzymatic and non-enzymatic) and their potential
cross-interaction. The understanding of the differential
responses of candidate genes to specific nutrients targeting
the NO pathway may form the basis for a risk-stratified
approach to nutritional treatments of chronic metabolic and
cardiovascular disorders.
Vita JA. (2011) Endothelial function. Circulation.124(25): e906-12.
Förstermann U et al. (2012) Nitric oxide synthases: regulation and
function. Eur Heart J. 33(7):829-37.
Lundberg JO, et al. (2009) Nitrate and nitrite in biology, nutrition and
therapeutics. Nat Chem Biol. 12:865-869.
Siervo M, et al. (2011) Measurement of in vivo nitric oxide synthesis
in humans using stable isotopic methods: a systematic review. Free
radical biology & medicine. 51(4):795-804.
Niu W, et al. (2011) An Updated Meta-Analysis of Endothelial Nitric
Oxide Synthase Gene: Three Well-Characterized Polymorphisms with
Hypertension. PLoS One. 6(9): e24266.
Yang J, et al. (2008) Associations of Hypertension and Its Complications with Variations in the Xanthine Dehydrogenase Gene. Hypertens
Res. 31(5):931-940.
George TW et al (2012). The Glu298Asp single nucleotide polymorphism in the endothelial nitric oxide synthase gene differentially affects
the vascular response to acute consumption of fruit and vegetable
puree based drinks. Mol Nutr Food Res. 56(7):1014-24.
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SA007
Impact of dietary polyphenols on peripheral and
cerebrovascular blood flow
D. Vauzour
Norwich Medical School, University of East Anglia, Norwich,
Norfolk, UK
Numerous epidemiological and human intervention studies
have suggested that regular consumption of polyphenol-rich
foods, such as fruits, vegetables, cocoa, tea and wine, may
exert beneficial effects on the peripheral and the cerebral vascular system. For example, a daily intake of cocoa derived
flavonoids, a subclass of polyphenol, was found to be the causal
factor in determining the relatively low incidence of hypertension and CVD incidence in the Kuna Amerinds in Panama.
In support of these findings, recent meta-analyses of combined
flavonoids, showed significant improvements in both acute
and chronic vascular reactivity as assessed by flow-mediated
dilation (FMD) and diastolic and systolic blood pressures. In
particular, diets rich in cocoa derived flavonoids are capable
of reducing cardiovascular risk by increasing nitric oxide
bioavailability and lowering blood pressure. In the context of
the CNS, brain imaging studies in humans have also demonstrated that the consumption of polyphenol-rich food may
enhance cortical blood flow (CBF). In particular, consumption
of cocoa flavonoids may improve CBF in healthy older adults
and in young adults in response to a cognitive task. More
recently, a greater hemodynamic response as assessed by
increased regional fMRI activation has also been reported in
older adult subjects with mild cognitive impairment following
consumption of Concord grape juice. These effects are particularly significant, as increased cerebrovascular function is
known to facilitate adult neurogenesis and to enhance vascularisation, two events important in the maintenance of cognitive performances. Since ageing is known to impair vascularisation, endothelial function and decreases endothelial
progenitor cells, the influence of dietary agents on the production of vascular derived factors and in the maintenance of
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Research Symposia
CBF may hold a clinical value in preventing both CVD risk and
cognitive decline.
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SA008
Role of microRNAs in the cardioprotective effects of
polyphenols: A nutrigenomic approach
D. Milenkovic
INRA, Centre de Clermont-Ferrand, Clermont-ferrand, France
Consumption of flavanol-rich foods is associated with a reduced
risk of cardiovascular diseases, which was linked to improvements in endothelial function. The specific flavanols involved
in these beneficial effects and underlying molecular mechanisms is still largely unknown. We have shown that exposure
of TNFα-activated endothelial cells to flavanol metabolites (4’O-methyl(−)-epicatechin, 4’-O-methyl(−)-epicatechin-7-β-Dglucuronide and (−)epicatechin-4’-sulfate) at physiologicallyrelevant concentrations decreased the adhesion of monocytes
to endothelial monolayers. Nutrigenomic analysis showed that
these metabolites modulate expression of genes involved in
the regulation of cell adhesion/junctions, focal adhesion or
cytoskeleton remodeling, and this by affecting phosphorylation levels of p65 and p38 of NF-κB and MAPK cell-signaling
pathways respectively. Together with cell signaling pathways,
microRNAs (short, endogenous, noncoding, single-stranded
RNAs) represent another class of molecular post-transcriptional
regulators of gene expression. Our nutrigenomic studies have
shown that exposure of endothelial cells to the same metabolites can also modulate the expression of miRNAs. Among differentially-expressed miRNAs are those involved in the regulation of inflammation or cell adhesion, such as miR-221 and
miR-181. Bioinformatic analysis shows that the potential target genes these miRNAs are also involved in regulation of cell
adhesion, cytoskeleton, focal adhesion, transendothelial migration. These miRNAs could exert post-transcriptional regulation
by inhibiting protein synthesis, as BIRC2, or by inducing mRNA
degradation, as WASP1.
These data suggest that miRNAs are important molecular targets of flavanol metabolites involved in the regulation of
expression of genes controlling adhesion and transendothelial migration processes. This original result contributes to
increase the knowledge about the mechanisms underlying the
protective effect of flavanols on vascular endothelium.
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SA009
Synaptic energy use and supply
D. Attwell
UCL, London, UK
A universal constraint on the evolution of brains is that the
nervous system’s computational power is limited by its energy
supply. By describing an energy budget for the grey matter of
the mammalian CNS, I will explain how key design features of
the brain are partly determined by the energy supply the brain
receives as oxygen and glucose, and how matching of brain
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energy supply to brain energy use underpins BOLD functional
magnetic resonance imaging. I will examine why the white
matter uses less energy than the grey matter, and whether
myelination really saves energy. I will present a simple account
of how information flow through synapses relates to the energy
used by the synapses and show that this explains why synapses
are unreliable transmitters of information. I will outline why
the primary locus of control of the brain’s energy supply, and
hence of the generation of BOLD fMRI signals, is in capillaries
rather than arterioles.
Wellcome Trust, Fondation Leducq, European Research Council
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SA010
Pannexin channels and ischaemia
R.J. Thompson and N.L. Weilinger
Hotchkiss Brain Institute, University of Calgary, Calgary, AB,
Canada
The pannexin ion channels are members of the gap junction
superfamily. Unlike their connexin cousins, pannexins function
as bona fide ion channels in the plasma membrane, and do not
likely form intercellular junctions. Pannexin-1 (Panx1) is activated during ischaemia, contributing to the anoxic depolarization (Thompson et al., 2008) and suggesting that Panx1
may be important for cell death. The mechanism of Panx1 activation involves the N-methyl-D-aspartate receptor (NMDAR)
and Src family kinases (SFKs) (Weilinger et al., 2012). Here we
tested the hypothesis that NMDAR-SFK-Panx1 signalling
induces neuronal death during ischaemia through mitochondrial dysfunction. We used patch-clamp recording and live cell
imaging from hippocampal brain slices / neuronal cultures prepared from isofluorane (inhaled) anesthetised rats or mice to
determine if there is a direct link between Panx1 and mitochondrial function. Anoxia opened Panx1 channels because
Panx1 antagonists or knockout substantially reduced the
anoxic depolarization. Block of the NMDAR with APV also
reduced the anoxic depolarization and the combined effect of
APV and Panx1 blockers was not summative. We designed a
small interfering peptide, Panx305-318 that mimics the SFK
consensus site on the Panx1 c-terminal. Panx305-318 potently
blocked ischaemia-induced Panx1 opening in brain slices and
cultured neurons. Interestingly, Panx1, and not NMDARs, was
critical for ischaemia-induced Ca2+-dysregulation and neuronal
death. Panx1 block with Panx305-318 attenuated mitochondrial depolarization and permeability transition pore opening,
measured by live cell fluorescent imaging. Taken together,
these data suggest that Ca2+ influx through NMDAR activated
Panx1 channels is specifically coupled to mitochondrial dysfunction and neuronal death during ischaemia.
Thompson, RJ, Jackson MF, Olah ME, Rungta RL, Hines DJ, Beazely MA,
MacDonald JF and MacVicar BA. (2008). Activation of pannexin-1
hemichannels augments aberrant bursting in the hippocampus. Science, 322: 1555-1559.
Weilinger NL, Tang PL and Thompson RJ (2012). Anoxic activation of
NMDA receptors opens pannexin-1 via Src family kinases. Journal of
Neuroscience. 32:12579-12588.
This work was supported by grants from Canadian Institues of
Health Research and Alberta Innovates - Health Solutions.
Research Symposia
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SA011
Hydrogen peroxide as a neuromodulator
B. O’Neill1,3, J.C. Patel1, D. Sverdlin1 and M.E. Rice1,2
1Neurosurgery, New York University School of Medicine, New York,
NY, USA, 2Neuroscience and Physiology, New York University
School of Medicine, New York, NY, USA and 3Psychiatry, New York
University School of Medicine, New York, NY, USA
Hydrogen peroxide (H2O2) is recognized as an intra- and intercellular signalling molecule that can influence processes from
embryonic development to cell death. Most research on H2O2
signalling has focused on relatively slow signalling, on the order
of minutes to days, via second messenger cascades. However,
H2O2 can also mediate subsecond signalling via ion channel
activation. We have examined rapid signalling by H2O2 in the
nigrostriatal dopamine (DA) system, which includes the substantia nigra pars compacta (SNc) and dorsal striatum, and
which plays critical roles in movement and motor learning
mediated by the basal ganglia. We have also examined H2O2
regulation of GABAergic neurons of the SN pars reticulata (SNr),
which are the primary output neurons of the basal ganglia. All
of our work has been conducted using ex vivo brain slices prepared from adult male guinea pigs or mice, after inducing deep
anaesthesia (50 mg/kg sodium pentobarbital, i.p.). Methods
include fast-scan cyclic voltammetry to detect DA release,
whole-cell recording to monitor neuronal activity, and fluorescence imaging to indicate H2O2 generation. In guinea pig
SNc DA neurons, endogenously generated H2O2 activates ATPsensitive K+ (KATP) channels that inhibit DA neuron firing
(Avshalumov et al. 2005). In guinea pig striatum, H2O2 generated downstream from glutamatergic AMPA receptors in
striatal medium spiny neurons (MSNs) acts as a diffusible messenger that inhibits axonal DA release, also via KATP channels
(Avshalumov et al. 2008; Patel et al. 2011). The source of
dynamically generated H2O2 is mitochondrial respiration (Bao
et al. 2009); thus, H2O2 provides a novel link between activity
and metabolism via KATP channels. Additional targets for
dynamic regulation by H2O2 include a subclass of transient
receptor potential channels, TRPM2. In contrast to the
inhibitory effect of H2O2 acting via KATP channels, TRPM2 channel activation is excitatory. In guinea pig brain slices, H2O2 elevation increases the excitability of striatal MSNs and increases
the firing rate of SNr GABAergic neurons via TRPM2 channel
activation (Bao et al. 2005; Lee et al. 2011, 2013). Notably,
however, emerging evidence indicates that dynamic regulation of DA release and neuronal activity by H2O2 differs in
mouse brain. Although evoked axonal DA release is modulated
by glutamate and GABA acting at AMPA and GABAA receptors
in the dorsal striatum of mice, as in guinea pigs, this regulation does not appear to be H2O2 dependent, indicated by a
lack of effect of catalase application in mouse striatal slices.
One underlying factor may be a species difference in nigrostriatal KATP channel expression. Using subunit-selective KATP
channel openers, we found that axonal DA release and DA neuron activity in guinea pig brain slices can be suppressed by activation of either SUR1-containing or SUR2-containing KATP channels (Avshalumov et al. 2005; Patel et al. 2011). In mouse
striatal slices, however, only SUR1-selective diazoxide, but not
SUR2-selective cromakalim leads to suppression of evoked DA
release. Another species difference in H2O2-dependent mod-
ulation of neuronal activity is that H2O2 elevation in mouse
midbrain slices leads to inhibition of SNr GABAergic neurons
via predominant KATP channel activation, as opposed to the
TRPM2-dependent excitation seen with H2O2 elevation in
guinea pig SNr neurons (Lee et al. 2011). This is particularly
surprising given the limited regulation of DA release by H2O2sensitive KATP channels in mouse striatum. Differences in the
functional activation of H2O2-dependent KATP and TRPM2 channels between guinea pigs and mice suggest divergent roles for
this regulatory process across species. The need for neuronal
regulation by a metabolic signal like H2O2 might depend on
unique behavioural demands across species that require differential patterns of ion channel expression. Other factors
include species differences in the generation or metabolism
of H2O2, for example by the glial antioxidant network – the
strength of which correlates with neuron-to-glia ratio, with
higher control in guinea pig (or human) vs. mouse. Regardless of these differences, however, responsiveness to H2O2 and
conservation of nigrostriatal SUR1 expression in diverse species
supports the idea that KATP channels are important regulators of DA neuronal function and DA release, particularly in
light of previous work showing greater sensitivity of SUR1- vs.
SUR2-containing channels to H2O2 and to metabolic stress.
Avshalumov et al. (2005). J Neurosci 25, 4222-4231
Avshalumov et al. (2008). J Neurophysiol 100, 1590-1601
Bao L et al. (2005). J Neurosci 25,10029-10040
Bao L et al. (2009). J Neurosci 29, 9002-9010
Lee CR et al. (2011). Front Syst Neurosci 5, 14
Lee CR et al. (2013). J Neurosci 33, 1157-1168
Patel JC et al. (2011). J Neurochem 118, 721-736
This work was supported by National Institutes of Health (USA)
grants NS036362, NS045325, NS063656, and DA007254 and
by the Attilio and Olympia Ricciardi Research Fund.
requirements.
SA012
Calcium regulation of mitochondrial function in neurons
I. Llorente-Folch1,2, C.B. Rueda1,2, I. Amigo1,3, C. Laura1,2,
P. Gonzalez-Sanchez1,2, P. Martinez-Valero1,2, I. Juaristi1,2,
B. Pardo1,2, A. del Arco4 and J. Satrustegui1,2
1Molecular Biology, Centro de Biologia Molecular Severo Ochoa,
Madrid, Spain, 2CIBER de Enfermedades Raras (CIBERER), Madrid,
Spain, 3Molecular Biology-UAM, Instituto de Investigación Sanitaria
Fundación Jiménez Díaz (IIS FJD), Madrid, Spain and 4Facultad de
Ciencias Ambientales y Bioquímica, Universidad de Castilla la
Mancha, Toledo, Spain
Calcium is thought to regulate respiration but it is unclear
whether this is dependent on the increase in ATP demand
caused by any Ca2+ signal or to Ca2+ itself. [Na+]i, [Ca2+]i and
[ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca2+ clearly showed that
Ca2+ stimulation of coupled respiration is required to maintain [ATP]i levels. Ca2+ may regulate respiration by activating
metabolite transport in mitochondria from outer face of the
inner mitochondrial membrane, or after Ca2+ entry in mitochondria through the calcium uniporter (MCU). Two Ca2+ regulated mitochondrial metabolite transporters are expressed
in
neurons,
the
aspartate/glutamate
exchanger
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Research Symposia
Aralar/AGC1/Slc25a12, a component of the malate/aspartate
shuttle, with a Kd for Ca2+ activation of 300 nM, and the ATP
Mg/Pi exchanger SCaMC3/Slc25a23, with S0.5 for Ca2+ of 300
nM and 3.4 μM, respectively. The lack of SCaMC3 results in a
smaller Ca2+ dependent stimulation of respiration only at high
workloads, as caused by veratridine, whereas the lack of
ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca2+ dependent responses to
all workloads (veratridine, K+ depolarization, carbachol). The
lack of ARALAR caused a reduction of about 65-70% in the
response to the high workload imposed by veratridine, and
completely suppressed the OCR responses to moderate (K+
depolarization) and small (carbachol) workloads, effects
reverted by pyruvate supply. For K+ depolarization, this occurs
in spite of the presence of large [Ca2+]mit signals and increased
reduction of mitochondrial NAD(P)H. These results show that
ARALAR/MAS is a major contributor of Ca2+ stimulated respiration in neurons by providing increased pyruvate supply to
mitochondria. In its absence and under moderate workloads,
matrix Ca2+ is unable to stimulate pyruvate metabolism and
entry in mitochondria suggesting a limited role of MCU in these
conditions.
Llorente Folch I et al. (2013) Calcium regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1 malate
aspartate shuttle in intact cortical neurons. J. Neurosci.33 13957,13971
This work was supported by the Ministerio de Economía grant
BFU2011-30456, by CIBERER (an initiative of the ISCIII), by
the Comunidad de Madrid grant S2010/BMD-2402 MITOLABCM (to J. S.), by ISCIII Grant PI080610 (to A. del A.), and by an
institutional grant from the Fundación Ramon Areces to the
Centro de Biología Molecular Severo Ochoa. C.B.R is a recipient
of a FPU fellowship from the Ministerio de Educación y Ciencia.
We thank Dr Hiromi Imamura, Kyoto University for kindly
providing the GO-ATeam plasmids. The authors thank Isabel
Manso and Bárbara Sesé for technical support, José Belio for
his help in preparing the figures and María Angeles Muñoz,
from the Unit of Optical and Confocal Microscopy, for her
inestimable support.
requirements.
SA013
Reactive oxygen species and inhibitory transmission
D. Bowie
McGill University, Montreal, QC, Canada
Neuronal communication imposes a heavy metabolic burden
in maintaining ionic gradients essential for action potential firing and synaptic signalling (1). Given its high energy requirements and limited energy reserves, the evolving brain has
developed metabolically-efficient wiring (2) and signalling
strategies (3, 4) to transmit information. How energy consumption is distributed among different cell types has yet to
be established. In this presentation, I will discuss recent work
highlighting the bioenergetics of inhibitory GABAergic
synapses (5) and the signalling role of reactive oxygen species
(mROS) derived from the mitochondria (6). Emerging data suggest that mROS may act as a homeostatic signalling molecule
coupling cellular metabolism to the strength of inhibitory transmission.
Harris,J.J., Jolivet,R., & Attwell,D. Synaptic energy use and supply. Neuron 75, 762-777 (2012).
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Mitchison,G. Axonal trees and cortical architecture. Trends Neurosci.
15, 122-126 (1992).
Levy,W.B. & Baxter,R.A. Energy efficient neural codes. Neural Comput.
8, 531-543 (1996).
Balasubramanian,V., Kimber,D., & Berry,M.J. Metabolically efficient
information processing. Neural Comput. 13, 799-815 (2001).
Buzsaki,G., Kaila,K., & Raichle,M. Inhibition and brain work. Neuron
56, 771-783 (2007).
Accardi,M.V. et al. Mitochondrial reactive oxygen species regulate the
strength of inhibitory GABA-mediated synaptic transmission. Nat. Commun. 5, 3168 (2014).
This work was supported by operating grants from the
Canadian
Institutes
of
Health
Research,
Brain@McGill–Neuroscience Center Zurich partnership and a
Van Gelder salary award from the Savoy Foundation.
requirements.
SA014
Epigenetic regulation of cardiovascular development and
disease
G.W. Dorn
Medicine, Washington University in St Louis, St Louis, MO, USA
Genetic programming determines differentiation of cardiomyocytes from mesodermal precursor cells, the proper
structure of developing embryonic hearts, and adapative or
maladaptive responses to cardiac injury/stress. Conventional
genetic regulatory mechanisms for these processes have been
defined, revealing central roles for a handful of cardiac transcription factors. Similarities in regulated gene expression
observed in embryonic versus injured or diseased adult hearts
became the conceptual foundation for the notion that recapitulated embryonic gene expression is a marker (and potential mediator) of heart disease in adults, the so-called “fetal
gene program”. Recent technological advances in genomics
have revealed, in addition to the established transcriptional
pathways, multiple epigenetic mechanisms that contribute to
the overall transcriptional landscape of developing and diseased hearts. We will review the mechanism by which noncoding RNAs, derived from what has in the past been considered “junk DNA”, post-transcriptionally (microRNAs) and
epigenetically (long non-coding [lnc] RNAs) orchestrate gene
and gene product expression. We will evaluate accumulating
evidence that DNA methylation, chromatin remodeling, and
histone modification are dynamic processes that modulate
gene expression in cardiac development and disease. Finally,
we will consider an integrated model in which these epigenetic mechanisms mutually interact to govern and direct transcription factor-mediated cardiac gene expression.
requirements.
Research Symposia
SA015
miRNAs and epigenetic regulation of vascular phenotype
R. Boon
Goethe University, Frankfurt, Germany
Non-coding RNAs, in particular microRNAs, have recently
gained much interest as key regulators of cellular functions.
In the vasculature, many miRNAs have been identified that
play a role in the endothelium or smooth muscle cells. We have
focused on miRNAs that are regulated by fluid shear stress in
endothelial cells, as well as miRNAs that are regulated by aging
in the cardiovascular system.
Using gene set enrichment analysis of microarray expression
profiling of shear stress exposed HUVECs, we found that the
sequence complementary to the miR-30 seed is enriched in
genes that are downregulated by shear stress. Consistently,
miR microarray analysis showed that exposure of human umbilical vein ECs (HUVECs) to prolonged laminar shear stress upregulates all members of the miR-30 family, (1.6- to 2.0-fold,
p<0.05). To determine the regulation of miR-30, we overexpressed the shear-induced transcription factor Krüppel-like factor 2 (KLF2). KLF2 overexpression induced a 1.7- to 2.1-fold
upregulation of the miR-30 family members (p<0.05). Since
miR-30 family members are predicted to affect inflammation
associated genes, we further characterized the function of miR30 family members. Overexpression of miR-30a and -30b in
HUVECs by transfection of precursor miRs (Pre-miR) reduced
the TNF-α-induced expression of the cell-cell adhesion molecules E-selectin, VCAM1 and ICAM1. Furthermore, miR-30 is
predicted to target the 3’UTR of Angiopoietin-2 (Ang2) and
overexpression of miR-30 inhibits the expression of Ang2, while
miR-30 inhibition using LNA-modified antisense oligonucleotides induces Ang2 expression. KLF2 overexpression also
reduces Ang2 levels. Stimulation with recombinant Ang2 alleviates the anti-inflammatory properties of miR-30 overexpression, indicating a causal role of Ang2 for the effects of miR30.
The miR-143/145 family is likewise induced by laminar shear
stress in endothelium. We show that miR-143/145 are induced
by KLF2 and are secreted in vesicles by endothelial cells. These
vesicles are taken up by smooth muscle cells and this induces
a contractile phenotype. Injection of miR-143/145-containing vesicles reduces atherosclerosis formation in mice in vivo.
Another miRNA that plays a role in smooth muscle cells is miR29, which is induced by aging and inhibits the expression of
extracellular matrix proteins. Inhibition of miR-29 augments
extracellular matrix production and reduced age-induced
aneurysm formation.
requirements.
SA016
of global chromatin remodeling in the setting of complex diseases is not understood. Common diseases like cardiovascular disease are the result of interplay between common genetic
variation and environmental factors—they are also the leading causes of death worldwide. A better understanding of the
molecular events that cause these diseases offers great potential to impact human health. Our group seeks to understand
the basic principles for genome packaging and regulation in
the adult heart, in particular, in the cardiac myocyte, which is
a non-dividing cell that is multi-nucleated and whose genome
is largely heterochromatic. The approaches we are taking in
mouse models include in vivo imaging of global chromatin patterns (on the scale of the whole genome and individual chromosomes, using DAPI and fluorescence in situ hybridization,
FISH) as well as investigation of specific loci for genes operative in the cardiac myocyte (using FISH and real-time transcription assays). In parallel studies, we are using sequencingbased genomics experiments, including chromatin
immunoprecipitation and bisulfite sequencing, to investigate
the role of specific proteins and DNA methylation, respectively,
in the regulation of cardiac gene expression. The dynamics of
chromatin rearrangement during disease are being investigated using pressure overload and isoproterenol (beta-adrenergic receptor agonist) models of cardiac hypertrophy and failure. Our studies are revealing the principles for cardiac-specific
genome packaging and transcriptome regulation, identifying novel molecular mechanisms that causally impact the
response of the heart to injury. Furthermore, by investigating
the role of genetic diversity in chromatin structure, we have
identified features of the epigenome that may be predictive
of disease severity prior to environmental insult. The goal of
these studies is to understand the logic for genome packaging in the adult heart and to use this knowledge to design diagnostic and therapeutic strategies for heart failure.
Franklin S, Chen H, Mitchell-Jordan S, Ren S, Wang Y, Vondriska TM.
Quantitative analysis of the chromatin proteome in disease reveals
remodeling principles and identifies HMGB2 as a regulator of hypertrophic growth. Mol Cell Proteomics. 2012; 11:M111.014258.
PMID:22270000
Mitchell-Jordan S, Chen H, Franklin S, Stefani E, Bentolila LA, Vondriska
TM. Features of endogenous cardiomyocyte chromatin revealed by
super resolution STED microscopy. J Mol Cell Cardiol. 2012. 53:552558. PMID:22846883
Chen H, Monte E, Parvatiyar M, Rosa Garrido M, Franklin S, Vondriska
TM. Structural considerations for chromatin state models with transcription as a functional readout. FEBS Lett. 2012. 586: 3548-3554.
PMID:22940112
Monte E, Mouillesseaux K, Chen H, Kimball T, Ren S, Wang Y, Chen JN, Vondriska TM, Franklin S. Systems proteomics of cardiac chromatin
identifies nucleolin as a regulator of growth and cellular plasticity in
cardiomyocytes. Am J Physiol. 2013;305:H1624-38. PMID: 24077883
This work is supported by the United States National Institutes
of Health, the American Heart Association and the Department
of Anesthesiology in the David Geffen School of Medicine at
UCLA.
requirements.
In vivomeasurements of chromatin dynamics reveal
principles for genomic packaging in health and disease
T. Vondriska
Anesthesiology, UCLA, Los Angeles, CA, USA and Physiology, UCLA,
Los Angeles, CA, USA
While changes in chromatin modifications and gene expression are well characterized in eukaryotic development, the role
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Research Symposia
SA017
MicroRNA-mediated mechansims of renal injury
J. Lorenzen
Hannover Medical School, Hannover, Germany
Ischemia-reperfusion (I/R) injury of the kidney is one of the primary causes of acute kidney injury. It is associated with severe
morbidity and mortality and thus represents a major socioeconomic health problem. It is a consequence of a variety of
different injurious insults in native kidneys (e.g. during cardiac
surgery). Moreover, it is commonly associated with the transplantation procedure and thus an unavoidable phenomenon
in transplanted kidneys 1. During ischemic acute kidney injury
a transient drop in blood flow to the kidney is followed by a
reperfusion period. Reperfusion itself, though vital to a restoration of kidney function, is associated with significant additional
cellular injury 2. We have recently summarized the deleterious events resulting from I/R-injury 3. The damage inflicted
by tissue ischemia is subsequently aggravated by a dramatic
surge in reactive oxygen and nitrogen species during reperfusion. These induce protein modifications, lipid oxidations,
and DNA double strand breaks finally culminating in endothelial dysfunction, neutrophil adherence to endothelium and
trans-endothelial migration, the release of inflammatory mediators, cellular calcium overload and eventually cell death 3. In
the kidney blood flow to the outer medulla is disproportionately reduced with respect to the reduction in total blood flow.
Thus, epithelial cell injury is mainly detected in the S3 segment
of the proximal tubule, located in the outer medulla. Interplay
of several events contributes to the cellular injury observed in
the kidney. The damaged endothelium interacts with and activates inflammatory cells through enhanced expression of adhesion molecules (e.g. ICAM-1, selectins) 4. This interaction in
turn contributes to obstruction of capillaries and postcapillary
venules, further activation and transmigration of leukocytes,
production of cytokines and inflammation in tubular epithelial cells 5. Capillary rarefaction in the inner stripe of the outer
medulla ensues, which due to the development of chronic
hypoxia is an important contributor to post-AKI tubule-interstitial fibrosis and progression to chronic kidney disease 4. Cell
polarity and cytoskeletal arrangement is severely impaired in
proximal tubular epithelial cells during ischemia 4. Important
phenotypical changes are loss of the proximal tubule brush
border as well as loss of polarity and derangement of adhesion molecules and other membrane proteins and disruption
of cell-cell interactions at adherent and tight junctions 4.
MicroRNAs (miRNAs) are currently under intense investigation
as powerful regulators of various diseases with potential critical impact on disease initiation and/or progression, including kidney disease 5. MiRNAs represent small non-coding RNA
transcripts with a length of ~22 nucleotides, which through
post-transcriptional binding of the 3’-untranslated region (UTR)
of mRNA targets lead to the repression of gene/protein expression and/or translational inhibition of protein synthesis 5. The
first miRNA, lin-4, was discovered while investigating genetic
loci responsible for temporal patterning in Caenorrhabdidits
elegans 5. Intriguingly, a single microRNA may alter the expression of a large number of target genes, thus influencing a specific pathology by regulating whole disease-specific pathways
and signalling cascades rather than a single gene. This unique
function underlines the immense importance of these small
molecules. The biogenesis of microRNAs follows a tightly regulated pattern 5. Following transcription in the nucleus miRNA
precursors are transported into the cytosol, where they are
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further processed by a ribonuclease, named DICER 5. Mature
miRNAs finally interact in the so called RNA-induced silencing
complex (RISC) with cognate sequences in the 3’-untranslated
region (3’-UTR) of their target mRNAs 8. MiRNA antagonists
(antimiRs) are available to specifically cleave mature miRNAs,
thereby silencing their cellular effects and thus serve as powerful novel therapeutics of many diseases 5. Our group is interested in the mechanisms of microRNA mediated kidney injury.
These short, noncoding RNA molecules are involved in a variety of different biological processes such as proliferation, apoptosis and differentiation. We aim to identify novel microRNAs in different mouse models of kidney injury and elucidate
involved signaling pathways. We are interested in modulating
pathological microRNA expression by RNA therapeutics,
termed antagomirs, which enable specific targeting and cleavage of miRNAs and thus modulation of pathological signaling
pathways in vivo. In addition, microRNAs are released into the
extracellular compartment (blood and urine) in patients. Thus,
circulating microRNAs may serve as a non-invasive tool to
detect and monitor disease activity 6. We also aim to investigate the release pattern of circulating microRNAs.
Bon D, Chatauret N, Giraud S, Thuillier R, Favreau F, Hauet T. New
strategies to optimize kidney recovery and preservation in transplantation. Nat Rev Nephrol. 2012; 8: 339-347.
Weight SC, Bell PR, Nicholson ML. Renal ischaemia—reperfusion injury.
Br J Surg. 1996; 83: 162-170.
Lorenzen JM, Batkai S, Thum T. Regulation of cardiac and renal
ischemia-reperfusion injury by microRNAs. Free Radic Biol Med. 2013;
.
Bonventre JV, Yang L. Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest. 2011; 121: 4210-4221.
Lorenzen JM, Haller H, Thum T. MicroRNAs as mediators and therapeutic targets in chronic kidney disease. Nat Rev Nephrol. 2011; 7:
286-294.
Lorenzen JM, Thum T. Circulating and urinary microRNAs in kidney
disease. Clin J Am Soc Nephrol. 2012; 7: 1528-1533.
requirements.
SA018
Novel mechanisms regulating cardiac class II histone
deacetylases
M. Avkiran
Cardiovascular Division, King’s College London, London, UK
The activity of myocyte enhancer factor 2 (MEF2) transcription factors is a critical driver of the pathological cardiac hypertrophy and remodeling processes that culminate in heart failure, in both acquired and genetic forms of the disease.
Extensive studies in recent years have revealed that class IIa
histone deacetylase (HDAC) isoforms, such as HDAC4 and
HDAC5, act as signal-responsive repressors of nuclear MEF2
activity in cardiac myocytes and that their spatial regulation
provides a key mechanism for the neurohormonal control of
such activity. The compelling scheme that has emerged is that
HDAC4 and HDAC5 are direct substrates for serine/threonine
protein kinases of the Ca2+/calmodulin-dependent kinase family, and that the phosphorylation of conserved residues that
flank nuclear localization signal domains in HDAC4/5 leads to
their exclusion from the nucleus and consequent de-repression of MEF2 activity. Recent studies suggest that protein
kinase A (PKA) may also regulate MEF2 activity through HDAC-
Research Symposia
mediated mechanisms, but in an opposing manner, by promoting HDAC5 nuclear retention or HDAC4 proteolytic cleavage. Furthermore, there is emerging evidence that HDAC
nuclear localization may be regulated additionally by a novel,
phosphorylation-independent mechanism, through redoxdependent modification of reactive cysteine residues in
HDAC4. Finally, HDAC5 may directly interact with HDAC2, a
class I HDAC that has itself been causally implicated in cardiac
hypertrophy and remodeling, and alter its function through
deacetylation. How these different pathways are integrated in
the neurohormonal regulation of HDAC localization and function in cardiac myocytes is unclear, with the picture further
complicated by the existence of regulatory crosstalk between
relevant kinase signaling pathways. This presentation will discuss emerging evidence on the mechanism(s) through which
clinically significant neurohormonal stimuli may trigger posttranslational regulation of HDAC isoforms, with particular focus
on the role of kinases, phosphatases and oxidative signalling.
requirements.
SA019
Overview of extrapituitary growth hormone and prolactin:
Autocrine/paracrine roles in health and disease
S. Harvey1, C.G. Martinez-Moreno1, M. Luna2 and C. Aramburo2
1Physiology,
University of Alberta, Edmonton, AB, Canada and
Celular y Molecular, Instituto de Neurobiolgia,
Universidad Nacional Autonoma de Mexico, Queretaro, Qro,
Mexico
2Neurobiologia
Growth hormone (GH) and prolactin (PRL) are both endocrines
that are synthesized and released from the pituitary gland into
systemic circulation. Both are therefore hormones and both
have numerous physiological roles mediated through a myriad of target sites and both have pathophysiological consequences when present in excess or deficiency. GH or PRL gene
expression is not, however, confined to the anterior pituitary
gland and it occurs widely in many of their central and peripheral sites of action (1,2). This may reflect “leaky gene” phenomena and the fact that all cells have the potential to express
every gene that is present in their genome. However, the presence of GH or PRL receptors in these extrapituitary sites of
GH and PRL production suggests that they are autocrine or
paracrine sites of GH and PRL action. These local actions often
occur prior to the ontogeny of pituitary somatotrophs and lactotrophs and they may complement or differ from the roles of
their pituitary counterparts. Many of these local actions are
also of physiological significance, since they are impaired by
a blockade of local GH or PRL production or by an antagonism of local GH or PRL action. These local actions may also
be of pathophysiological significance, since autocrine or
paracrine actions of GH and PRL are thought to be causally
involved in a number of disease states, particularly in cancer.
Autocrine GH for instance, is thought to be more oncogenic
than pituitary GH and selective targeting of the autocrine moiety may provide a therapeutic approach to prevent tumor progression. In summary, GH and PRL are not just endocrine hormones, as they have autocrine and/or paracrine roles in health
and disease.
Harvey S (2010). Endocrine 38, 335-359
Harvey S, Aramburo C, & Sanders EJ (2012). Endocrine 41, 19-30
Supported by NSERC of Canada and CONACyT of Mexico
requirements.
SA020
Growth hormone production and role in the reproductive
system of female chicken
A. Hrabia
Animal Physiology and Endocrinology, University of Agriculture in
Krakow, Krakow, Poland
Growth hormone (GH) produced mainly by pituitary gland
plays a key role in growth regulation, cell differentiation and
metabolism control. Accumulating evidence in mammals indicates that it is also involved in the regulation of reproductive
functions through endocrine, autocrine or paracrine pathways.
In recent years, research has been undertaken tending towards
the explanation the role of GH in local regulation of development and functions of the ovary and oviduct in birds.
Protein and mRNA expression of GH and its receptors (GHR)
was found in individual compartments of the chicken ovary.
GH-immunoreactivity was more intense in the granulosa layer
than in the theca layer. Moreover, secretion of GH by the granulosa cells of the largest preovulatory ovarian follicles was
revealed. These observations suggest that avian ovary is an
extrapituitary site of GH synthesis and GH-responsive organ.
Subsequent studies have shown that the role of GH in the ovary
is associated with the regulation of fundamental processes
occurring in it, such as steroidogenesis, proliferation and apoptosis. It was observed that recombinant chicken GH (cGH)
injected during sexual maturation caused increase in progesterone content in the chicken ovary just before and after maturation as well as increase in estradiol content before maturation. In vitro experiments revealed that cGH directly
stimulated estradiol secretion by chicken prehierarchical ovarian follicles whereas inhibited release of estradiol and elevated
secretion of progesterone by yellow hierarchical follicles. Farther, cGH lowered LH-stimulated secretion of estradiol by the
theca layer of the three largest preovulatory follicles. Locally
produced GH was able to stimulate the synthesis of progesterone by the granulosa cells of the largest follicle, which was
blocked by cGH antibody. Up-regulation of cytochrome P450scc mRNA expression (a rate-limiting enzyme in progesterone synthesis) was also shown in hen ovarian follicles. Injections of chickens with cGH from 10 weeks of age until
maturation resulted in increased weight of the ovary, number of ovarian follicles and number of proliferating cells in the
ovarian stroma and prehierarchical follicles. These results support earlier suggestion that GH is involved in regulation of proliferation process since injections of ovine GH to the laying
hens increased the number of small follicles in the ovary. Concomitantly, cGH administration significantly diminished apoptotic cell number in the ovarian stroma and white follicles during puberty.
Most recent results indicate that the avian oviduct, like the
ovary, is an extrapituitary site of GH production and a target
organ for GH. The expression of GH mRNA and protein has
been found in oviductal segments (differing in structure and
functions). Similarly mRNA expression and protein localization
of GHR have been demonstrated in the chicken oviduct. Expression of GHR was lower in the infundibulum than in the magnum, isthmus and shell gland. This may indicate that GH participates in the regulation of egg component synthesis in these
parts. In addition, GHR immunoreactivity was particularly pres-
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Research Symposia
ent in the mucosa lining the oviduct, what further suggests
involvement of GH in the production of egg constituents.
Exogenous GH treatment to chickens resulted in an increase
in mRNA expression of ovalbumin, major egg-white protein
synthesized in the magnum, and ovocalyxins 32 and 36
(eggshell matrix proteins) produced in the shell gland as well
as increase in the eggshell thickness. The action of GH in the
avian oviduct appears to be also related to the regulation of
apoptotic cell death, since injections of cGH for several weeks
during puberty caused inhibition of cell apoptosis as identified by TUNEL assay, and reduced expression and activity of
selected caspases, markers of apoptosis, in the magnum of
chicken oviduct.
In summary, the results so far clearly indicate that GH is
involved in general mechanism responsible for activation and
functioning of avian ovary and oviduct. The action of GH may
reflect the effect of pituitary GH, but evidence that GH is produced in the reproductive system and parallel localized with
its receptors strongly suggests that GH may also act as
paracrine and/ or autocrine factor.
Supported by grants: DS-3243/KFiEZ, MNiSZW – 2 P06D 034
28 and NCN - 2011/01/B/NZ4/03665
requirements.
for prostate cancer. Our data show that the activation of Stat5
signaling correlates with the occurrence of pathological
basal/stem cell clusters in prostate tumors. Immunohistochemical analyses of these basal/stem cell clusters and immediately adjacent luminal cells demonstrated higher proliferation, Stat5 signaling, and expression of the stem/progenitor
cell marker Sca-1 (stem cell antigen 1) compared to physiological basal cell clusters that are occasionally detected in wildtype prostates. These results suggest that basal cells clusters
in Pb-PRL mice are more numerous and also display altered differentiation status. In addition, FACS analysis of isolated
prostate cells revealed the existence of a new subpopulation
of Sca-1-positive luminal cells that was amplified in Pb-PRL compared to wild-type prostates. These cells might represent luminal progenitors originating from the amplified basal/stem cell
compartment and could be relevant in the initial steps of PRLinduced prostate tumorigenesis (7). Despite the obvious link
between Stat5 signaling and histopathological features of PbPRL prostate tumors, our ongoing data indicate that mouse
as well as human basal/stem cells are not direct targets of PRL.
This suggests that the pathogenic phenotypes of Pb-PRL
prostates are in fact mediated by paracrine messengers that
we aim to identify.
Wu, Z. S. et al. (2011) J Clin.Endocrinol.Metab 96, E1619-E1629
Li, H. et al.(2004) Cancer Res. 64, 4774-4782
Goffin, V. et al. (2011) Nat.Rev.Urol. 8, 597-607
Kindblom, J. et al. (2003) Endocrinology 144, 2269-2278
SA021
Rouet, V. et al. (2010) Proc Natl.Acad.Sci.U.S.A 107, 15199-15204
Goldstein, A. S. et al (2010) Science 329, 568-571
Locally expressed growth hormone and prolactin in cancer
Sackmann Sala et al., AM. J. Pathol., in revision
V. Goffin and L. Sackmann Sala
Institut Necker Enfants Malades, Inserm and University Paris
Descartes, Paris, France
Although prolactin (PRL) and growth hormone (GH) are prototypic pituitary hormones, they are also produced in several
extrapituitary sites, including male and female reproductive
tissues. However, the physiological relevance of these locallyproduced hormones in these tissues is far from being well
understood due to unknown extrapituitary PRL/GH gene regulation and detection/quantification issues. The amplification
of autocrine/paracrine PRL and/or GH signaling have been suggested to participate in tumorigenesis of various tissues, including prostate, mammary and endometrial cancers (1;2). Intriguingly, the molecular mechanisms of action of autocrine GH
have been suggested to exhibit some differences compared
to circulating (endocrine) GH, which could ultimately favor
tumor progression and resistance to treatments.
Within the past few years our Laboratory has focused on the
mechanisms of action of locally-produced PRL on prostate
tumorigenesis (3). We use Pb-PRL transgenic mice that express
rat PRL under the control of the prostate-specific probasin promoter leading to high PRL secretion by luminal epithelial cells
(4). These mice develop benign prostate hyperplasia (BPH)
slowly progressing to prostate intraepithelial neoplasia (PIN)
that in some instances further evolve to locally invasive adenocarcinomas. Besides strong phosphorylation of Stat5, canonical signaling pathway of the PRL receptor, the major hallmark
of these tumors involves anarchic amplification of the p63-positive basal epithelial cell compartment (5). This compartment
is (one of) the niche(s) of prostate stem cells, whose involvement in prostate cancer initiation and recurrence has been
recently assessed (6). Therefore, understanding the mechanism of basal/stem cell amplification due to local hyperprolactinemia may be of broad interest to develop new treatments
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Grant support: Fondation ARC (PDF20101202327 and
SFI20101201635), Fondation de France (2011-00020359), La
Ligue contre le Cancer (13/75-63), INCa (n° 2013-107)
requirements.
SA022
Extra-pituitary prolactin and prolactin-like hormones in
zebrafish and chickens
Y. Wang, J. Li, G. Bu and L. Zhu
College of Life Sciences, Sichuan University, Chengdu, Sichuan,
China
It is well documented that prolactin (PRL) plays important roles
in controlling vertebrate reproduction, growth and development, metabolism, osmo-regulation, immunomodulation, and
behavior. PRL is synthesized mainly by pituitary lactotrophs
and regulates these physiological processes through an
endocrine route in vertebrates; however, there has been
increasing evidence that in mammals, PRL is also expressed
in the extra-pituitary tissues such as brain, immune cells, skin
and mammary gland, and plays important autocrine/paracrine
roles in these tissues (Ben-Jonathan et al. 1996, Harvey et al.
2012). In contrast, evidence for the extra-pituitary expression
of PRL in non-mammalian vertebrates was limited. Here, we
summarize our recent progress on the expression of PRL gene
(PRL) and a novel PRL-like gene (PRL-L) in the extra-pituitary tissues of chickens (Gallus gallus) and zebrafish (Danio rerio). Our
data shows that: 1) cPRL mRNA is expressed in several extrapituitary tissues of chickens, including testes and spinal cord
Research Symposia
(detected by quantitative real-time RT-PCR), and its expression
in the testes is stage-dependent with relatively high mRNA levels noted at the post-hatch stage, whereas cPRL mRNA seems
to be highly expressed in the spinal cord at all stages examined (from embryonic day 14 to adult stage); 2) cPRL mRNA
is also expressed in the skin of chicken embryos or post-hatching chicks, and its expression is likely associated with feather
development; 3) a novel prolactin-like protein (cPRL-L) encoded
by PRL-L gene (EU327778) is present in chickens. This putative
PRL-L shares 30–35% amino acid sequence identity with PRL
of chickens, rats and humans. Functional study further proved
that recombinant cPRL-L protein produced in E. coli can activate chicken PRL receptor (cPRLR) expressed in human hepatocellular carcinoma (HepG2) cells, indicating that cPRL-L functions as an additional ligand for cPRLR; 4) Unlike cPRL, cPRL-L
is widely expressed in adult chicken extra-pituitary tissues with
only minimal expression detected in the pituitary gland by RTPCR. In day-7 chicken embryos, the mRNA expression of cPRLL, but not cPRL, was also detected in all extra-pituitary tissues
examined; 5) Congruent to our findings in chickens, a functional prolactin-like protein (zfPRL-L, EU327779) was also identified in zebrafish, and RT-PCR assay revealed that zfPRL-L mRNA
is expressed in the extra-pituitary tissues including brain, gill,
muscle, ovaries and testes. Taken together, the wide expression of PRL and/or PRL-L in the extra-pituitary tissues of chickens (or zebrafish) suggests their potential paracrine/autocrine
roles in these tissues of non-mammalian vertebrates.
Ben-Jonathan, N et al. (1996). Endocr Rev 17, 639-669.
Harvey, S et al. (2012). Endocrine 41, 19-30.
This work was supported by grants from the Natural Science
Foundation of China (30971569, 31172202, and 31271325 )
requirements.
SA023
Prolactin and the skin
R. Paus1,2 and E. LangaN1,3
1Dermatology Research Centre, University of Manchester,
Manchester, UK, 2Dept. of Dermatology, University of Muenster,
Muenster, Germany and 3Dept. of Dermatology, University of
Luebeck, Luebeck, Germany
Over the past decade, evidence has accumulated that mammalian integument, including human skin, is an important
extrapituitary site of prolactin (PRL) expression and hormone
activity, since both murine and human skin not only express
functional receptors, but also PRL itself. This is well in line with
the long-standing recognition that the mammary gland is an
epidermally derived skin appendage, in whose development
and remodelling during lactation PRL is appreciated to play an
important role, and that PRL is known to impact on hair growth
in several mammalian species. Since integumental structures
far antedate the appearance of a pituitary gland during evolution, it is reasonable to assume that PRL functions in the skin
of current day mammals are indicative of ancestral functions
of this pleiotropic peptide hormone, some of which may still
be conserved and operative in human skin. However, it remains
unclear whether PRL generated in human skin is systemically
released or primarily serves intracutaneous auto-/paracrine
functions. Moreover, the functional significance of intracutaneous PRL expression in skin health and disease (e.g. psoriasis, lupus erythematosus) remains quite unclear.
Here we summarize our own limited excursion into cutaneous
PRL biology, which has helped to generate some intriguing
pointers that guide future work at this interdisciplinary
research frontier that borders on epithelial biology, neuroendocrinology and clinical dermatology. Following up our older
findings on the hair cycle-dependent expression pattern and
hair cycle-inhibitory effects of PRL in mice, we subsequently
showed that PRL also inhibits the growth of male occipital scalp
hair follicles (HFs). Yet, in female frontotemporal human HFs
PRL has hair growth-stimulatory properties and also elicits a
strikingly distinct gene expression response than in male HFs.
Thus, quite like with androgens and estrogens, the response
of human skin to PRL shows fundamental gender- and/or location dependent differences.
Regarding the controls of PRL and PRLR expression in human
skin, data from the organ culture of female human scalp HFs
suggest that classical stimulators of pituitary PRL secretion,
i.e. thyrotropin-releasing hormone (TRH) and 17-beta-estradiol, both of which are also produced by the pilosebaceous
unit itself, upregulate intracutaneous PRL and PRLR expression, just as they do in the pituitary gland. Instead, dopamine,
the dominant pituitary (inhibitory) control of PRL expression
and secretion does not change the follicular expression of PRL
protein in situ. Interestingly, the proinflammatory cytokine,
interferon-gamma, increases PRL immunoreactivity in the
epithelium of organ-cultured human HFs, while tumour necrosis factor -α decreases both PRL and PRLR protein expression
in situ. This indicates that the central and peripheral regulation of PRL in humans, namely in the pituitary gland and the
skin, shows important similarities, but is distinct. This may be
important in inflammatory dermatoses (e.g. psoriasis, lupus
erythematosus), and their management (e.g., with TNF-alpha
antagonists).
PRL has also surfaced as a novel neuroendocrine regulator of
human keratin expression and epithelial stem cell biology in
situ, since PRL differentially regulates a defined subset of keratins and keratin-associated proteins in human scalp HFs. For
example, PRL up-regulates expression of keratins K5 and K14
and of the epithelial stem cell-associated keratins K15 and K19,
whereas it inhibits K6 and K31 expression. Since a pure competitive PRL receptor antagonist alone also modulates keratin expression, “tonic stimulation” by endogenously generated PRL may be required for normal expression levels of
selected keratins.
Taken together with the recognized role of PRL as immunomodulator and in regulating epithelial cell proliferation/apoptosis/
electrolyte transport and preliminary evidence that PRL may
negatively regulate selected aspects of wound healing, these
recent insights into PRL as modulator of keratin expression and
epithelial stem cell function in human skin and/or HFs underscore that the systematic exploration of PRL functions in
human skin is likely to uncover novel, translationally relevant
PRL functions in peripheral tissue physiology and pathology.
requirements.
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Research Symposia
SA024
Normal function of the skeletal muscle microvasculature
and its endothelium
A.J. Wagenmakers
Research Institute for Sport and Exercise Sciences, Liverpool John
Moores University, Liverpool, UK
The skeletal muscle microcirculation is in full control of the
delivery of nutrients and oxygen to skeletal muscle fibers. The
presence of a monolayer of endothelial cells on the luminal
side supports this function. The total ECL surface area in an
adult human has been estimated at > 700 m2. The microvasculature contributes 99% of this with most of the ECL being
present in the dense network of muscle capillaries [1]. The
rationale for this distribution is that the ECL in muscle controls
the transendothelial transport of nutrients, oxygen and hormones into the interstitial fluid that surrounds the muscle
fibres. With multiple terminal arterioles and 20-40 capillaries
serving one muscle fibre, the microvasculature is acutely and
chronically managing to match energy supply to the demand
of individual muscle fibers. The transport capacity depends on
the available surface area. About 10% of the muscle capillaries is perfused in the resting state. The additional recruitment
during exercise is large and required to ensure that the supply of blood-borne fuels and oxygen meets the high energy
demand of the contracting muscle fibres.
Textbooks traditionally assume that activation of the insulin
signalling cascade in skeletal muscle fibres is the primary control mechanism for glucose uptake. There now is compelling
evidence that the delivery of insulin and glucose to the muscle interstitium is a conditional early event in muscle glucose
uptake [2]. Experiments using contrast-enhanced ultrasound
to estimate the volume of blood present in the muscle
microvasculature, have shown that physiological increases in
insulin lead to rapid increases in the microvascular blood volume in skeletal muscle. In rats these increases occur as early
as 5-10 min after the start of a physiological insulin infusion
and precede both activation of the muscle insulin signalling
cascade and increases in muscle glucose uptake, which are
seen after 15-30 min [3]. This increase in microvascular blood
volume has also been observed after ingestion of a mixed meal
and during light exercise and is taken to primarily reflect the
recruitment of capillaries that were not perfused before. This
conclusion is supported by estimates of the permeability surface area product (PSA) for insulin and glucose in human studies. The PSA went up 2-fold during an oral glucose tolerance
test and 11-fold during a physiological insulin infusion, with
equal-fold increases seen for skeletal muscle glucose uptake
[4].
Important information on the underlying mechanisms has
come from studies in cultured endothelial cells, which identified an endothelial insulin signalling cascade, in which activation of Akt leads to ser1177 phosphorylation and activation
of eNOS and therefore an increase in the production of the
potent vasodilator nitric oxide (NO). In vivo NO then acts upon
the smooth muscle cell layer of skeletal muscle arterioles [5]
and leads to vasodilatation and recruitment of additional muscle capillaries. An important in vivo observation [3] was that
pre-treatment of rats with the eNOS inhibitor L-NAME prevented the insulin induced increase in microvascular blood volume in skeletal muscle and reduced both glucose uptake and
activation of the insulin signalling cascade in skeletal muscle.
Other signals found to activate eNOS via ser1177 phosphorylation are fluid shear forces exerted on a cultured EC mono-
14P
layer and exposure of cultured EC’s to vascular endothelial
growth factor (VEGF) and may be relevant for the angiogenic
effect of exercise and determine perfusion rates of skeleletal
muscle during exercise [6].
Topics covered in this symposium: Anton Wagenmakers will
give an introduction to explain the normal function of the
endothelium of the microvasculature in skeletal muscle and
the impact that impairments in insulin, VEGF and exercise shear
stress induced eNOS activation are expected to have on insulin
sensitivity, angiogenesis and the regulation of muscle perfusion during exercise. Michelle Keske will present her latest data
on the impaired microvascular responses that are seen in obesity, ageing and other insulin resistant states. Jefferson Frisbee will report the changes seen in the spatial distribution and
time responses in the microvascular perfusion of skeletal muscle in rats with the metabolic syndrome. Matthew Cocks on
the basis of his latest exercise training interventions will then
encourage you to adopt a more physically active lifestyle to
keep the density and vasodilatory potential of your skeletal
muscle as high as possible and thus prevent impaired glucose
tolerance and chronic disease in later life [7]. Ed Eringa will
present recent data demonstrating that perivascular adipose
tissue within skeletal muscle alters vascular function and
impairs insulin-induced vasodilatation in muscle of obese individuals.
Wolinsky H (1980). Circ Res 47,301-311.
Barrett EJ et al. (2009). Diabetologia 52, 752-764.
Vincent MA et al. (2004). Diabetes 53, 1418-1423.
Gudsbjornsdottir S et al. (2003). J Clin Endocrinol Metab 88, 45594564.
Rattigan S et al. (2006). Essays Biochem 42, 133-144.
Wagenmakers AJM et al. (2006). Essays Biochem 42, 193-210.
Wagenmakers AJM and Cocks M (2013). Physiology News 93, 22-26.
requirements.
SA025
Muscle microvascular blood flow responses in insulin
resistance and ageing
M.A. Keske, D. Premilovac, R.M. Dwyer, S.M. Richards and
S. Rattigan
University of Tasmania, Hobart, TAS, Australia
Insulin resistance plays a key role in the pathogenesis of type
2 diabetes and precedes the onset of diabetes. Skeletal muscle is the major site for insulin-mediated glucose storage and
is thereby a key influence on postprandial blood glucose levels [1]. Insulin resistance is characterised by impaired insulinmediated glucose disposal in skeletal muscle and is often associated with endothelial dysfunction. Although multiple
mechanisms underlie development of insulin resistance, our
research over the past 15 years implicates microvascular dysfunction in skeletal muscle as a major cause. We have shown
that insulin enhances flow to the microvasculature in muscle
facilitating access of glucose and insulin to the myocyte, thus
contributing to enhanced glucose disposal [2]. Obesity [3],
insulin resistance (induced by high fat or high sodium diets for
example) [4] and ageing [5] are associated with impaired
microvascular responses in skeletal muscle. Impairments in
insulin-mediated microvascular perfusion in muscle can directly
cause insulin resistance [6], and this event occurs early in the
Research Symposia
aetiology of this condition. Understanding the mechanisms
involved in the development of microvascular dysfunction in
muscle is crucial to prevent the progression of insulin resistance, and it also provides a novel treatment strategy in which
microvascular function can be targeted preventing the development of type 2 diabetes.
Defronzo RA (1988). Diabetes 37, 667-687.
Vincent MA et al. (2004). Diabetes 53, 1418-1423.
Clerk LH et al. (2006). Diabetes 55,1436-1442.
St-Pierre P et al. (2010). Diabetes Obes Metab 12, 798-805
Durham WJ et al. (2010). FASEB J. 24, 4117-27
Premilovac D et al. (2013). Cardiovasc Res 98, 28-36.
This work was funded by the National Health and Medical
Research Council of Australia, Australian Research Council,
Heart Foundation of Australia, and National Institutes of Health.
requirements.
SA026
Altered perfusion control within skeletal muscle with
metabolic syndrome: Does a shifted attractor define
peripheral vascular disease?
J.C. Frisbee1,2, J.T. Butcher1,2, P.D. Chantler3,2, S.J. Frisbee4,2
and F. Wu5,2
1Physiology
and Pharmacology, West Virginia University HSC,
Morgantown, WV, USA, 2Center for Cardiovascular and Respiratory
Sciences, West Virginia University HSC, Morgantown, WV, USA,
3Exercise Physiology, West Virginia University HSC, Morgantown,
WV, USA, 4School of Public Health, West Virginia University HSC,
Morgantown, WV, USA and 5Novartis Institutes for Biomedical
Research, East Hanover, NJ, USA
A key clinical outcome for peripheral vascular disease (PVD)
in patients is a poor ability for skeletal muscle to resist fatigue
with elevated metabolic demand. Our investigations have
revealed that the most commonly interrogated sites of physiological dysfunction (e.g., altered dilator reactivity, endothelial function, constrictor reactivity, etc.) are not robust predictors of poor functional outcomes. Given this, we approached
the issue of vasculopathy with elevated PVD risk from the perspective of hemodynamic control and mass transport and
exchange using a vertically-integrated, multi-scale approach.
These studies build on previous work in the skeletal muscle
microcirculation of the obese Zucker rat (OZR) model of the
metabolic syndrome to integrate existing knowledge into a
greater understanding of the links between impaired muscle
perfusion and performance (1). In OZR cremaster muscle, we
demonstrated that perfusion distribution at microvascular
bifurcations (γ) was consistently more heterogeneous than in
controls; increasing from ~0.46-0.54 to ~0.57-0.61 in OZR.
Further, this shift in γ was spatially consistent and impacted
perfusion distribution throughout the network. However, the
underlying mechanistic contributors were spatially divergent
as altered adrenergic constriction was the major contributor
to altered γ at proximal microvascular bifurcations, and exhibited a steady decay in terms of its significance with longitudinal distance, while endothelial dysfunction was a stronger contributor to altered γ in distal bifurcations with no discernible
role proximally (despite the dysfunction being clearly demonstrable). Using measured values of γ, simulations predict that
alterations to γ in OZR caused more heterogeneous perfusion
distribution in distal arterioles than in controls; an effect that
could only be rectified by combined adrenoreceptor blockade
and improveed endothelial function. Using tracer washout, we
determined that this effect was also demonstrable using the
in situ gastrocnemius muscle of OZR. (2,3) To minimize the
functional implications of this increased spatial perfusion heterogeneity, a likely compensatory mechanism could be an
increased temporal switching at arteriolar bifurcations to minimize downstream perfusion deficits. Using the in situ cremaster muscle, we determined that temporal activity (the
cumulative sum of absolute differences between successive
values of γ, taken every 20 seconds) was lower in OZR than in
control animals, and this difference was present in both proximal (1A-2A) and distal (3A-4A) arteriolar bifurcations. While
adrenoreceptor blockade improved temporal activity in 1A2A arteriolar bifurcations in OZR, this was without impact in
the distal microcirculation, where only interventions against
oxidant stress and thromboxane A2 were effective. (4) Analysis of the attractor for γ indicated that it was not only elevated
in OZR versus controls, but also exhibited reductions in range,
indicative of a loss of system flexibility and the ability to adapt
to imposed physiological, pharmacological, and/or pathological challenges. It must be noted that these cumulative
upstream impairments to hemodynamic control then enter
into a capillary network that has experienced a rarefaction of
22-25%, thus further compromising the processes of mass
transport/exchange. (5) In our most recent work, we have
determined the ultimate hemodynamic consequence of these
cumulative upstream perfusion problems, using both the cremaster and gastrocnemius preparations to determine
microvascular hematocrit (HMV) in the skeletal muscle of OZR.
The use of a multiple tracer washout approach demonstrated
that aggregate HMV in OZR skeletal muscle tended to be
reduced as compared to controls, although this effect was not
pronounced. However, based on an imaging/counting
approach in the cremaster muscle, HMV in OZR was demonstrated to be much more variable than in controls, with many
capillaries have extremely low hematocrit levels (6). These
results suggest that intramuscular perfusion distribution in
the OZR model of the metabolic syndrome is altered to result
in an extremely variable and excessively stable distribution of
blood and erythrocyte flow in the distal microcirculation. Combined with a growing inability of the microcirculation to control its own perfusion in response to any imposed challenge,
these may represent increasingly significant contributors to
the manifestation of poor muscle performance in this condition.
Frisbee JC, Goodwill AG, Butcher JT, Olfert IM. (2011) Exp Physiol.
96:369-83.
Wu F, Beard DA, Frisbee JC. (2011). J Physiol. 589:4527-43.
Frisbee JC, Wu F, Goodwill AG, Butcher JT, Beard DA. (2011). Am J Physiol Regul Integr Comp Physiol. 301:R975-86.
Butcher JT, Goodwill AG, Stanley SC, Frisbee JC. (2013). Am J Physiol
Heart Circ Physiol. 304:H547-58.
Goodwill AG, Frisbee SJ, Stapleton PA, James ME, Frisbee JC. (2009).
Microcirculation. 16:667-84.
J.T. Butcher, S.C. Stanley, S.D. Brooks, P.D. Chantler, F. Wu, J.C. Frisbee. (2014). Microcirculation (in review)
American Heart Association (IRG 14330015), the National
Institutes of Health (RR 2865AR, P20 GM103434, T32 HL
90610), a West Virginia University Research Development
Grant and support provided through Center for Cardiovascular
and Respiratory Sciences at the West Virginia University Health
Sciences Center.
15P
Research Symposia
requirements.
requirements.
SA027
SA028
Effect of different training modes on skeletal muscle
microvascular density and endothelial enzymes controlling
NO production
Perivascular adipose tissue control of insulin-induced
vasoreactivity in skeletal muscle
M. Cocks
School of Sport and Exercise Sciences, Liverpool John Moores
University, liverpool, UK
Impairments in microvascular perfusion that occur as a result
of capillary rarefaction or reduced insulin mediated dilation of
terminal arterioles have been proposed to limit nutrient and
insulin delivery to skeletal muscle in sedentary individuals, obesity, metabolic syndrome and ageing, via a mechanism involving reductions in endothelial NO production. As such the mechanisms that underlie these impairments, and how lifestyle
interventions influence them, are of great scientific and clinical significance.
Current knowledge on the enzymes that determine NO
bioavailability is primarily based on measurements in isolated
blood vessels from animal models. A lack of suitable techniques
has meant there is a paucity of data regarding the mechanisms
that lead to impaired microvascular perfusion in human skeletal muscle. As such our lab has recently developed immunofluorescence microscopy methods which allow quantitation
of the protein content and phosphorylation state of enzymes
within the endothelium of the microvasculature of human muscle (1).
Using these techniques our lab has demonstrated that the
microcirculation in human muscle is highly responsive to
increases in physical activity with substantial changes in capillary density and content and activity of endothelial enzymes
that control muscle perfusion occurring. In support of this,
endurance training (ET) in lean healthy individual results in
increased capillarization and elevated eNOS protein content
(2). Interestingly, ET in obese individuals causes similar
increases in capillarization and eNOS content, but also results
in decreased NADP(H)oxidase content, which, is not apparent in lean individuals. Such adaptations are not restricted to
endurance-type activities. High intensity interval training (HIT)
induces comparable improvements in capillarization and
endothelial enzymes to ET in both lean (1) and obese individuals (unpublished data). However, 6wks of HIT in lean individuals increased eNOS protein content of the muscle microvascular endothelium more than ET (2; P < 0.05). These findings
add to the growing support that HIT is an effective time-saving training method. Furthermore, unpublished data suggest
that resistance training induces smaller improvements in
endothelial enzymes than ET and HIT. It is becoming increasingly apparent that precise regulation of skeletal muscle perfusion by the microvasculature is crucial for maintaining
healthy skeletal muscle function. As such interventions aimed
at improving microvascular perfusion are crucial, with the
above data suggesting that exercise training is an essential
lifestyle modification capable of maintaining or improving
skeletal muscle endothelial function. However, further research
is necessary to fully elucidate the microvascular adaptations
which occur following exercise training, and what form of training is optimal as a therapeutic strategy.
Cocks M et al. (2012). Microcirculation 19, 642-651
Cocks, M., et al. (2013). J Physiol 591, 2682-2690
16P
E.C. Eringa
Laboratory for Physiology, Institute for Cardiovascular Research
(ICaR-VU), VU University Medical Center (VUMC), Amsterdam,
Netherlands
The muscle microcirculation regulates delivery of nutrients
and oxygen to myocytes, acutely and chronically matching
supply and demand. Consequently, microvascular characteristics such as resistance, surface area and flow regulate myocellular glucose uptake(1) and exercise tolerance. Microvascular
endothelium plays a critical role in these functions through
control over vascular resistance, angio- and arteriogenesis, distribution of blood flow and inflammation. In man, insulin-stimulated perfusion of capillaries in muscle is closely related to
insulin-stimulated glucose uptake(1;2). In rodent models,
insulin-induced NO synthesis has been shown to regulate glucose uptake through transendothelial transport (TET) of insulin
from blood to the muscle interstitium. The balance between
secretion of NO and endothelin-1 by endothelial cells is an
important mediator of these effects.
Insulin’s effects on muscle perfusion are impaired in obesity
and are regulated by bioactive products of adipose tissue,
adipokines(3). Adipokines such as adiponectin, fatty acids and
cytokines, activate distinct signaling mediators in vascular
endothelium, including 5’AMP-activated protein kinase, protein kinase C theta (PKCθ) and c-jun N-terminal kinase, respectively.
It has been recently established that perivascular adipose tissue (PVAT), found around arteries, veins and the proximal part
of microvascular beds (4), is a functionally relevant source of
adipokines. In the aorta and coronary circulation, PVAT is a site
of inflammation in atherosclerosis (5). In resistance arteries,
PVAT determines vascular tone through direct actions on
smooth muscle (6), and through indirect effects on insulin signaling in vascular endothelium (7). These effects of PVAT are
effectuated by 5’AMP activated protein kinase (AMPK) and
inflammatory signaling through c-jun N-terminal protein kinase
(JNK). Infiltration of inflammatory cells and the resultant signaling cascades play critical roles in impairment of vasodilator
effects of PVAT in obesity(6,7). A mediating role of PVAT in the
relationship between obesity and insulin resistance in man is
further supported by our recent finding that vasodilator effects
of muscle PVAT are impaired in obese subjects.
In conclusion, recent clarification of the complex relationships
between local adipose tissue, microvascular endothelium and
muscle cells provides new avenues for prevention of type 2
diabetes and cardiovascular disease in the near future.
Kubota T, Kubota N, Kumagai H, Yamaguchi S, Kozono H, Takahashi
T, Inoue M, Itoh S, Takamoto I, Sasako T, Kumagai K, Kawai T,
Hashimoto S, Kobayashi T, Sato M, Tokuyama K, Nishimura S, Tsunoda M, Ide T, Murakami K, Yamazaki T, Ezaki O, Kawamura K, Masuda
H, Moroi M, Sugi K, Oike Y, Shimokawa H, Yanagihara N, Tsutsui M,
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and Kadowaki T. Impaired Insulin Signaling in Endothelial Cells Reduces
Insulin-Induced Glucose Uptake by Skeletal Muscle. Cell Metabolism
13:294-307,2011.
Research Symposia
Meijer RI, de Boer MP, Groen MR, Eringa EC, Rattigan S, Barrett EJ,
Smulders YM and Serne EH. Insulin-induced microvascular recruitment
in skin and muscle are related and both are associated with whole
body glucose uptake. Microcirculation 19:494-500,2012.
Eringa EC, Bakker W and van H, V. Paracrine regulation of vascular
tone, inflammation and insulin sensitivity by perivascular adipose tissue. Vascul Pharmacol 56:204-209,2012.
Yudkin JS, E Eringa and Stehouwer CD. “Vasocrine” signalling from
perivascular fat: a mechanism linking insulin resistance to vascular disease. Lancet 365:1817-1820,2005
Mazurek T, Zhang L, Zalewski A, Mannion JD, Diehl JT, Arafat H, SarovBlat L, O’Brien S, Keiper EA, Johnson AG, Martin J, Goldstein BJ, Shi Y.
Human Epicardial Adipose Tissue Is a Source of Inflammatory Mediators. Circulation. 108:2460-2466,2003.
Greenstein AS, Khavandi K, Withers SB, Sonoyama K, Clancy O,
Jeziorska M, Laing I, Yates AP, Pemberton PW, Malik RA, Heagerty AM.
Local inflammation and hypoxia abolish the protective anticontractile
properties of perivascular fat in obese patients. Circulation 119:16611670,2009
Meijer RI, Bakker W, Alta CL, Sipkema P, Yudkin JS, Viollet B, Richter
EA, Smulders YM, van H, V, Serne EH and Eringa EC. Perivascular Adipose Tissue Control of Insulin-Induced Vasoreactivity in Muscle Is
Impaired in db/db Mice. Diabetes 62:590-598,2013.
requirements.
SA029
porters that mediate the translocation of inorganic and small
organic anions.
Biophysically, prestin acts as an area motor by alternating
between two conformations that occupy different cross-sectional areas within the membrane. Joint conformational transitions of the tightly packed prestin motors in the plasma membrane of the OHC are driven by changes in membrane potential
and result in macroscopic cellular length changes. The molecular mechanism underlying electromotility is probably related
to the ion transport cycle in SLC26 transporters, as non-mammalian prestin orthologs are anion antiporters and prestindriven electromotility is strictly anion-dependent. Beyond these
observations, however, the molecular mechanisms underlying motor activity and transport by SLC26 proteins remained
essentially unknown, as structural information for the SulP
transporter family is lacking.
We have now derived a structural model of prestin and of a
transport-competent ortholog by combining homology modeling, molecular dynamics and substituted cysteine accessibility scanning. The model provides a structural explanation
for the anion dependence of prestin’s electromechanical activity and rationalize the relevance of previously identified protein domains required for electromotility. It provides a structural framework for deciphering the mechanistic principles of
both, electromechanical activity of prestin and anion transport by SLC26/SulP transporters.
requirements.
SLC26 transporters and Epithelial Fluid and HCO3- secretion
S. Muallem
SA031
Molecular Physiology and Therapeutics Branch, NIDCR/National
Institutes of Health, Bethesda, MD, USA
-
HCO3 secretion is a key function of secretory epithelia and
involves HCO3- entry at the basolateral membrane and exit
across the luminal membrane. In most epithelia the bulk of
HCO3- entry is mediated by the Na+-HCO3- co-transporter
NBCe1-B and HCO3- exit is mediated by the combined and regulated action of CFTR and members of the SLC26 transporters.
The function and regulation of the various SLC26 transporters
expressed in epithelia is beginning to emerge. In this presentation, first, the properties of several SLC26 transporters will
be reviewed and second, we will discuss regulation of slc26a6
by the IP3 Receptor-binding Protein Released with IP3 (IRBIT)
in the context of synergism in epithelial fluid and HCO3- secretion.
requirements.
SA030
Slc26 members in the auditory system
D. Oliver
Dept. of Physiology, Philipps University, Marburg, Germany
The high acuity of hearing in mammals requires an active
mechanical process boosting vibration in the inner ear.
‘Cochlear amplification’ is provided by high-frequency cellular
motility of auditory outer cells (OHCs). This ‘electromotility’
is generated by prestin (SLC26A5), an OHC-specific membrane
protein acting as an electro-mechanical transducer. Intriguingly, Prestin belongs to the SLC26/SulP family of anion trans-
Diverse physiological functions of Slc26 members in the
gastrointestinal tract: Lessons learned from genetic diseases
and gene-deficient mice
U. Seidler
Hannover Medical School, Hannover, Germany
The gastrointestinal tract expresses a multitude of Slc26 family members, several of them with ill-defined function. This
presentation will focus on the three Slc26 isoforms that are
highly expressed in the apical membrane of gastrointestinal
epithelial cells, but with different segmental expression and,
most importantly, different anion transport properties, as discussed by the first speaker.
The Slc26a9 isoform is strongly expressed in the luminal membrane of the stomach, is also found in oesophagus and duodenum, the lung, and specialized cells of the kidney, pancreas,
reproductive tract and neuronal system. Its genetic deletions
results in a complete loss of gastric acid secretion and extensive fundic hyperplasia within a few weeks after birth. Duodenal HCO3- secretion is also altered in Slc26a9 knockout mice,
particularly at young ages. Polymorphisms in this gene carry
a higher risk of meconium ileus in cystic fibrosis newborns and
a worse prognosis for CF diabetes.
The Slc26a6 isoform is particularly strongly expressed in the
upper small intestine, the renal proximal tubule, the pancreas,
but at relatively lower levels in various other organs. Its genetic
deletion results in reduced small intestinal HCO3- and oxalate
secretion, the development of oxalate kidney stones in some
mouse strains and small intestinal fluid and nutrient absorptive defects. A polymorphism in the Slc26a6 gene is associated
with increased urinary oxalate excretion and hypertension.
The Slc26a3 isoform is very highly expressed in the large intestine and the gallbladder and at lower levels in the small intes-
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Research Symposia
tine, the pancreas and the reproductive system. Mutations in
Slc26a3 are the molecular basis for congenital chloride-losing
diarrhoea. These patients suffer from lifelong diarrhoea. One
of the associated findings in long term follow up of larger
patient cohorts is a high incidence of both acute and chronic
intestinal inflammation. The Slc26a3-deficient mouse proved
to be a good model for studying potential barrier defects in
these mice that may explain the underlying pathophysiology,
such as defective ion transport, an altered mucus barrier, and
changes in the intestinal microbiome.
requirements.
requirements.
SA032
SA033
The role of IRBIT in synergistic activation of the SLC26
transporters by Ca2+ and cAMP signaling
Role of SLC26A9-mediated Cl- secretion in mucus obstruction
and airway inflammation
S. Park
M.A. Mall
Ewha Womans Univerisity, Seoul, Republic of Korea
University of Heidelberg, Heidelberg, Germany
A central function of epithelia is the control of the volume and
electrolyte composition of bodily fluids through vectorial transport of electrolytes and the obligatory H2O. In exocrine glands,
fluid and electrolyte secretion is carried out by both acinar and
duct cells, with the portion of fluid secreted by each cell type
varying among glands. All acinar cells secrete isotonic, plasmalike fluid, while the duct determines the final electrolyte composition of the fluid by absorbing most of the Cl- and secreting HCO3 -. Ductal fluid and HCO3 - secretion are mediated by
the basolateral membrane Na+-HCO3 - cotransporter NBCe1-B
and the luminal membrane Cl-/HCO3 - exchanger slc26a6 and
the Cl- channel CFTR. The function of the transporters is regulated by multiple inputs, which in the duct include major regulation by the WNK/SPAK pathway that inhibit secretion and
the IRBIT/PP1 pathway that antagonize the effects of the
WNK/SPAK pathway to both stimulate and coordinate the
secretion. An important concept in biology is synergism among
signaling pathways to generate the final physiological response
that ensures regulation with high fidelity and guards against
cell toxicity. While synergism is observed in all epithelial functions, the molecular mechanism mediating the synergism is
not known. Recent work reveals a central role for IRBIT that
integrates and synergizes the function of the Ca2+ and cAMP
signaling pathways in activation of epithelial fluid and electrolyte secretion. In the resting state IRBIT is sequestered by
the high level of IP3Rs at the apical pole. Minimal stimulation
of Gs-coupled cAMP generating receptors results in PKA-mediated phosphorylation of the IP3Rs at specific serine residues
that increases the apparent affinity of the IP3Rs to IP3 and at
the same time reduces their apparent affinity to IRBIT. Now,
weak stimulation of Gq-coupled GPCRs that generates minimal amount of IP3 results in dissociation of IRBIT from the
IP3Rs and its translocation to CFTR and slc26a6 to activate
them and stimulate fluid and electrolyte secretion. These findings provide a general molecular pathway for synergism
between the Ca2+ and cAMP signaling pathways with multiple
implications to disease states, including the autoimmune disease Sjogren’s syndrome and cystic fibrosis.
Mucus obstruction and airway inflammation are key pathological features of chronic airway diseases including cystic fibrosis (CF) and asthma. Previous studies in human primary airway
epithelial cultures and transgenic mouse models demonstrated
that airway surface dehydration caused by defficient CFTRmediated Cl+ secretion and/or increased Na+ absorption mediated by epithelial Na+ channels (ENaC) constitutes an important mechanism leading to impaired mucociliary clearance and
lung disease in CF.
Besides restoration of CFTR function, activation of alternative
Cl+ channels may improve airway surface hydration in CF airways. Recent evidence suggests that epithelial Cl+ secretion
is increased in Th2-mediated airway inflammation, however,
the molecular identity of the underlying Cl+ conductance was
not known. SLC26A9 is a member of the SLC26 family of anion
transporters predominantly expressed in epithelia of the lung
and upper GI tract. Recent studies showed that SLC26A9 functions as a Cl+ channel that contributes to constitutive and cAMPdependent Cl+ secretion in human bronchial epithelial (HBE)
cells. Based on its functional properties in transduced cells and
expression pattern in mouse airways, we hypothesized that
SLC26A9 may function as an alternative Cl+ channel that may
contribute to airway surface liquid (ASL) homeostasis in health
and in Th2-mediated airway inflammation.
To test this hypothesis, we compared transepithelial ion transport in freshly excised bronchial tissues, lung morphology and
airway mucus content in wild-type versus Slc26a9-deficient
mice under physiological conditions and after intratracheal
instillation of IL-13 to model Th2-mediated airway inflammation. Further, in a human study population of 661 children with
asthma (i.e. a prototypical Th2-dominated airway disease) and
658 healthy controls, we tested if polymorphisms in the
SLC26A9 gene are associated with asthma.
In bioelectric studies in native airway tissues, lack of SLC26A9
had no effect on basal, cAMP-mediated or Ca2+-activated Cl+
conductance (CaCC) under physiological conditions. In airways
from wild-type mice, constitutive as well as Ca2+-activated Cl+
secretion were ~2-fold increased after intratracheal instillation
of IL-13. The IL-13 induced constitutive Cl+ conductance was
completely abrogated, whereas up-regulation of CaCC was preserved in native airway tissues from SLC26A9-deficient mice.
While IL-13 induced goblet cell metaplasia and mucus overproduction to similar levels in both genotypes, lack of SLC26A9mediated Cl+ secretion was associated with airway mucus
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Research Symposia
obstruction in IL-13 treated SLC26A9-deficient mice that did
not occur in wild-type controls.
Testing of effects of genetic variants within the SLC26A9 locus
on asthma risk identified a single nucleotide polymorphisms
(SNP) in the 3’UTR of SLC26A9 (rs2282430) that was associated with asthma. In silico analyses predicted that the A allele
of rs2282430 strengthens binding of hsa-miR-632 to the
SLC26A9 3’UTR. Functional testing of precursor hsa-miR-632
on the wild-type 3’UTR sequence and the SNP in HEK293 cells
using luciferase reporter assays demonstrated that hsa-miR632 reduced luciferase activity in cells co-transfected with
the SNP compared with the wild-type 3’UTR. These results suggest that the SNP in the SLC26A9 3’UTR identified in our
genetic studies reduced protein expression and indicate that
binding of hsa-miR-632 is involved in this process.
Our studies demonstrate that SLC26A9 constitutes a Cl+ channel in native airway epithelia that is quiescent under physiological conditions, but constitutively active to promote Cl+
secretion and airway surface hydration in Th2-mediated airway disease. The observation that genetic deletion of SLC26A9
produced significant airway mucus obstruction in allergic airway disease indicates that SLC26A9-mediated Cl+ secretion is
essential for maintaining ASL homeostasis required for effective MCC in the presence of mucus hypersecretion. These
results suggest that SLC26A9 may serve as a novel therapeutic target to improve airway surface hydration in CF and potentially other mucostatic airway diseases.
requirements.
molar) concentrations. By virtue of engaging enzyme-coupled
receptors, these low NO concentrations, which are well below
the affinity of NO for its receptors (10 nM), are able to stimulate physiological (submicromolar) rises in cGMP concentration. By contrast, when populations of densely-packed neurones are active simultaneously, NO is able to act more like a
volume transmitter, coordinating the behaviour of intermingled cells even though they lack any anatomical connectivity
with the NO-producers.
Batchelor, A.M., Bartus, K., Reynell, C., Constantinou, S., Halvey, E.J.,
Held, K.F., Dostmann, W.R., Vernon, J. & Garthwaite, J. (2010) Exquisite sensitivity to subsecond, picomolar nitric oxide transients conferred on cells by guanylyl cyclase-coupled receptors. Proc. Natl. Acad.
Sci. U. S. A, 107, 22060-22065.
Roy, B., Halvey, E.J. & Garthwaite, J. (2008) An enzyme-linked receptor mechanism for nitric oxide-activated guanylyl cyclase. J. Biol. Chem.,
283, 18841-18851.
Wood, K.C., Batchelor, A.M., Bartus, K., Harris, K.L., Garthwaite, G.,
Vernon, J. & Garthwaite, J. (2011) Picomolar nitric oxide signals from
central neurons recorded using ultrasensitive detector cells. J. Biol.
Chem., 286, 43172-43181.
requirements.
SA035
Activation of neuronal ANO1 (TMEM16A) channels by
localized Ca2+ signals
N. Gamper
SA034
Localised versus volume transmission by nitric oxide
J. Garthwaite
Wolfson Inst for Biomed Res, University College London, London,
UK
Nitric oxide (NO) functions as a transmitter/diffusible second
messenger in most tissues, its formation from L-arginine typically being triggered by a rise in Ca2+(via calmodulin). NO
exerts physiological effects by binding to specialized guanylyl
cyclase-coupled receptors, resulting in cGMP generation in target cells. Despite having many context-dependent roles and
being implicated in numerous disease states, there has been
a lack of conceptual understanding of how NO operates at the
cellular and subcellular levels. From this perspective, many
uncertainties remain, including the NO concentrations attained
following activation of NO synthases (prototypically in neurones or endothelial cells) by different stimuli, their timecourses, how far the signals spread in physiologically relevant
concentrations (whatever those may be), what constitutes a
pathological NO concentration, and the mechanisms responsible for NO consumption in different tissues, how rapidly they
operate, and where. We have adopted several approaches
aimed at gaining a more concrete, quantitative, understanding of this widespread signalling pathway. These approaches
have included analyzing NO receptor function, allowing it to
be encapsulated in a quantitative model (Roy et al., 2008), realtime imaging of cellular NO signal transduction in target cells
(Batchelor et al., 2010), and the use of ultrasensitive detector
cells to record NO generation from native sources (Wood et
al., 2011). The current picture is that, when generated in a
synapse, NO is likely to act only very locally, probably within
the bounds of that synapse, and to exist only in very low (pico-
Faculty of Biological Sciences, University of Leeds, Leeds, UK
Peripheral nociceptive (‘pain’) neurons are primary sensors of
tissue damage. One group of chemical stimuli that can excite
these neurons and cause pain is the ligands of pro-inflammatory G protein coupled receptors (GPCR) that activate phospholipase C and induce release of Ca2+ from the intracellular
stores. This class of GPCR includes bradykinin B2 receptor and
protease activated receptor-2 (PAR2). Among several cellular
targets of such receptors is Ca2+ activated Cl- channel ANO1
(TMEM16A), which depolarizes and excites nociceptive neurons as these accumulate high intracellular Cl- concentrations
(1, 2). Since many types of Ca2+ signals exit in neurons, nociceptive neurons must be able to identify Ca2+ signals originating specifically from the potentially damaging stimulus
while ignoring ‘irrelevant’ Ca2+ signals. We found that ANO1
in nociceptive dorsal root ganglion (DRG) neurons exists as a
part of a signaling complex that also includes B2 and/or PAR2
receptors (3). The complex is assembled at the junctions
between plasma membrane and endoplasmic reticulum (ER)
and ensures close apposition (and physical association) of
plasma membrane ANO1 channels and ER’s IP3 receptors; the
later serve as sources of Ca2+ for the activation of ANO1 in
response to PAR2 or B2 receptor triggering. We further hypothesize that these ANO1-containing junctional microdomains
may create a local environment that is poorly accessible for
cytosolic Ca2+ ions that do not originate from the ER, thus
protecting ANO1 from ‘irrelevant’ Ca2+ signals. Indeed, we
found that ANO1 channels couple poorly to the Ca2+ influx
through the voltage-gated Ca2+ channels or TRPV1. Disruption of these ANO1-containing complexes (e.g. by disrupting
interaction of ANO1 with the IP3 receptors) results in ‘promiscuous’ activation of ANO1 by global cytosolic Ca2+ signals
which, in turn, makes nociceptive DRG neurons more excitable
(3). Similar ANO1 signaling complexes may exist in other neu-
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Research Symposia
rons and non-neuronal cells. For example, in smooth muscle
cells a close association of endogenous Ca2+ activated Cl- channels and ryanodine receptors has been hypothesized (4). In
sum, we suggest that in nociceptive DRG neurons and possibly in some other cell types there are specific signaling complexes, which bring together ANO1, GPCRs, and ER-localized
IP3 receptors. These complexes may serve to ensure the fidelity
of GPCR signaling.
requirements.
SA037
Cho H et al. (2012). Nat Neurosci 15, 1015-1021.
Signalling domains involved in the modulation of the slow
afterhyperpolarising potassium current sI(AHP) and the
firing properties of hippocampal pyramidal neurons
Jin H et al. (2013). Sci Signal 6, ra73.
P. Pedarzani
Rao R et al. (2008) J Gen Physiol 132:145-160.
NPP, UCL, London, UK
requirements.
A slow afterhyperpolarization (sAHP) follows bursts of action
potentials in hippocampal pyramidal neurons and is mediated
by a Ca2+-dependent K+ current known as sI(AHP). This current
presents slow kinetics of activation and decay, with time constants in the range of seconds. The sI(AHP) is voltage-independent: its activation requires changes in the intracellular
Ca2+ concentration generated by the opening of voltage-gated
Ca2+ channels during action potentials. Calcium-induced calcium release from ryanodine-sensitive stores further contributes to the activity-dependent potentiation of this current.
In hippocampal neurons, sI(AHP) can be easily distinguished
from the SK-channel mediated I(AHP): the two currents present different kinetics, functional coupling to calcium sources
and pharmacological profiles, with the bee-venom toxin
apamin inhibiting I(AHP), but not sI(AHP). The sAHP is responsible for the late phase of spike frequency adaptation and leads
to a strong reduction in action potential firing. A distinctive
feature of sI(AHP) is its modulation by several neurotransmitters and second messenger pathways. In particular, various
monoamine transmitters (noradrenaline, dopamine, histamine
and serotonin) suppress sI(AHP) through a signaling pathway
that involves cAMP as a second messenger and the activation
of protein kinase A (PKA). Additionally, the sI(AHP) is tonically modulated by the basal level of activity of PKA and a serine/threonine protein phosphatase, suggesting that the sAHP
channels might be part of a signalling complex. To identify the
components of the signaling domains underlying the
monoaminergic and basal modulation of sI(AHP), we first
focused on specific adenylyl cyclases. In particular, we
addressed the role of the calcium-stimulated adenylyl cyclases,
AC1 and AC8, by using genetically modified mice lacking both
AC1 and AC8 (DKO). Suppression of the sI(AHP) by beta-adrenergic receptor agonists and serotonin was similar in CA1 pyramidal neurons from wildtype and DKO mice. Activation of NMDA
receptors by high frequency (100 Hz) synaptic stimulation used
for the induction of long-term potentiation at the Schaffer collateral-CA1 synapse leads to a PKA-dependent, transient inhibition of the postsynaptic sAHP (1). The NMDA receptor-mediated suppression of sAHP is abolished in DKO mice. Similar
results were obtained in response to a stimulation protocol
pairing postsynaptic depolarization with 5 Hz presynaptic stimulation. We conclude that the sAHP channels might be part
of distinct signalling domains in CA1 pyramidal neurons. The
calcium-sensitive adenylyl cyclases AC1 and AC8 do not seem
to be an essential component of the signalling complex utilized by some monoaminergic transmitters to suppress
sI(AHP), but they are essential for the modulation of sAHP by
NMDA receptors in response to synaptic stimuli that induce
long-term synaptic plasticity.
Liu B et al. (2010). J Clin Invest 120, 1240-1252.
SA036
Fast and slow regulation of M-type K+ channels by
AKAP79/150 signaling complexes
J. Zhang and M.S. Shapiro
Physiology, Univ. Texas Health Sci. Ctr., San Antonio, TX, USA
M-type K+ channels, comprised of KCNQ2-5 (Kv7.2-7.5) subunits, play key roles in the regulation of neuronal excitability.
We study the regulation of M-channel activity and transcriptional expression by A-kinase-anchoring protein (AKAP)79/150.
Our FRET and functional studies suggest AKAP79/150 action
correlates with the PIP2-depletion mode of neuronal M current suppression, and is disrupted by functional calmodulin.
The rapid modulation of M current involves recruitment of PKC
to the channels, their subsequent phosphorylation, and likely
decreased affinity of the channels for PIP2. The complex orchestrated by AKAP79/150 also includes M1 muscarinic receptors,
thus constituting a “signalosome” spanning the initiating
receptor to channel target. We have also discovered a novel
mechanism regulating KCNQ2/3 transcriptional expression by
neuronal activity, involving activation of calcineurin (CaN) and
Nuclear Factor of Activated T-cells (NFAT) transcription factors,
orchestrated by AKAP79/150. The signal requires Ca2+ influx
through L-type Ca2+ channels and both local and global Ca2+
elevations. AKAP79/150 and the complex it organizes play a
distinct role in activity-dependent M-channel transcription,
which may potentially serve throughout the nervous system
to limit over-excitability associated with disease states such as
epilepsy. We are now utilizing “super-resolution” stochastic
optical reconstruction microscopy (STORM) offering sub-diffraction (~20 nm) resolution, to directly visualize these
AKAP79/150 signaling complexes and interactions between
AKAP79/150, ion channels and receptors in neurons. STORM
uses dyes that can cycle between a dark and a fluorescent state
thousands of times, thus enabling detection of the precise
localization of the center of these scattered spots given by
cumulative integration of each cycle. Using multi-color STORM
to simultaneous image AKAP150, KCNQ2-3 or KCNQ5 channels, and receptors, we observe AKAP150 to form signaling
clusters with the channels and receptors at the single-complex
level. We have also obtained evidence that AKAP79/150 links
different channel types together, raising the possibility of their
functional, as well as physical, coupling.
Supported by National Institutes of Health NINDS
20P
Blitzer RD, Wong T, Nouranifar R, Iyengar R & Landau EM. (1995). Postsynaptic cAMP pathway gates early LTP in hippocampal CA1 region.
Neuron 15, 1403-1414.
Research Symposia
requirements.
SA038
An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels. Everett
KL,
Cooper
DMF.
PLoS
ONE
8(9):
e75942.
doi:10.1371/journal.pone.0075942 (2013)
requirements.
Ca2+ and cAMP microdomains centred on adenylyl cyclases
D.M. Cooper
SA039
Pharmacology, University of Cambridge, Cambridge, UK
Ca2+ and cAMP are the archetypical second messengers that
regulate cell physiology and which have established the paradigms for second messenger signalling. It is clear that these
two second messengers modulate each other’s activities at
numerous levels to yield responsive and elegant control over
cellular processes. A particularly important and approachable
interaction is the modulation by Ca2+ arising from store-operated Ca2+-entry of adenylyl cyclase activities in non-excitable
cells. The same detail is not available for the interaction that
arises by Ca2+ arising from L-type channel and adenylyl cyclases
(ACs), although such modulation certainly occurs in both neurons and cardiomyocytes.
Powerful tools have been developed for the dissection of these
interactions in live cells by using fluorescent sensors for both
Ca2+ (based on GCamps) and cAMP (based on EPAC) which
are tethered to adenylyl cyclases. Tethering ACs with these
sensors has no effect on their responsiveness or targetting.
Application of these sensors have uncovered dynamic behaviours of both Ca2+ and cAMP in the domains of the ACs that
are distinct from transitions of these messengers in the cellular cytosol. Indeed Ca2+-sensitive ACs report quite distinct
Ca2+ and cAMP signals to those reported by Ca2+-insensitive
ACs in response to the same signal.
These sensors have also been used to dissect the composition of the complex that mediates the selective sensitivity of
the Ca2+-stimulated AC, AC8, to store-operated Ca2+-entry
(SOCE). Application of these sensors in combination with siRNA
knockdown of target proteins (along with other complex dissection techniques, such as FRET analysis between target proteins, mutagenesis, etc) has allowed the description of an intimate, direct-binding complex that forms between Orai1, the
channel component of SOCE and the AC8.
A newer version of the cAMP sensor, which is resistant to fluctuations in pH associated with L-type channel activity, has now
been developed which will allow the cAMP and Ca2+
microdomains in excitable cells to be approached.
Adenylyl cyclases and the interaction between calcium and cAMP signalling. D.M.F. Cooper, N. Mons and J.W. Karpen. Nature 374, 421-424
(1995).
Organization and Ca2+ regulation of adenylyl cyclases in cAMP
microdomains. Willoughby, D and Cooper, DMF. Physiological Reviews
87, 965-1010 (2007)
Probing functional modules within the central oxytocin
system
V. Grinevich
University of Heidelberg and German Cancer Research Center,
Heidelberg, Germany
Oxytocin (OT) is exclusively expressed in hypothalamic neurons, which extensively project their axons to a wide range of
forebrain and hindbrain regions. Taking in consideration enormous variety of behavioral effects of OT, executed by only 8000
neurons (rat), it was tempting to speculate about existence
of distinct functional modules within the central OT system,
controlling distinct (at least extreme) behaviors. To probe this
possibility we developed and applied new genetic technique
named“virus-mediated genetic activity-induced tagging”
(vGAIT), which is based on transcriptional activity of immediate early gene c-fos. The use of vGAIT inbehaving animals allows
to genetically tag OT neurons, which were active upon a certain behavior, such as fear. Therefore, employing the paradigm
of contextual fear conditioning, we observed an expression
of vGAIT-mediated channel rhodopsin 2 (ChR2) in ~ 15% of OT
neurons, which were historically activated by associative fear
learning. More importantly, we can demonstrate ‘mimicry’ by
eliciting an unfreezing behavior response upon activating OTfibers expressing ChR2 in the central amygdala, which originate from a small fraction of experience-activated OT neurons.
Furthermore, the strength and duration of the effect were
comparable with blue-light inducedmobility observed in animals with expression of ChR2 in all OT neurons. In conclusion,
we have identified a minimal OT neuronal assembly, which is
sufficient to illicit a behavior response by an artificial stimulation of experience activated tagged neurons. The universality
of this assembly for other types of fear (i.e. social or innate
fears) as well as deciphering OT modules for the distinct form
of behaviors, such as empathic and pro-social behaviors,
remain further investigations.
requirements.
SA040
Distinct pools of cAMP centre on different adenylyl cyclase isoforms
in pituitary-derived GH3B6 cells. S. Wachten, N Masada, LJ Ayling, A
Ciruela, VO Nikolaev, MJ Lohse and DMF Cooper. J Cell Science 123,
95-106 (2010)
Oxytocin/vasopressin signaling as ancient modulator of
associative learning in C. elegans
Direct demonstration of discrete Ca2+ microdomains associated with
different adenylyl cyclase isoforms. D Willoughby, S Wachten, N
Masada & DMF Cooper J Cell Science 123, 107-117 (2010)
I. Beets, T. Janssen, L. Temmerman, L. Frooninckx and
L. Schoofs
Direct binding of AC8 and Orai1 underlies dynamic Ca2+-regulated
cAMP signalling. D Willoughby, KL Everett, ML Halls, J Pacheco, P Skroblin, L Vaca, E Klussmann and DMF Cooper Science Signaling (2012) 5,
ra29
KU Leuven, Leuven, Belgium
The neuropeptides oxytocin (OT) and vasopressin (VP) are wellknown for their peripheral hormonal effects, and act as key
neuromodulators in the mammalian brain influencing social
behavior and cognition. Homologous neuropeptides are widely
distributed among the invertebrate lineage as well, suggest-
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Research Symposia
ing an ancient origin for this neuropeptide family. Whereas
hormonal effects of OT/VP-related signaling such as on reproduction and myoactivity seem to be well conserved, potential
neuromodulatory effects remain largely unexplored in invertebrates, keeping their evolutionary origin in the dark.
The nematode Caenorhabditis elegans has a small nervous system of only 302 neurons, but despite its small size harbors a
wide plethora of over 250 bioactive peptides. Using a combined bio-informatic and reverse pharmacology approach, we
have identified a signaling system related to OT and VP signaling in C. elegans. Similar to most invertebrates, C. elegans
has a single OT/VP-related peptide – designated nematocin –
that dose-dependently activates a G protein-coupled receptor
(NTR-1). In vivo localization highlights a putative role of nematocin signaling in modulating the sensory neural circuit, and
knockout of nematocin or its receptor in C. elegans caused
defects in gustatory associative learning. We found that nematocin is released from specific interneurons activating its receptor on a gustatory neuron of the learning circuit, and interacts
with serotonergic and dopaminergic neurotransmission. In
addition, we have identified several other evolutionary conserved neuropeptide systems in C. elegans including tachykinin
and neuromedin related signaling and are currently characterizing their modulatory role in behavioral plasticity.
Our study suggests that neuropeptides of the oxytocin/vasopressin family are ancient modulators of neural circuits underlying behavioral plasticity, and sheds light on the cellular and
molecular networks behind these effects.
requirements.
SA041
Neural mechanisms of social relationships: Implications for
novel treatments for autism
L.J. Young
Psychiatry, Emory University, Atlanta, GA, USA
Social relationships contribute to both mental and physical
wellbeing, and early social experiences can influence in the
quality of relationships throughout the life span. Studies in
monogamous prairie voles have provided insights into the neural mechanisms of the formation and maintenance of social
bonds, as well as to the consequences of social loss. Oxytocin
and vasopressin play important roles in several aspects of social
cognition and behavior in animal models, including social
recognition, maternal nurturing and social bonding. Studies
in mice suggest that these neuropeptides increase the salience
of social stimuli, enhancing the neural processing of social
cues. Species differences in oxytocin and vasopressin receptors expression in the mesolimbic dopamine reward pathway
are associated with species differences in social bonding in
voles. Oxytocin and vasopressin act in the nucleus accumbens
and ventral pallidum, respectively, during pair bond formation
to link the neural encoding of the social stimuli of the partner
with the rewarding aspects of mating. We have identified
genetic polymorphisms that predict neuropeptide receptor
expression in the brain, which in turn predict social behavior,
including susceptibility to the impact of early social stressors
on adult social attachment. Loss of a bonded partner results
in activation of the stress axis and the development of depressive-like behavior in male prairie voles. The social loss induced
depression is mediated by corticotropin releasing factor (CRF),
which suppresses oxytocin secretion in the nucleus accum-
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bens. Infusion of either a CRFR2 antagonist or oxytocin into
the nucleus accumbens prevents social loss-induced depression. This CRF induced negative affect following separation
from the partner may serve to maintain the social bond. There
are remarkable parallels between those studies in voles and
recent studies in humans which suggest that these mechanisms are highly conserved from rodent to man. In humans,
intranasal delivery of oxytocin enhances eye gaze into the eyes
of others, the ability to infer the emotions of others from facial
cues, empathy, and socially reinforced learning. These results
suggest that the oxytocin system may be a viable target for
drugs to improve social functioning in autism.
requirements.
SA042
Oxytocin receptors and social behaviour: Quantity does
matter
M. Busnelli
Institute of Neuroscience, CNR, Milan, Italy and Dept. of Medical
Biotechnology and Translational Medicine, University of Milan,
Milan, Italy
The neuropeptide oxytocin (OXT) regulates a number of socioemotional behaviors in mice, including parental care, pairbonding, social memory, and social aggression by activating
OXT receptors (OXTR). Both distribution and number of OXT
receptors in the brain affect the type and degree of behavioural responses. Many factors, such as early life stress (Lukas
et al., 2010) and poor social rearing conditions (Ahern and
Young, 2009), have been shown to cause a reduction in brain
OXTRs linked to an impairment in social functioning.
Moreover, prolonged pharmacological treatments with neurotrasmitters or neuropeptides can produce variations of receptor expression (receptors desensitization or up-regulation) in
the brain. When C57BL/6J male mice were daily treated with
intranasal OXT (0.15 IU and 0.3 IU) for 15 days (Huang et al.,
2013) we observed a greater reduction of OXTRs (-20%) in limbic brain areas involved in social behavior (i.e. amygdala) compared to a lesser reduction of OXTRs (-10%) in areas involved
in memory and social cognition (i.e. hippocampus, lateral septum). This receptor down-regulation was accompanied by an
impairment in social behaviors towards same and opposite sex
and towards familiar and unfamiliar subjects.
We have also observed that genetically manipulated mice, with
a total absence of OXTRs (Oxtr-/-; Sala et al. 2011) in the brain
exhibit specific social deficits, increased aggression and
impaired cognitive flexibility. However, heterozygous (Oxtr+/) mice, with approximately 50% fewer oxytocin receptors
(OXTRs) in the brain than the wild type, show impaired social
behaviours but not increased aggression or cognitive inflexibility.
Our study indicates that the reduction of the OXTRs affects
specific behaviours in a dose-dependent manner: social behaviour is sensitive to even a partial reduction in OXTR expression,
whereas increased aggression and impairment in cognitive
flexibility occures when the OXTRs are completely absent.
Lukas M, Bredewold R, Neumann ID, Veenema AH. 2010 Maternal separation interferes with developmental changes in brain vasopressin
and oxytocin receptor binding in male rats. Neuropharmacology;58:78–87.
Research Symposia
Ahern TH, Young LJ. 2009 The impact of early life family structure on
adult social attachment, alloparental behavior, and the neuropeptide systems regulating affiliative behaviors in the monogamous prairie
vole (Microtus ochrogaster). Front Behav Neurosci;3:17
Bosch OJ, Neumann ID (2012). Horm Behav 61, 293-303
blood can prove fatal, the regulated excretion of CO2 via breathing is extremely important for life. The accepted explanation
for the nature of this regulation involves the indirect effect of
CO2 on blood pH being detected by chemosensors in the brain
stem that are highly sensitive to pH. Our work challenges this
explanation and suggests that molecular CO2 can be directly
detected.
We discovered that ATP release in the central chemosensitive
areas of the brain stem was a key signalling step in response
to elevated CO2 (1). By following the mechanisms of ATP
release we documented that hemichannels of connexin26
(Cx26) were an important conduit for CO2-dependent ATP
release (2, 3). Furthermore our evidence suggested that CO2
interacted directly with Cx26 to open the hemichannel and
allow the efflux of ATP (3). The ATP, once released, can excite
specific neurons in the brain stem through activation of receptors selectively sensitive to ATP (4, 5). ATP release is thus the
essential link between the opening of Cx26 by CO2, neuronal
activation, and the consequent neurophysiological responses
that result in enhanced breathing.
Most recently we have provided compelling genetic evidence
for the molecular mechanism of direct sensing of CO2 via Cx26
(6). The three known CO2-sensitive connexins (Cx26, Cx30,
Cx32) possess a “carbamylation motif” that is absent from a
CO2-insensitive (Cx31) connexin. This motif comprises Lys125
and four further amino acids that orient Lys125 towards
Arg104 of the adjacent subunit of the connexin hexamer. By
introducing the carbamylation motif into Cx31, we created a
mutant hemichannel (mCx31) that was opened by increases
in CO2. Mutation of the carbamylation motif in Cx26 and
mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the
hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing
of CO2. In our latest studies, my laboratory has used conditional transgenesis to delete Cx26 from specific lineages of
cells in intact mice, such as glial or neural crest-derived cells.
These deletions greatly reduce CO2-dependent ATP release
from the chemosensitive areas of the brain stem. Whole body
plethysmography recordings from awake mice show that their
sensitivity to variations of inspired CO2 is also greatly reduced.
Our studies have helped to establish direct sensing of CO2 via
connexin hemichannels as a new chemosensory transduction
principle in the field in mammalian physiology.
Bosch OJ (2013). Phil Trans R Soc B, 368 20130085
Gourine AV et al (2005) Nature 436, 108-111.
Bosch OJ (2011) . Horm Behav 59, 201-212
Huckstepp RT et al (2010) J Physiol 588, 3901-3920.
Sala M, Braida D, Donzelli A, et al. Mice heterozygous for the oxytocin
receptor gene (Oxtr(+/-)) show impaired social behaviour but not
increased aggression or cognitive inflexibility: evidence of a selective
haploinsufficiency gene effect. J Neuroendocrinol. 2013; 25: 107-18
Sala M, Braida D, Lentini D. et al. Pharmacologic rescue of impaired
cognitive flexibility, social deficits, increased aggression, and seizure
susceptibility in oxytocin receptor null mice: a neurobehavioral model
of autism. Biol Psychiatry. 2011;69(9): 875-82
Huang H, Michetti C, Busnelli M, et al., Chronic and Acute Intranasal
Oxytocin Produce Divergent Social Effects in Mice. Neuropsychopharmacology. 2013 in press
Telethon Foundation (GGP12207), FIRB
RBPR05JH2P004 and ISS (RF-2010-2311148)
Italnanonet
requirements.
SA043
Mother’s love is driven by brain oxytocin - and vasopressin,
too
O. Bosch
University of Regensburg, Regensburg, Germany
Maternal behavior is the most important pro-social female
behavior. Its establishment requires remarkable brain adaptations peripartum like increased activity of the brain oxytocin
system. Recently, we characterized the brain vasopressin system as further important mediator of the fine-tuned regulation of maternal care (Bosch and Neumann, 2012) and maternal aggression (Bosch, 2013) in rodents. While vasopressin and
oxytocin act pro-maternal, these neuropeptides have opposite effects on anxiety (Bosch, 2011). To increase our understanding of how maternal behavior and, consequently, the relationship between mother and infant is regulated provides
important insights into possible dys-regulation of maternal
attachment as seen, for example, in postpartum mood disorders.
requirements.
Huckstepp RT et al (2010) J Physiol 588, 3921-3931.
Thomas T & Spyer KM (2000) J Physiol 523, 441-447.
Wenker IC et al (2012) J Physiol 590, 2137-2150.
Meigh L et al (2013) eLife 2:e01213.
SA044
CO2 signalling via changes in connexin hemichannels and
ATP release
I thank the MRC for generous support.
requirements.
N. Dale
School of Life Sciences, University of Warwick, Coventry, UK
The precise control of breathing is fundamental to the survival
of all terrestrial vertebrates. Breathing fulfils two essential functions: provision of oxygen to support metabolism; and removal
of CO2, the unavoidable by-product of metabolism. CO2 readily combines with H2O to form HCO3- and H+. Because an
increase by only 100 nM of the hydrogen ion concentration in
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Research Symposia
SA045
requirements.
Carbon dioxide - dependent modulation of NFκB
C.T. Taylor1,2, C.C. Scholz2, J. Rodríguez2, B. Bahar3, A. von
Kriegsheim2 and E. Cummins1
1School
of Medicine and Medical Science, UCD, Dublin, Ireland,
2Systems Biology Ireland, University College Dublin, Dublin, Ireland
and 3School of Agriculture, Food Science and Veterinary Medicine,
University College Dublin, Dublin, Ireland
Carbon dioxide (CO2) is increasingly being appreciated as an
intracellular signaling molecule that affects inflammatory and
immune responses. Elevated arterial CO2 (hypercapnia) is
encountered in a range of clinical conditions, including chronic
obstructive pulmonary disease and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. The
current state of the art is that hypercapnia is detrimental in
the context of a pathogenic infectious challenge (1), however,
it may be beneficial against a background of destructive inflammation (2). Thus, CO2 can act at the axis of immunity and
inflammation through its role as a signaling molecule contributing to suppression of innate immune responses and
attenuation of pro-inflammatory signaling. The molecular
mechanisms underpinning CO2-dependent modulation of
immune/inflammatory signaling are not fully understood but
are at least in part regulated through modulation of the NFκB
pathway. NFκB is a master regulator of immune and inflammatory signaling (3). We have recently demonstrated a CO2dependent cellular re-organisation of NFκB family members,
particularly those associated with non-canonical NFκB signaling (IKKα (4) and RelB (5)). In this study, we characterize the
CO2-dependent modulation of RelB, identifying a CO2-dependent cleavage from its C-terminal that is dependent on a 20
amino-acid motif. Furthermore, using semi-quantitative mass
spectrometry, we characterize the RelB interactome under
conditions of elevated CO2 to examine CO2- dependent
changes in protein-protein interactions and gain mechanistic
insight into the molecular mechanisms underpinning CO2 sensitivity of the NFκB pathway. A better understanding of the
molecular mechanisms underpinning CO2 signaling in the context of inflammation and immunity could be of clinical benefit regarding the treatment of patients experiencing hypercapnia and for the development of new therapeutic strategies
for the manipulation of inflammatory signaling.
Gates, K. L., Howell, H. A., Nair, A., Vohwinkel, C. U., Welch, L. C., Beitel, G. J., Hauser, A. R., Sznajder, J. I., and Sporn, P. H. (2013) Hypercapnia Impairs Lung Neutrophil Function and Increases Mortality in
Murine Pseudomonas Pneumonia. Am J Respir Cell Mol Biol
Cummins, E. P., Selfridge, A. C., Sporn, P. H., Sznajder, J. I., and Taylor, C. T. (2013) Carbon dioxide-sensing in organisms and its implications for human disease. Cell Mol Life Sci
Ghosh, S., and Hayden, M. S. (2012) Celebrating 25 years of NF-kappaB research. Immunol Rev 246, 5-13
Cummins, E. P., Oliver, K. M., Lenihan, C. R., Fitzpatrick, S. F., Bruning, U., Scholz, C. C., Slattery, C., Leonard, M. O., McLoughlin, P., and
Taylor, C. T. (2010) NF-kappaB links CO2 sensing to innate immunity
and inflammation in mammalian cells. J Immunol 185, 4439-4445
Oliver, K. M., Lenihan, C. R., Bruning, U., Cheong, A., Laffey, J. G.,
McLoughlin, P., Taylor, C. T., and Cummins, E. P. (2012) Hypercapnia
induces cleavage and nuclear localization of RelB protein, giving insight
into CO2 sensing and signaling. J Biol Chem 287, 14004-14011
Work in the author’s lab is supported by a principal investigator
award (CTT) from Science Foundation Ireland ( CTT, EPC). CCS,
JR and AvonK are supported by a Science Foundation Ireland
award to Systems Biology Ireland.
24P
SA046
CO2 sensing and behavior in insects
I.C. Grunwald Kadow
Sensory Neurogenetics Research Group, Max-Planck Institute of
Neurobiology, Martinsried, Germany
Odors play important roles in the life of many animals including insects. The atmospheric gas CO2 presents an interesting
and for humans relevant example, because CO2 is an attractive cue used by disease vectors such as mosquitoes to find
their hosts for blood feeding. However, CO2 can elicit strong
attraction but also avoidance behavior depending on the insect
species. In contrast to mosquitoes, the vinegar fly Drosophila
melanogaster is strongly repelled by even low levels of CO2
present for instance in an odor released by stressed flies. On
the other hand, it is also produced by rotting fruit the main
food source of these flies.
CO2 is detected by a pair of dedicated 7-TM receptors, Gr21a
and Gr63a, present on the olfactory organs of insects. Upon
activation of CO2 sensory neurons, specific neural circuits are
activated in the brain and trigger the avoidance behavior. We
and others have recently shown that CO2 is processed by parallel neural pathways in a context-dependent manner. A hungry fly might use this parallel processing to overcome its aversion to CO2 in the context of foraging and feeding.
Furthermore, our results suggest that innate CO2 behavior is
modulated by the neurotransmitter dopamine.
In my talk, I will present our latest efforts to understand how
CO2 is modulated and processed in a context-dependent manner in the periphery and central brain.
requirements.
SA047
Bicarbonate/pHi sensitive soluble Adenylyl Cyclase (sAC)
regulates lysosomal pH
N. Rahman1,3, F.R. Maxfield2, T.A. Milner3, J. Buck1 and
L.R. Levin1
1Pharmacology, Weill Cornell Medical College, New York, NY, USA,
2Biochemistry,
Weill Cornell Medical COllege, New York, NY, USA
and 3Brain and Mind, Weill Cornell Medical COllege, New York,
NY, USA
In age-related neurodegeneration, damaged proteins and
organelles accumulate within neurons. Normally, these proteins and organelles would be degraded in the lysosomes by
cathepsins, which are optimally active at acidic pH, but with
aging, age-related diseases and lysosomal storage disorders,
elevation of lysosomal pH hinders normal degradation and
impairs autophagy leading to neuronal dysfunction. The molecular processes and signaling pathways that regulate lysosomal
pH are not understood. While it has been shown that addition
of exogenous cAMP can lower lysosomal pH, the relationship
between cAMP and pH is unclear. Lysosomal pH is dependent
upon the V-ATPase, and bicarbonate-regulated, soluble adenylyl cyclase (sAC) is essential for pH-dependent V-ATPase mobi-
Research Symposia
lization in both epididymis and kidney. We now show sAC to
be essential for lysosomal acidification. In sAC null fibroblasts
and primary neurons, lysosomal acidification is impaired resulting in diminished cathepsin activity, substrate proteolysis and
autophagosome clearance during autophagy. The lysosomal
acidification defect in sAC null cells is rescued by exogenous
cAMP. Thus, sAC appears to be an essential source of cAMP
which regulates lysosomal pH. These results suggest sAC serves
as a previously unappreciated sensor of lysosomal pH, and
define it as a possible target for designing novel treatment
strategies for neurodegenerative disorders.
This work is supported by NIH grants, RO1 GM62328 (JB and
LRL) and 1F31NS081930-01A1 (NR)
requirements.
SA048
The influence of elevated carbon dioxide on cell signalling
and physiology
M. Cann1, M. Turner2, M. Gray2 and Z. Cook1
1SBBS, Durham University, Durham, UK and 2Instutute for Cell and
Molecular Biosciences, Newcastle University, Newcastle, UK
Elevated carbon dioxide is generally detrimental to animal cells
suggesting an interaction with core processes in cell biology.
We demonstrate that elevated carbon dioxide blunts G-protein activated cAMP signalling. The effect of carbon dioxide is
independent of changes in intracellular and extracellular pH,
independent of the mechanism used to activate the cAMP signalling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated
that the effect of elevated carbon dioxide on cAMP levels
required the activity of the inositol triphosphate receptor. Consistent with these findings, carbon dioxide caused an increase
in steady state cytoplasmic calcium concentrations not
observed in the absence of the inositol triphosphate receptor
or under non-specific acidotic conditions. We examined the
well-characterized cAMP dependent inhibition of the isoform
3 Na+/H+ antiporter (NHE3) in kidney epithelial cells to demonstrate a functional relevance for carbon dioxide mediated
reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated carbon dioxide abrogated the inhibitory
effect of cAMP on NHE3 function via an inositol triphosphate
receptor dependent mechanism. We further expand on these
findings through a discussion of the influence of carbon dioxide on the physiology of airways cells.
requirements.
SA049
The physiology of water and electrolyte transport in gut
and kidney
J. Leipziger
Dept. of Biomedicine, Physiology, Aarhus University, Aarhus,
Denmark
loop (TAL). Novel experimental evidence will be presented that
highlight an important role of the TAL in renal acid base physiology. The thick ascending limb of the loop of Henle drives
the urinary concentration ability and at the same time permits
the excretion of extra water via its ability to dilute the urine.
It is critical for the absorption of the divalent cations Ca2+ and
Mg2+. By blocking the apical NKCC2 co-transporter the loop
diuretic furosemide triggers a marked diuresis and saluresis.
In addition, furosemide causes an acute and pronounced urinary acidification and long term application of this diuretic
triggers systemic metabolic alkalosis. This furosemide effect
is currently explained by an increased Na+ load to the collecting duct, which leads to an increased lumen-negative electrical voltage. This in turn facilitates H+ secretion via the apical
H+ ATPase in α-intercalated cells. The direct role of the TAL on
urinary acidification has not been thoroughly addressed. In
this presentation data will be presented that identify the TAL
as a major site of H+ secretion after the application of
furosemide. This TAL-dependent H+ secretion/urinary acid
excretion is of substantial magnitude and can be specifically
blocked by inhibiting the NHE3 Na+/H+ antiporter. These novel
results have major implications to understand the pathophysiology of distal renal tubular acidosis (dRTA), a heterogeneous clinical syndrome with the inability to acidify the urine.
In dRTA the furosemide test is believed to test the ability of
the collecting duct to acidify the urine.
requirements.
SA050
What we see is what we believe. Imaging of kidney
metabolism and transport in health and disease
A. Hall
University of Zurich, Zurich, Switzerland
The major function of the kidney tubule is to transport large
amounts of fluid from the glomerular filtrate back into the
blood-stream. This imposes high energy demands on tubular
cells, which are densely populated with mitochondria to generate sufficient ATP via aerobic metabolism. Transport and
metabolism are thought to be tightly coupled in kidney
tubules, but the mechanisms via which they are related are
poorly understood. Mitochondria are complex and dynamic
organelles that have a number of other important functions
beyond ATP production. A range of insults can damage mitochondria in kidney tubules - including ischemia, diabetes, toxic
drugs and ageing - leading to solute transport defects and progressive declines in kidney function. We have developed live
imaging techniques using multiphoton microscopy (MPM) to
study the function and behavior of mitochondria in tubular
cells in intact kidney tissue, both under physiological conditions and in disease models. Using this approach we have discovered that intrinsic mitochondrial function varies along the
nephron, and have made novel insights into mechanisms of
mitochondrial dysfunction in kidney tubular diseases.
Hall AM, Rhodes GJ, Sandoval RM, Corridon P and Molitoris BA. In
vivo multiphoton imaging of mitochondrial structure and function in
acute kidney injury. Kidney International 2013 Jan;83(1):72-83.
Hall AM, Crawford C, Unwin RJ, Duchen MR and Peppiatt-Wildman CM.
Multiphoton imaging of the functioning kidney. The Journal of The
American Society of Nephrology 2011 Jul;22(7):1297-304.
This presentation addresses the current knowledge of the
transport physiology of the thick ascending limb of Henle’s
25P
Research Symposia
requirements.
SA051
Inner ear and renal tubular physiology: Kidney and ear listen
to the same music
F. Karet
University of Cambridge, Cambridge, UK
Despite their geographical separation, the renal tubule and
the cochlea share a number of anatomical and physiological
features, including basement membrane structure and ion
transport mechanisms. The consequence of cochlear failure is
sensorineural hearing loss, whereas a variety of different phenotypes accompany tubular pathobiology. In concert, a number of syndromes are the result of the same abnormality at
the two sites, and these will form the basis of this talk.
Wellcome Trust, Cambridge Biomedical Research Centre.
requirements.
SA052
Renal Fanconi syndromes. Genetics and physiology
M. Khosravi
Nephrology, Royal Free & UCL, London, UK
The kidney plays a crucial role in maintaining fluid and electrolyte homeostasis. In adults, selective filtration by the renal
glomerulus produces more than 150 liters of ultrafiltrate every
day; 99% of which must be reabsorbed by highly specialized
segments of the renal tubule. Impaired function of the first
post- glomerular segment, the proximal tubule, can cause lifethreatening losses of fluids, electrolytes, and low-molecularweight nutrients.
Such dysfunction, known as renal Fanconi’s syndrome most
often occurs in children as part of multisystem metabolic diseases such as cystinosis, Dent’s disease, the Fanconi–Bickel
syndrome, the oculocerebrorenal syndrome (Lowe’s syndrome), tyrosinemia type I, Wilson’s disease, fructose intolerance, galactosemia, the ARC (arthrogryposis, renal dysfunction, and cholestasis) syndrome, and mitochondrial
disorders.
In both children and adults, acquired Fanconi’s syndrome may
result from toxic effects of anti–human immunodeficiency
virus drugs, heavy metals, antibiotics, valproic acid, glue sniffing, or exposure to suramin, fumaric acid, or ifosfamide.1,2
Finally, there have been rare cases of severe, isolated renal Fanconi’s syndrome in which the molecular, biochemical, and cellular defects remain unknown.3 Elucidation of these defects
might provide a better understanding of the mechanisms of
tubular reabsorption and lead to new therapeutic interventions.
One possible cause of renal Fanconi’s syndrome involves
impaired mitochondrial function, because the high metabolic
activity of the proximal tubule makes it particularly susceptible to restrictions in energy output.4 Mitochondrial production of ATP, essential for generating the energy dependent ion
gradients that drive renal tubular reabsorption, is impaired in
acute kidney injury.5
26P
We have recently gained further insight into a mechanism of
impaired tubular function by identifying a defective mithochondrial protein through clinical and genetic characterization
of a family with isolated autosomal dominant Fanconi’s syndrome.
We linked the phenotype of this family’s Fanconi’s syndrome
to a single locus on chromosome 3q27, where a heterozygous
missense mutation in EHHADH segregated with the disease.
The p.E3K mutation created a new mitochondrial targeting
motif in the N-terminal portion of EHHADH, an enzyme that
is involved in peroxisomal oxidation of fatty acids and is
expressed in the proximal tubule. Immunocytofluorescence
studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired
mitochondrial oxidative phosphorylation and defects in the
transport of fluids and a glucose analogue across the epithelium. 1H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members.
Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature
of the mutation rather than haploinsufficiency.
Mistargeting of peroxisomal EHHADH disrupts mitochondrial
metabolism and leads to renal Fanconi’s syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein
adds to the spectrum of monogenic mechanisms of Fanconi’s
syndrome.
Hall AM, Hendry BM, Nitsch D, Connolly JO. Tenofovir associated kidney toxicity in HIV-infected patients: a review of the evidence. Am J
Kidney Dis 2011;57: 773-80.
Ciarimboli G, Holle SK, Vollenbröcker B, et al. New clues for nephrotoxicity induced by ifosfamide: preferential renal uptake via the human
organic cation transporter 2. Mol Pharm 2011;8:270-9.
Kleta R. Fanconi or not Fanconi? Lowe syndrome revisited. Clin J Am
Soc Nephrol 2008;3:1244-5.
Szeto HH, Liu S, Soong Y, et al. Mito- chondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury. J Am Soc
Nephrol 2011;22:1041-52.
Hall AM. Pores for thought: new strategies to re-energize stressed
mitochondria in acute kidney injury. JAmSoc Nephrol 2011;22:986-9.
Enriko D. Klootwijk, Ph.D., Markus Reichold, Ph.D., Amanda
Helip-Wooley, Ph.D., Asad Tolaymat, M.D., Carsten Broeker,
M.Sc., Steven L. Robinette, B.Sc., Joerg Reinders, Ph.D.,
Dominika Peindl, M.D., Kathrin Renner, Ph.D., Karin Eberhart,
Ph.D., Nadine Assmann, M.Sc., Peter J. Oefner, M.D., Ph.D.,
Katja Dettmer, Ph.D., Christina Sterner, M.Sc., Josef Schroeder,
Ph.D., Niels Zorger, M.D., Ph.D., Ralph Witzgall, M.D., Ph.D.,
Stephan W. Reinhold, M.D., Ph.D., Horia C. Stanescu, M.D.,
Ph.D., Detlef Bockenhauer, M.D., Ph.D., Graciana Jaureguiberry,
M.D., Holly Courtneidge, M.Sc., Andrew M. Hall, M.D., Ph.D.,
Anisha D. Wijeyesekera, Ph.D., Elaine Holmes, Ph.D., Jeremy
K. Nicholson, Ph.D., Kevin O’Brien, M.S.N., Isa Bernardini, M.Ed.,
Donna M. Krasnewich, M.D., Ph.D., Mauricio Arcos-Burgos,
M.D., Ph.D., Yuichiro Izumi, M.D., Ph.D., Hiroshi Nonoguchi,
M.D., Ph.D., Yuzhi Jia, M.D., Ph.D., Janardan K. Reddy, M.D.,
Mohammad Ilyas, M.D., Robert J. Unwin, M.D., Ph.D., William
A. Gahl, M.D., Ph.D., Richard Warth, M.D., Ph.D., and Robert
Kleta, M.D., Ph.D. Drs. Klootwijk, Reichold, Helip-Wooley, Gahl,
Warth, and Kleta contributed equally to this work.
requirements.
Research Symposia
SA053
A day in our specialized renal tubular clinic. How physiology
supports the NHS (and vice versa)
D. Bockenhauer
Great Ormond Street Hospital, London, UK
Rare inherited diseases provide a unique window into specific
aspects of physiology. In turn, our knowledge of physiology
can help understand the molecular basis of these disease,
enable precise diagnosis and provide a platform for the development of improved treatments.
At Great Ormond Street, we have a specialized renal tubular
clinic caring for more than 200 patients with mostly Mendelian
disorders of tubular function.
I will use EAST syndrome as an example of successful collaboration between clinic and physiology that led to the first recognition of this disorder, the identification of the molecular basis
and novel observations of the specific clinical consequences.
These investigations now allow us to provide affected patients
and their families a specific diagnosis, as well as clinical and
genetic counseling.
Moreover, we have been able to establish animal models of
the disease, enabling the testing of potential treatments.
requirements.
SA054
Evolution and environmental stressors: The role of
neuroplasticity in the sensitization of hypertension
tension which was demonstrated by giving access to 2% NaCl
as the sole drinking fluid after the DELAY period.
The enhancements in experimentally-induced hypertension
as a result of exposure to subsequent stimuli are examples of
response sensitization. Functional studies indicate that the
sensitized hypertensive response is mediated by the central
nervous system (CNS) and that the heightened responses are
associated with sustained molecular changes in the central
regions involved in blood pressure regulation. We have found
that mRNA or protein of several biochemical factors which
serve as putative markers of neuroplasticity (e.g., brain-derived
neurotrophic factor, cAMP response element-binding protein)
are increased in structures lying along the lamina terminalis,
which comprise a forebrain region that is part of the neural
network that controls blood pressure. Importantly in addition
to these indices that are generally associated with memorial
processes, there are sustained changes in this region of components of the brain-renin-aldosterone system.
Taken together these findings indicate that stressors present
at an earlier time can produce enduring changes in the CNS
and suggest that these molecular changes result in increased
responding to subsequent hypertension-eliciting challenges.
Because of the observations of increased activity in the brain
renin-angiotensin-aldosterone system in our sensitization studies, discussion will focus on the role of early activation of the
systemic renin-angiotensin-aldosterone system and its influence on the central system as key mediators of stressors that
have long-term influences on the regulation of blood pressure
and pathogenesis of hypertension.
This work was supported in part by the National Institutes of
Health grants HL-14388, HL-98207, and MH-80241.
requirements.
A.K. Johnson
Psychology, University of Iowa, Iowa City, IA, USA
Approximately one-third of individuals over the age of 20 in
developed countries have hypertension, and high blood pressure is recognized to be a major risk factor for coronary heart
disease and stroke. The causes of the onset and the progressive increase in blood pressure over the course of a lifetime to
eventually become diagnosed as frank hypertension are not
known. Recently it has become more apparent that although
heredity is a factor, the sum of the contributions of the identified genes affecting blood pressure cannot account for the
incidence of hypertension. This is consistent with what has
been described as the “missing heritability” of other complex
diseases and has led others to propose that hypertension
results from epigenetic mechanisms involving the interaction
of genes X environment.
In recent studies we developed an experimental approach in
rats to explore the effects of antecedent physiological and psychological challenges (stressors) on the hypertensive response
elicited by a subsequent treatment following an imposed intervening period of delay. This procedure is referred to as the
INDUCTION-DELAY-EXPRESSION paradigm. Using this experimental design we have found that initial challenges with low,
non-pressor doses of either angiotensin II or aldosterone given
during INDUCTION significantly amplified the hypertensive
response seen in a low-dose-angiotensin II-slow pressor model
of hypertension which was given during EXPRESSION (i.e., test
phase). The same antecedent treatments with either
angiotensin II or aldosterone also enhanced salt sensitive hyper-
SA055
The role of neurohumoral factors in association between
cardiovascular diseases and depression
E.V. Bouzinova1, O. Wiborg1, S.L. Christiansen1, C. Aalkjaer2
and V.V. Matchkov2
1Translational Neuropsychiatry Unit, Aarhus University, Risskov,
Denmark and 2Department of Biomedicine, Aarhus University,
Aarhus, Denmark
Depression is associated with a doubled risk for cardiovascular disease and patients with circulatory disorders often develop
a severe depression. Mechanisms underlying both pathologies
in the developed state are similar: dysfunctions of the hypothalamic-pituitary-adrenal axis and the autonomic nervous system, an activation of the renin-angiotensin-aldosterone system and pro-inflammatory cytokines, as well as cardiovascular
dysregulations. All these factors are capable for inducing both
depression and changes in structure and function of the heart
and blood vessels thereby creating a malignant cycle which
usually leads to the chronic diseases (Fig. 1). Research on these
mechanisms is complicated and often impossible in humans,
but an animal model of depression provides opportunities for
it. A Chronic Mild Stress (CMS) model of depression in rats is
established in our group at the TNU at the Aarhus University.
The model resembles closely the pathology in human population. The CMS model utilizes finding from epidemiological
study about crucial role of environmental factors, mainly stressful live events, in the etiology of depression. The CMS para-
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digm is built on the chronic exposure to unpredictable mild
stressors of small laboratory animals. The behavioural changes
observed in this model are consistent with depression symptoms, e.g. abnormal physical activity and an inability to experience pleasure from normally pleasurable stimuli - anhedonia,
shown by a reduction in the consumption of sucrose solution
in a sucrose consumption test (SCT), where animals are
exposed to a bottle of palatable sucrose solution once a week
during 1 h. Anhedonia-like behaviour is defined as the reduction in sucrose intake for more than 30% compare to baseline
(before CMS). Importantly, not all rats subjected to CMS
develop anhedonia. The model demonstrates differentiated
response to CMS and some animals are resilient to the CMS
paradigm (fall in SCT no more than 10%), mirroring gradual
stress coping abilities of humans (for review (1).
Using the model we have shown that the low quality of maternal care, in early life, increases the susceptibility to stress exposure in adulthood. Our results indicate that high, as opposed
to low maternal care, predisposed rats to a differential stresscoping ability. Moreover, low maternal care offspring had lower
weight gain and lower locomotion, with no additive effect of
stress. Subchronic exposure to chronic mild stress induced an
increase in faecal corticosterone metabolites, which was only
significant in rats from low maternal care dams (2).
Rats responded to CMS with anhedonic behaviour have an
increased level of corticosterone (CORT) and two peaks in diurnal CORT secretion (3).
Decrease in expression of glucocorticoid receptors (GR) in different areas of hippocampus was observed in anhedonic rats
compare to control and resilient rats (p<0.001 by 2-way
ANOVA) after 4 weeks of CMS by semiquantitative analysis of
protein expression detected by GR M-20 antibody. Similar to
the systemic CORT concentration, effect of CMS on GR expression was not distinguishable after 8 weeks of CMS.
Multi-Plex cytokine detection system revealed 14 products different between CMS groups and/or time of CMS. Results were
subjected to principle component analysis. If to consider the
positive loadings on both factors as an activation of immune
response, than control rats demonstrate negative activation
at 4 and 8 weeks of CMS. In contrast, resilient rats demonstrate
strong activation of immune response. Anhedonic rats demonstrate increase in the activation of immune system between
4 and 8 weeks of CMS.
We found in myograph studies the extended peripheral effect
of increased systemic CORT. Depression-like symptoms were
associated with increased neuronal monoamine reuptake in
small arteries, which compensates for stress-induced suppression of CORT-sensitive extra-neuronal reuptake. These
changes are due to an increased expression of neuronal noradrenaline transporter and a decreased expression of extra-neuronal organic cation co-transporter 2 (4).
Activation of immune system and increased concentration of
pro-inflammatory cytokines in circulation are known factors
affecting the endothelial functions. Depression-like symptoms
are associated with reduced endothelium-dependent relaxation due to suppressed EDH-like relaxation despite the upregulation of the NO and COX-2-dependent pathways in small
arteries. We suggest that this suppression of relaxation might
be another reason for elevated vasoconstriction and thus TPR
in depression (5).
Increased heart rate in anhedonic rats and no changes in arterial blood pressure between three groups suggest that rats
with depression-like behaviour compensate the decrease in
cardiac output with an increase in total peripheral resistance
(4).
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Figure 1. The vicious cycle of cardiovascular disease and depressive disorder. Several factors are common for both disorders: dysfunctions of the
hypothalamic-pituitary-adrenal axis and the autonomic nervous system
(sympathetic/parasympathetic), an activation of the renin-angiotensinaldosterone system and pro-inflammatory cytokines, as well as cardiovascular dysregulations. Importantly, all these factors are capable of inducing
both depression and changes in structure and function of the heart and
blood vessels thereby creating a malignant cycle which usually leads to the
chronic diseases.
Wiborg O (2013). Cell Tissue Res. 2013 Oct;354(1):155-69.
Henningsen K et al, 2012. Behav Pharmacol. 2012 Dec;23(8):735-43.
Christiansen S et al., 2012. Stress. 2012 Nov;15(6):647-57
Bouzinova EV et al., 2012. Psychosom Med. 2012 Apr;74(3):278-87
Bouzinova EV et al., 2014. Psychosom Med. 2014. In Press
Work was supported by the Lundbeck foundation
requirements.
SA056
Sex-related differences in cardiovascular action of crowding
stress in young normotensive and hypertensive rats
I. Bernatova, A. Puzserova and P. Balis
Institute of Normal and Pathological Physiology, Slovak Academy
of Sciences, Bratislava, Slovakia
Although the modern lifestyle in industrial cities with a high
population density is a risk factor in the development of
lifestyle-related diseases, including cardiovascular and mental
disorders, the causal role of stress in the aetiology of cardiovascular diseases is not understood satisfactorily. Furthermore
age, sex and family history of hypertension represent important issues that may interact with environmental factors and
participate in development of cardiovascular disorders.
The aim of this study was to investigate the influence of chronic
social stress produced by crowding on blood pressure (BP),
vascular function and nitric oxide (NO) production in young
normotensive Wistar-Kyoto (WKY), borderline hypertensive
(BHR) and spontaneously hypertensive (SHR) rats of both
sexes.
Five-week males and females of WKY, BHR (F1 offspring of SHR
dames and WKY sires) and SHR were exposed to stress produced by crowding (5 rats/cage, 70 cm2/100g, n=10) for two
weeks as described previously (1). Controls were kept 4
rats/cage (200 cm2/100g, n=8). Blood pressure (BP) was determined by tail-cuff method. Vascular function (endothelium-
Research Symposia
dependent acetylcholine (ACh)-induced relaxation and its components) of the femoral artery was investigated using wire
myograph. NO synthase activity was determined by conversion of [3H]-L-arginine in the aorta and brainstem. Results were
analysed by ANOVA and Bonferroni post-hoc test and they
are presented as mean±SEM.
Basal BP of 5-week-old male and female WKY, BHR and SHR
was 104±2 and 103±2 mmHg, 127±3 and 115±4 mmHg and
140±3 and 137±2 mmHg, respectively (p<0.001 among
strains). Two-week crowding accelerated BP increase in both
BHR males and females (p<0.01) vs. basal which was not seen
in WKY and SHR. In SHR similar time-related elevation of BP
was observed in control and stressed groups at the end of
experiment. However, a significant BP elevation was seen in
SHR males after the 1st week of stress (vs. control), which was
not observed in females. There was significantly higher BP in
males vs. females during the experiment (131±2 vs. 125±2
mmHg, p<0.0001). Stress significantly reduced body weight
gain (p<0.0001) and elevated plasma corticosterone (p<0.01)
and BP (p<0.001) vs. control rats, however these effects were
the most pronounced in BHR strain. Blood glucose was reduced
in stressed males while no changes were observed in females
vs. non-stressed rats. NO synthase activity in the aorta of control BHR and SHR females was significantly higher vs. WKY
(p<0.05) and stress elevated NOS activity in females of all
strains investigated vs. control. In males, aortic NO production
was elevated only in control SHR vs. WKY and interestingly
no effect of stress was seen in young males. However, stress
reduced NO production in the brainstem of both males and
females of WKY and BHR while no effect was observed in SHR
of both sexes.
Endothelial dysfunction was observed in SHR strain vs. both
WKY and BHR. Stress did not affect vascular function in females
significantly in any strain despite quantitative alterations of its
NO-dependent and NO-independent components. However,
in males stress paradoxically improved endothelium-dependent ACh-induced relaxation in SHR vs. control, which was associated with an elevation of NO-independent component of
relaxation without changes in NO-dependent relaxation.
In conclusion, results showed that the influence of chronic
social stress produced by crowding in young rats depends on
the genetic predisposition to hypertension (in both males and
females). WKY rats were able to adapt to stress while BHR were
the most vulnerable to stress-induced elevation of BP. In contrast, genetic predisposition to hypertension is a dominant factor in hypertension development in SHR. Finally, differences
in stress-induced time course of BP increase, mechanism of
vascular function and NO production were observed in young
males and females.
Slezak P et al.(2014). BioMed Research International, vol. 2014, Article ID 413629, 11 pages, doi:10.1155/2014/413629
This study was supported by the Slovak Research and
Development Agency, grant No. APVV-0523-10 and Slovak
Grant Agency for Science, grant No. 2/0084/14.
requirements.
SA057
Work stress and development of atherosclerosis
N. Eller
Occupational and Environmental Medicine, Bispebjerg University
Hospital, Copenhagen, Denmark
Ischaemic heart disease (IHD) is one of the Western world’s
most widespread diseases and causes of death. Among the
many risk factors for this condition, work-related psychosocial
factors increase the risk of IHD independently of the conventional coronary risk factors (1). Because psychological stressors activate the physiological stress response, a patho-physiological explanation may be that long-term or repeated
physiological stress due to work-related psychosocial factors
may be atherogenic. Usually, theoretical stress models are used
as the bases for self-report questionnaires, i.e., the Demand
Control Model (DC model), and the Effort Reward Imbalance
Model (ERI model), as these models (particularly the DC model)
have been frequently examined, and several papers have
shown associations between psychosocial load according to
the models and different endpoints. The use of theoretically
based stress models may be a strength of a design, but at the
same time, it is important that the models do not rule out
the exploration of other stressors. During the last 20 years or
more, the labour market has changed, and fewer people are
employed in production (e.g., industry and farming), whereas
more people are employed in education, administration, health
care, and knowledge production. This shift means that the
stress models used in the 1980s might require further development to be used today.
In the epidemiological setting, the physiological stress
response can be measured non-invasively through the use of
salivary cortisol and analysis of heart rate variability (HRV) (2).
Cortisol is easily measured in the saliva, and saliva samples can
be stored at room temperature for a long time before the cortisol decays, making this method appropriate for use in epidemiological studies, but great intra and between individual
variations are the reason for methodological problems.
Research using salivary cortisol as either an effect measure or
an explanatory variable has recently been reviewed. The conclusion was that study design is of the utmost importance, but
that the deviation measures, i.e., CAR or diurnal slope (decrease
in cortisol from morning to evening), appear to be rather consistently associated with psychological factors, whereas a flat
diurnal curve (low morning cortisol and high evening cortisol)
appears to be associated with ill-health. Several reviews have
concluded that work-related psychosocial factors are associated with salivary cortisol levels. Work stress may be considered to be a rather low stressor and so with presumably little
physiological effect. Subsequently, the positive association
between psychological strain at work and increased levels of
cortisol may be interpreted as a normal physiological activation.
HRV is defined as the spontaneous fluctuations in the sinus
rhythm due to processes in the body. By analysing the variability of heart rhythms observed in an electrocardiogram
(ECG), the activity in the autonomous nervous system can be
evaluated. HRV, in the form of reduced values for time-domain
analysis, is known to be associated with, among other things,
heart disease and to predict illness and death. The association between HRV and IMT is still controversial, and future
research on this issue is needed.
The use of acute myocardial infarction (AMI) as an endpoint
in stress research has been problematic. However, today, the
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individual’s level of atherosclerosis can be directly estimated
by ultrasound examination of the walls of the arteries in the
neck, and the intima media thickness of the vessel wall (IMT)
may be considered to be a valid alternative for cardiovascular
events as outcome. IMT and progression in IMT have both been
linked to work-related psychosocial factors (3-5).
The literature indicate that atherosclerosis may, independent
of other risk factors, be provoked by psychosocial strain. In a
population in which conventional coronary risk factors have
low prevalence, i.e. younger people, greater focus on psychosocial factors might be important for the prevention of atherosclerosis. The use of physiological measurements for stress
will be of great significance in connection with understanding
work stress and the long-term effects of this condition.
SA059
Nonreceptor tyrosine kinases and the actin cytoskeleton in
vascular smooth muscle
J. Ohanian and V. Ohanian
Institute of Cardiovascular Sciences, Univeristy of Manchester,
Manchester, UK
There is considerable interest in the possibility that late life
depression refractory to conventional medication is associated
with abnormalities of the arterial circulation. Studies from our
laboratory have demonstrated increased intimal medial thickening in medium sized arteries as well as impaired endothelium dependent relaxation and increased vascular hypertrophy in small resistance size vessels from patients with
depression but with a similar cardiovascular risk profile to
match control subjects. Depressed patients had significantly
more dilated Virchow-Robin spaces in the basal ganglia as
imaged by magnetic resonance. The overall impression is that
such patients might well benefit from vasculo-protective measures to improve endothelial function or retard the progression
of atherosclerosis in an attempt to improve depression scores
and overall mood.
Maintenance of normal peripheral vascular resistance and perfusion of vital organs depends on the structure and contractile function of small arteries within the vasculature, and vasoconstrictor hormone regulation of small artery contractility
is an important determinant of vascular resistance and blood
pressure. Changes in these properties of small blood vessels
lead to cardiovascular disease. For instance, in response to prolonged stress e.g. maintained high blood pressure, smooth
muscle cells within small arteries exhibit exaggerated contractility and remodelling of the vessel wall, as seen in hypertension or migration and proliferation as in arteriosclerosis.
An important role for the actin cytoskeleton in cellular
responses; contraction, proliferation and migration, is now
recognised. It is also established that a dynamic cytoskeleton
is necessary for smooth muscle cell migration, and recent evidence implicates the reorganisation of the actin cytoskeleton
and its interaction with the cellular contractile machinery as
essential for smooth muscle contraction and relaxation. Two
distinct pathways regulate cytoskeletal reorganisation in
response to agonists: p38 mitogen activated protein kinase
(MAPK) and nonreceptor tyrosine kinases such as Src family,
p125 focal adhesion kinase (p125FAK), and proline-rich tyrosine kinase-2 (PYK2). p38 MAPK regulates the actin cytoskeleton through the small heat shock protein 27 (Hsp27). By binding to and capping actin filaments, Hsp27 inhibits F-actin
polymerisation. This property of Hsp27 is a function of its phosphorylation state such that phosphorylated Hsp27 does not
bind to actin allowing reorganisation of the cytoskeleton. Nonreceptor tyrosine kinases regulate the actin cytoskeleton
through the tyrosine phosphorylation of cytoskeletal regulatory proteins, including adaptor proteins such as paxillin and
its homologue hydrogen peroxide-inducible clone-5 (Hic-5). G
protein-coupled receptor agonists activate nonreceptor tyrosine kinases, and in airway and vascular smooth muscle tissues, the tyrosine phosphorylation of paxillin and Hic-5 respectively, is associated with force generation. Although Hic-5 is a
homologue of paxillin, unlike the latter it is not activated by
integrins or phosphorylated by p125FAK. However, PYK2, a
calcium-dependent homologue of p125FAK, does phosphorylate Hic-5 suggesting different cellular functions for the two
adaptor proteins. Hic-5 has been identified in vascular smooth
muscle tissues, and is phosphorylated by PYK2 when stimulated by noradrenaline. Furthermore, through its ability to
interact with Hsp27, Hic-5 plays an important role in the regulation of smooth muscle actin dynamics. This talk will describe
the complex molecular mechanisms that regulate actin
cytoskeleton reorganisation in vascular smooth muscle, concentrating on tyrosine kinases and their downstream substrates, with a particular emphasis on their role in contractility.
requirements.
requirements.
Eller NH, Netterstrom B, Gyntelberg F, Kristensen TS, Nielsen F, Steptoe A, et al. Work-related psychosocial factors and the development
of ischemic heart disease: a systematic review. Cardiol Rev 2009
Mar;17(2):83-97.
Chandola T, Heraclides A, Kumari M. Psychophysiological biomarkers
of workplace stressors. Neurosci Biobehav Rev 2010;35(1):51-7.
Bots ML, Evans GW, Riley WA, Grobbee DE. Carotid Intima-Media Thickness Measurements in Intervention Studies. Design Options, Progression Rates, and Sample Size Considerations: A Point of View. Stroke
2003 Nov 13.
Eller NH, Netterstrom B. Psychosocial factors at home and at work and
four-year progression in intima media thickness. Int J Behav Med
2007;14(1):21-9.
Krause N, Lynch JW, Kaplan GA, Cohen RD, Salonen R, Salonen JT.
Standing at work and progression of carotid atherosclerosis. Scand J
Work Environ Health 2000 Jun;26(3):227-36.
requirements.
SA058
Structural and functional properties of the circulation in
late life depression
A. Heagerty1, A. Greenstein1, R. Paranthaman2, R. Malik1,
A. Burns1 and R. Baldwin1
1The University of Manchester, Manchester, UK and 2Royal Bolton
Hospital, Bolton, UK
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SA060
Interaction between reactive oxygen species, Src family
kinases, and the RhoA/Rho-kinase pathway in response to
constrictor stimuli in rat pulmonary artery
C. Mackay, Y. Shaifta, V. Snetkov, J.P. Ward and G. Knock
Asthma, Allergy& Lung Biology, King’s College London, London,
UK
Oxidative stress is associated with a number of cardiovascular
diseases including pulmonary hypertension (Liu et al., 2006).
Previous studies have shown that prostaglandin-F2α and
hypoxia both activate the Src family of non-receptor tyrosine
kinases (SrcFK) and enhance SrcFK-dependent total cellular
tyrosine phosphorylation and contraction, via activation of
Rho-kinase (Knock et al., 2008a, Knock et al., 2008b). We further hypothesise that SrcFK mediate these responses through
interaction with reactive oxygen species (ROS) production.
Isometric tension and protein phosphorylation responses were
recorded in freshly isolated intra-pulmonary arteries (IPA) of
male Wistar rats. Intra-cellular translocation of RhoA and measurement of ROS production were measured in primary cultures of rat pulmonary arterial smooth muscle cells (PASMC).
ROS was measured using the chemi-luminescent probe L-O12.
RhoA translocation was visualised using PASMC stably transfected with GFP-tagged RhoA. Data are expressed as mean ±
SEM, and compared by ANOVA.
SrcFK auto-phosphorylation (at tyr-416) was enhanced by
exogenous H2O2 (10μM) in a time-dependent manner (2min:
152 ± 21% of control, n=10, p<0.05 vs. control; 5min: 161 ±
22% of control, n=8, p<0.01 vs. control) and by the superoxide producing quinone LY83583 (1μM) in a time-dependent
manner (30sec: 162 ± 15% of control, n=8, p<0.01 vs. control;
1 min: 142 ± 15% of control, n=8, p<0.05 vs. control; 5min:
144 ± 23% of control, n=8, p<0.05 vs. control).
The thromboxane analogue U-46619 (100nM, 30min) also
enhanced SrcFK auto-phosphorylation (155 ± 14% of control,
n=10, p<0.01 vs. control), and enhanced phosphorylation of
the Rho-kinase substrate myosin phosphatase targeting subunit-1 (MYPT-1, at thr-855; 143 ± 9% of control, n=16, p<0.01
vs. control) and myosin light-chain-20 (MLC20, at ser-19; 189
± 21% of control, n=20, p<0.01 vs. control). Pre-incubation with
the antioxidant tempol (3mM, 10min) significantly inhibited
U46619-induced Src phosphorylation (95 ± 13% of control,
n=6, p<0.01 vs. U46619 alone) U46619-induced MYPT-1 phosphorylation (120 ± 5% of control, n=8, p<0.05, vs. U46619
alone) and U46619-induced MLC20 phosphorylation (117 ±15%
of control, n=10, p<0.05, vs. U46619 alone). Tempol had no
effect on basal phosphorylation of any of the three proteins
(Src, n=7; MYPT-1, n=7; MLC20, n=10). The SrcFK antagonist
PP2 (30μM, 10min) also inhibited U46619-induced SrcFK phosphorylation (61 ± 4% of control, n=5, p<0.01 vs. U46619 alone),
U46619-induced MYPT-1 phosphorylation (103 ± 8% of control, n= 13, p<0.01 vs. U46619 alone) and U46619-induced
MLC20 phosphorylation (112 ± 11% of control, n=15, p<0.01
vs. U46619 alone). PP2 significantly inhibited basal phosphorylation of SrcFK (51 ± 19% of control, n=4, p<0.05 vs. control),
but had no effect on basal MYPT-1 or MLC20 phosphorylation.
U46619 induced contractile responses in IPA and these
responses were significantly inhibited by the antioxidants tempol (3mM) (25 ± 5% relaxation, n=11, p<0.01 vs. DMSO control) and ebselen (10μM) (30 ± 4% relaxation, n=12, p<0.01 vs.
DMSO control).
U46619 (100nM, 30min) induced sustained increases in ROS
production in PASMC (216 ± 51% of control, n=8, p<0.01 vs.
control) and this was inhibited by 3mM tempol (22 ± 6% of
control, n=7, p<0.01 vs.U46619 alone) and 10μM ebselen (79
± 33% of control, n=5, p<0.01 vs. U46619 alone).
U46619 triggered reversible cytosol to cell periphery translocation of GFP-tagged RhoA in live PASMC (n=>25 cells). This
translocation was prevented by pre-incubation with 1μM ebselen (n=3), 3mM tempol (n=4) and 10μM PP2 (n=3).
Our data show that: U46619 induces ROS production in
PASMC; that exogenous ROS activate SrcFK; and that U46619induced SrcFK, RhoA and Rho-kinase activity and contraction
in IPA and PASMC are all ROS and Src-dependent. Src may therefore be acting as a ROS-sensitive intermediary for the activation of the RhoA/Rho-kinase Ca2+-sensitization pathway in
response to vasoconstrictor stimuli in rat pulmonary artery.
KNOCK, G. A., SHAIFTA, Y., SNETKOV, V. A., VOWLES, B., DRNDARSKI,
S., WARD, J. P. & AARONSON, P. I. 2008a. Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat
pulmonary artery. Cardiovasc Res, 77, 570-9.
KNOCK, G. A., SNETKOV, V. A., SHAIFTA, Y., DRNDARSKI, S., WARD, J.
P. & AARONSON, P. I. 2008b. Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery. Cardiovasc Res, 80, 453-62.
LIU, J. Q., ZELKO, I. N., ERBYNN, E. M., SHAM, J. S. & FOLZ, R. J. 2006.
Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox). Am J Physiol Lung Cell Mol Physiol, 290, L2-10.
Funding: British Heart Foundation, Wellcome Trust
requirements.
SA061
Regulation of RhoA signaling by tyrosine phosphorylation
of Rho exchange factors
C. Guilluy, G. Chadeuf, P. Pacaud and G. Loirand
U1087, Inserm, Nantes, France
RhoA is a member of the Rho protein family that has been identified as an essential regulator of vascular smooth muscle cell
functions. Through the activation of its target Rho kinase, RhoA
is the major regulator of the tonic component of vascular
smooth muscle cell contraction and plays a critical role in the
control of vascular smooth muscle differentiation, proliferation and migration. Aberrant activation of RhoA/Rho kinase
signaling is involved in the pathogenesis of several vascular
pathologies, including hypertension, coronary artery spasm,
effort angina, atherosclerosis and restenosis.
RhoA acts as molecular switches, that cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine
nucleotide exchange factors (GEFs) mediate the activation of
RhoA by promoting the release of GDP in exchange for GTP.
GTPase-activating proteins that stimulate intrinsic GTPase activity of RhoA, hydrolyze GTP to GDP then turn off activation.
Rho GEFs are multidomain proteins that connect RhoA to
numerous upstream receptors and signaling molecules. In particular, the subfamily of Rho GEFs that contains regulator of
G-protein signaling (RGS) domain (Arhgef1, Arhgef11,
Arhgegf12) mediates the activation of RhoA by G-protein-coupled receptors. Recent data and results, in particular on the
regulation of Arhgef1 will be described to show that Rho GEF
activity is regulated by phosphorylation by tyrosine kinases
in vascular smooth muscle cells.
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Research Symposia
This work was supported by grants from the French Agence
Nationale de la Recherche (ANR-11-BSV1-013-01), the
Fondation pour la Recherche Médicale (DEQ20090515416)
and and the Fondation de France (2013 00038590).
requirements.
SA062
Role of Src in airway smooth muscle responses to
bronchoconstrictors and TGF-β
N. Irechukwu, Y. Shaifta, K. O’Brien, J.P. Ward and G. Knock
Asthma, Allergy& Lung Biology, King’s College London, London,
UK
The Src-family of non-receptor tyrosine kinases (SrcFK) are
important regulators of cell migration and proliferation, but
also contribute to smooth muscle contractility. In asthma,
airway hyper-responsiveness has been associated with the
actions of inflammatory mediators, such as transforming
growth-factor-β (TGF-β), on smooth muscle function. We
hypothesised that SrcFK may contribute to airway smooth muscle responses to acute bronchoconstrictor stimuli, and to the
longer term enhancement of these responses by TGF-β.
Isometric tension was measured in isolated bronchioles of male
Wistar rats. The component of contraction not dependent on
changes in intracellular Ca2+ concentration ([Ca2+]i), was measured in α-toxin permeabilised bronchioles. Measurement of
[Ca2+]i (Fluo-4 kit, Molecular Probes), protein phosphorylation
and protein expression, were all performed in primary cultures
of human airway smooth muscle cells (ASM), obtained from
healthy volunteers by deep endobronchial biopsy. Statistical
comparisons were by one-way ANOVA unless otherwise stated.
The bronchoconstrictors carbachol (Cch) and bradykinin (BK)
both induced concentration-dependent (0.01-100μM) contractile responses in isolated rat bronchioles. Maximum contraction (Bmax) and LogM[EC50] (PD2) values were obtained
using Hill curve for CCh data and 2-site saturation curve for
BK data. These responses were variably sensitive to inhibition
by antagonists of Rho-kinase (Y27632, 10μM) and SrcFK (PP2,
30μM), with Cch-induced responses being partially but significantly sensitive to Y27632 (Bmax: control= 210 ±26% vs.
Y27632= 160 ± 26%, p<0.01; PD2: control= -5.82 ± 0.1 vs.
Y27632= -5.4 ± 0.1, p<0.01; n=6, paired t-tests) and PP2 (Bmax:
control= 178 ± 7.6% vs. PP2= 141 ±9.8%, p<0.01; PD2: control= -5.8 ± 0.1 vs. PP2= -5.55 ± 0.1, p<0.05; n=8, paired t-tests).
BK-induced contractions were abolished by Y27632 (n=4) while
PP2 significantly reduced amplitude of both high and low affinity components (Bmax1: control= 12 ± 4% vs. PP2= 1.7 ± 0.5%,
p<0.05; Bmax2: control= 24 ± 6% vs. PP2= 6.8 ± 2%, p<0.05;
n=11, paired t-test).
SrcFK auto-phosphorylation at tyr416 was enhanced in human
ASM by both 1μM BK (323 ± 91% of control, n=7, p<0.05 vs.
control) and 100μM CCh (170 ± 30% of control, n=8, p<0.05
vs. control). This phosphorylation was nearly abolished by PP2
in both cases (n=6-8, p<0.01 vs. BK or Cch alone). BK also
enhanced phosphorylation of myosin phosphatase targeting
subunit-1 (MYPT-1) at thr696 (206 ± 33% of control, n=20,
p<0.01 vs. control) and myosin light-chain-20 (MLC20) at ser19 (172 ± 31% of control, n=16, p<0.01 vs. control). Both phosphorylation responses were significantly inhibited by Y27632
(MYPT-1: n=13, p<0.01 vs. BK alone; MLC20: n=9, p<0.05 vs.
BK alone) and PP2 (MYPT-1: n=15, p<0.05 vs. BK alone; MLC20:
32P
n=15, p<0.05 vs. BK alone). Cch did not alter phosphorylation
of either protein.
Cch also induced concentration-dependent contractile
responses in α-toxin permeabilised rat bronchioles and these
were abolished by Y27632 (n=6) and inhibited by PP2 (Bmax:
control= 61 ± 8% vs. PP2= 38 ± 6%, p<0.05; PD2: control= -5.1
± 0.1 vs. PP2= -4.6 ± 0.1, p<0.05; n=9, unpaired t-test). Both
BK (n=12) and Cch (n=12) induced increases in [Ca2+]i in human
ASM. However, neither response was significantly affected by
SrcFK inhibition with PP2 (paired t-test).
Prior Incubation of bronchioles with TGF-β (30ng/ml, 18hr) in
serum-free media specifically enhanced the low affinity component of the BK-induced contraction when compared to incubation in media alone (Bmax2: media= 21 ± 5%, n=17 vs. TGFβ= 49 ± 12%, n=15, p<0.05, unpaired t-test). Cch-induced
contraction was not significantly altered by TGF-β incubation
(TGF-β n=8 vs. media only n=13). SrcFK inhibition did not prevent the potentiating effect of TGF-β on BK contraction, in fact,
contractile responses to both BK and Cch were generally less
sensitive to PP2 after 18hr incubation (with or without TGF-β).
In human ASM, incubation with TGF-β (10ng/ml, 24hr)
enhanced the protein expression of MLC20 (253 ± 29% of control, n=6, P<0.05 vs. control), MYPT-1 (199 ± 25% of control,
n=6, p<0.05 vs. control) and the RhoA guanine nucleotide
exchange factor ARHGEF-1 (201 ± 35% of control, n=6, p<0.05
vs. control). It also significantly reduced the protein expression of Src (83 ± 12% of control, n=16, p<0.05 vs. control),
but had no effect on RhoA (n=6) or Rho-kinase (n=6) expression.
Our results suggest that Src contributes to the bronchoconstrictor effects of BK and Cch, acting primarily via Rho-kinase
dependent Ca2+-sensitization, involving enhanced MYPT-1 and
MLC20 phosphorylation. Prior treatment with TGF-β enhances
BK-induced constriction and the expression of several contractile proteins, but this is not associated with an enhanced
dependence of contractile responses on SrcFK.
Funding: Wellcome Trust and British Heart Foundation
requirements.
SA063
New and notable proteins supporting ER/SR Ca2+-release
H. Takeshima
School of Pharm Sci, Kyoto University, Kyoto, Japan
During physiological contraction in striated muscle cells, the
electrical signal of depolarization is converted into the Ca2+
release signal mediated by ryanodine receptors in the sarcoplasmic reticulum. In order to function as efficient intracellular stores, the sarcoplasmic reticulum is equipped with synergistically acting Ca2+ uptake, storage and release machinery,
comprising of cytoplasmic, integral-membrane and luminally
located proteins. Although major Ca2+-handling proteins have
already been identified and extensively studied, there are still
as-yet-unknown proteins in the sarcoplasmic reticulum and
their roles remain to be clarified in future studies. We utilized
a unique immuno-proteomic approach to generate a monoclonal antibody library that targets major proteins localized to
skeletal muscle membrane systems in order to identify proteins with a key role in enabling efficient Ca2+ store functions.
In the course of screening novel membrane proteins, we identified several important sarcoplasmic reticulum proteins includ-
Research Symposia
ing junctophilins and TRIC (trimeric intracellular cation) channels. Junctophilin subtypes form junctional membrane complexes between the plasma membrane and the endo/sarcoplasmic reticulum in excitable cells. TRIC channel subtypes
act as novel monovalent cation-specific channels on intracellular membrane systems in various cell types. Our observations from knockout mice have provided evidence that both
junctophilins and TRIC channels support ryanodine receptormediated Ca2+ release in striated muscle cells. Moreover, mutations and polymorphisms in the human junctophilin and TRIC
channel genes are associated with hypertension risk and
genetic diseases including cardiac hypertrophy and osteogenesis imperfecta. This communication will focus on the physiological and pathological roles of junctophilin and TRIC channel subtypes.
requirements.
SA064
Critical role of TRIC-channels: What can be revealed by
single-channel studies?
E. Venturi1, A. Matyjaszkiewicz2, F. O’Brien1, M. Nishi3,
H. Takeshima3 and R. Sitsapesan1
1Department of Pharmacology, University of Oxford, Oxford, UK,
2Bristol
Centre for Complexity Sciences and Department of
Engineering Mathematics, University of Bristol, Bristol, UK and
3Graduate School of Pharmaceutical Sciences, University of Kyoto,
Kyoto, Japan
TRIC-A and TRIC-B are two homologous proteins found in the
sarcoplasmic/endoplasmic reticulum (ER/SR) of various tissues
and they function as monovalent cation-selective ion-channels
(1, 2). Genetic deletion studies have indicated a crucial role of
these channels in intracellular Ca2+-handling in different cell
types. Mice lacking both TRIC-A and TRIC-B die of heart failure
at embryonic stages and display serious defects in SR Ca2+release (1) while knockout of the TRIC-B isoform severely
impairs intracellular Ca2+-homeostasis in alveolar cells and
results in neonatal death (3). A TRIC-B deletion mutation has
also been recently linked to the genetic disorder, osteogenesis imperfecta (4). Identifying the biophysical properties and
the possible physiological regulators of the TRIC channels is
essential for understanding their individual functions in different tissues but investigation is complex because both isoforms are present in most tissues. We have therefore isolated
SR vesicles from Tric-a- knockout mouse skeletal muscle and
incorporated them into artificial membranes under voltageclamp conditions as previously described (5) in order to investigate regulatory gating mechanisms of native TRIC-B channels. We obtained TRIC-B single-channel recordings in K+
containing solutions in the presence of 10 μM free Ca2+ at
various pH and at holding potentials in the range ±50 mV.
Our single-channel data suggests that TRIC-B channels always
open to the full open state via transitions to sub-conducting
open states. We demonstrate that TRIC-B gating to the full
open state and to sub-conductance open states is steeply voltage-dependent. At negative potentials, Po in the full open state
was approximately 0 and few current fluctuations to sub-conductance levels were observed. Steep channel activation
occurred at positive potentials between 0 and +20 mV while
no further increase in Po was observed as the holding potential was raised above +20 mV. At physiologically relevant mem-
brane voltages (0±10 mV), sub-conductance opening events
accounted for approximately 75% of the total current flowing
through TRIC-B channels, indicating that sub-conductance state
gating may significantly contribute to physiological SR cation
currents. In addition to voltage, we find that pH is another
modulator of TRIC-B function. Cytosolic and luminal acidification markedly inhibited channel activity while alkaline pH on
either channel side activated TRIC-B. For example, mean Po at
+30 mV was decreased from 0.285±0.074 in symmetrical pH
7.2 to 0.099±0.028 (SEM, **p<0.01, n=8) after cytosolic pH
was lowered to 6.2. This change in gating reflected a substantial increase in the proportion of openings to sub-conductance states.
Our data suggests that TRIC-B channels would be constitutively
active at physiological SR membrane potentials and that only
a small voltage change across the SR would be sufficient to
produce a maximal increase in Po. Further experiments are
required to fully understand the complex mechanisms regulating TRIC-B channel opening and its precise role in the physiology and pathophysiology of SR Ca2+-release.
Yazawa, M., C. Ferrante, J. Feng, K. Mio, T. Ogura, M. Zhang, P. H. Lin,
Z. Pan, S. Komazaki, K. Kato, M. Nishi, X. Zhao, N. Weisleder, C. Sato,
J. Ma, and H. Takeshima. 2007. TRIC channels are essential for Ca2+
handling in intracellular stores. Nature 448:78-82.
Pitt, S. J., K. H. Park, M. Nishi, T. Urashima, S. Aoki, D. Yamazaki, J.
Ma, H. Takeshima, and R. Sitsapesan. 2010. Charade of the SR K+-channel: two ion-channels, TRIC-A and TRIC-B, masquerade as a single K+channel. Biophys. J. 99:417-426.
Yamazaki, D., S. Komazaki, H. Nakanishi, A. Mishima, M. Nishi, M.
Yazawa, T. Yamazaki, R. Taguchi, and H. Takeshima. 2009. Essential
role of the TRIC-B channel in Ca2+ handling of alveolar epithelial cells
and in perinatal lung maturation. Development 136:2355-2361.
Volodarsky, M., B. Markus, I. Cohen, O. Staretz-Chacham, H. Flusser,
D. Landau, I. Shelef, Y. Langer, and O. S. Birk. 2013. A Deletion Mutation in TMEM38B Associated with Autosomal Recessive Osteogenesis
Imperfecta. Hum. Mutat. 34:582-586.
Venturi, E., A. Matyjaszkiewicz, S. Pitt, K. Tsaneva-Atanasova, M. Nishi,
D. Yamazaki, H. Takeshima, and R. Sitsapesan. 2013. TRIC-B channels
display labile gating: evidence from the TRIC-A knockout mouse model.
Pflugers Arch - Eur J Physiol:1-14.
This work was funded by the British Heart Foundation.
requirements.
SA065
Role of junctional proteins in skeletal muscle strength
F. Zorzato1,3, B. Mosca1, O. Delbono2, M. Messi2,
L. Beergamelli1, R. Lopez3, M. Nishi4, H. Takeshima4, C. Paolini5
and F. Protasi5
1University of Ferrara, Ferrara, Italy, 2Department of Physiology
and Pharmacology, Wake Forest University School of Medicine,
Winston-Salem, NC, USA, 33) Departments of Biomedicine and
Anesthesiology, Basel University Hospital, Switzerland, Basel,
Switzerland, 44) Department of Biological Chemistry, Graduate
School of Pharmacological Sciences, Kyoto University, Kyoto, Japan
and 5CeSI & DNI,, Univ. G. d’Annunzio, Chieti, Italy
Skeletal muscle constitutes approximately 40% of body mass,
and age-induced decrease of muscle strength impinge on daily
activities and on normal social life in the elderly. Loss of muscle strength has been recognised as a debilitating and life
threatening condition also in cachexia in cancer patients and
in clinical conditions associated with prolonged bed rest. Skele-
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Research Symposia
tal muscle dihydropyridine receptors (Cav1.1) act as Ca2+ channels and voltage sensors to initiate muscle contraction by activating ryanodine receptors, the Ca2+ release channels of the
sarcoplasmic reticulum. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Cav1.1 and
the sarcoplasmic reticulum Ca2+ storage protein calsequestrin
(CASQ1).
We hypothesized that JP45 and CASQ1 form a signalling pathway which modulates Cav1.1 channel activity. We tested this
in flexor digitorum brevis (FDB) muscle fibres from JP45 and
CASQ1 double knock-out mice (DKO). Our results show that
Ca2+ transients evoked by tetanic stimulation in DKO fibres,
result from massive Ca2+ influx due to enhanced Cav1.1 channel activity. This enhanced activity causes an increase of muscle strength both in vitro and in vivo. We conclude that skeletal muscle contraction is strengthened through the modulation
of Cav1.1 channel activity by JP45 and CASQ1, making these
proteins candidate targets for strategies against the decay of
skeletal muscle strength.
This work was supported by funds from Swiss Muscle
foundation, A.F.M., S.N.F and Department of Biomedicine
University Hospital Basel. This study was also supported by
Research Grant no. GGP08153 from the Italian Telethon ONLUS
Foundation to F.P. and grants from the NIH/NIA (AG13934 and
AG15820) to O.D.
requirements.
SA066
Manipulation of SR Ca2+-release in heart failure through
mechanical intervention
M. Ibrahim and C. Terracciano
Imperial College London, London, UK
The contraction of cardiomyocytes is regulated by electrical
signals propagated by an exquisite system of membrane invaginations termed transverse (t)-tubules. This system ensures
maximal and synchronous contraction. Loss of this system in
heart failure is thought to be a key mechanism impairing cellular contractility. Artificial loss of the t-tubules induced by detubulating agents results in profound impairments of contractility.
Clinical studies have shown the ability, in selected patients,
of mechanical unloading using left ventricular assist device
therapy to induce regression of pathological remodeling in
heart failure. So called reverse remodeling has been described
at many levels.
I will discuss the hypothesis that the t-tubule system is regulated by mechanical loading conditions in a dynamic and timedependent manner.
Reversibility of T-tubule remodelling in heart failure: mechanical load
as a dynamic regulator of the T-tubules. Ibrahim et al Cardiovasc Res
2013
SA067
Triadin regulation of the ryanodine receptor complex
I. Marty
Grenoble Institut des Neurosciences, Inserm U836, La Tronche,
France
Skeletal muscle contraction is triggered when a membrane
depolarisation induces a massive increase in intracellular calcium concentration. This process called “excitation-contraction (E-C) coupling” relies on a multimolecular protein complex, the calcium release complex (CRC) organized around the
sarcoplasmic reticulum calcium channel, the ryanodine receptor (RyR1) in the reticulum membrane and the voltage dependant calcium channel, the dihydropyridine receptor DHPR, in
the plasma membrane. Among the proteins involved in the
efficient function of the CRC, calsequestrin (CSQ), triadin and
junctin are sarcoplasmic reticulum proteins able to interact
with RyR1 and regulate calcium release. A similar complex
exists in cardiac muscle, but with cardiac specific isoforms
(RyR2, CSQ2 …).
Triadin is a multiprotein family and the different isoforms could
have different functions. Whereas the two main skeletal muscle isoforms, Trisk 95 and Trisk 51, are specifically localized in
the skeletal muscle triad where they are associated to RyR1,
Trisk 32, a cardiac and skeletal muscle isoform, is localized in
skeletal muscle not only in the triad but in the whole reticulum, and regulates calcium releases via IP3R. In order to identify the function of triadin, triadin KO mice lines have been
developed. These mice have both skeletal muscle and cardiac
defects. Based on the phenotype of these mice, we search for
mutation in the triadin gene in patients with a rare cardiac disease, Catecholaminergic Polymorphic Ventricular Tachycardia
(CPVT), and without mutation identified in the classical genes
related to this disease, the cardiac ryanodine receptor RYR2
and the cardiac calsequestrin CASQ2. We identified the first
mutations in the TRDN gene, and demonstrated that these
mutations resulted in the absence of protein, therefore the
patients are all natural triadin KO. To further understand the
function of triadin, we expressed triadin in a non muscle cell
line, and observed that this expression resulted in modification of the endoplasmic reticulum, associated with an alteration of the microtubule network. We identified the amino
acids responsible of this effect. Our current hypotheses for the
functions of triadin are i) anchoring of CSQ to the triad, ii)
anchoring of the reticulum to the microtubule network and
iii) alteration of membrane curvature allowing RyR-DHPR contact.
requirements.
SA068
Two hands, one brain: The role of the ipsilateral hemisphere
in motor skill learning
Cesare Terracciano, Julia Gorelik and Magdi Yacoub
J. Diedrichsen
requirements.
Institute of Cognitive Neuroscience, University College London,
London, UK
A dominant idea in motor control is that skill learning is supported by plasticity in sensory-motor regions of the hemisphere contralateral to the trained hand. In contrast, the role
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Research Symposia
of the ipsilateral hemisphere is often considered to be that it
provides inhibitory input to these regions. Hence, it has been
suggested that it may be beneficial to suppress these regions
for improving motor skill learning. Using evidence from functional magnetic resonance imaging (fMRI) and transcranial
direct current stimulation (tDCS) studies, I will argue that the
ipsilateral hemisphere has a much more specific role in supporting motor skill acquisition, and that facilitatory intervention in these motor regions may provide beneficial effects for
skill learning and stroke recovery.
requirements.
SA069
Magnetic Resonance Spectroscopy as a tool to study the
role of GABA in motor plasticity and learning
C. Stagg
Bosnell R, Kincses T, Stagg CJ, Tomassini V, Kischka U, Jbabdi S, Woolrich M, Andersson J, Matthews PM and Johansen-Berg H. Motor practice promotes increased activity in brain regions functionally disconnected after subcortical stroke. Neurorehabilitation and Neural
Repair.25. 607-16. 2011
Dennis A, Bosnell R, Dawes J, Howells K, Cockburn J, Kischka U,
Matthews PM, Johansen-Berg H. Cognitive context determines premotor and prefrontal brain activity during hand movement in patients
after stroke. Stroke. 42. 1056-61. 2011
Crofts JJ, Higham DJ, Bosnell R, Jbabdi S, Matthews PM, Behrens TEJ,
Johansen-Berg H. Network analysis detects changes in the contralesional hemisphere following stroke. Neuroimage. 54. 161-9. 2011
Johansen-Berg H, Rushworth MFS, Bogdanovic M, Kischka U,
Wimalaratna S and Matthews PM. (2002) The role of the ipsilateral
premotor cortex in recovery of motor function after stroke. Proceedings of the National Academy of Sciences USA 99 14518-14523
Johansen-Berg H, Dawes H, Guy C, Wade D and Matthews PM. (2002)
Correlation between motor improvements and altered FMRI activity
after rehabilitative therapy. Brain 125, 2731-2742
requirements.
FMRIB Centre, University of Oxford, Oxford, UK
In this talk I will explore the role of GABAergic signalling in
motor plasticity. I will discuss the results of studies using Magnetic Resonance Spectroscopy (MRS), a non-invasive technique
that allows accurate quantification of neurotransmitters within
a localised region of the brain, which suggest that decreases
in inhibitory signalling occur during plasticity induction. I will
go on to present evidence suggesting that this decrease in inhibition can be related to behaviour on a subject by subject basis.
I will finish by highlighting recent evidence that modulation
of inhibitory processing is important in the maintenance of
plasticity in long-term plasticity such as that seen after recovery from stroke.
requirements.
SA070
Imaging and stimulating motor recovery after stroke
H. Johansen-Berg
University of Oxford, Oxford, UK
This talk will discuss ways in which functional and structural
imaging has contributed to our understanding of recovery after
stroke. It will also consider how this knowledge can be used
to design novel rehabilitation interventions. Examples will be
provided from FMRI studies which have shown that activity
during movement of an affected limb is altered after stroke.
In addition, studies using diffusion MRI will provide insights
into structural disconnection after stroke, and how this may
constrain recovery. Finally, recent evidence will be presented
showing positive effects of using non invasive brain stimulation as an adjunct to rehabilitation.
O’Shea J, Boudrias MH, Stagg, CJ, Bachtiar V, Kischka U, Blicher JU,
Johansen-Berg H. Predicting behavioural response to TDCS in chronic
motor stroke. Neuroimage. 85:924-33.
Stagg CJ, Bachtiar V, OShea J, Allman C, Bosnell RA, Kischka U,
Matthews PM, Johansen-Berg H. Cortical activation changes underlying stimulation-induced behavioural gains in chronic stroke. Brain. 135;
276-84. 2012
SA071
Combining training and immobilisation
E.E. Raffin and H.R. Siebner
DRCMR, Hvidovre, Denmark
Background: The primary motor cortex (M1) is part of the neural substrates of skills learning but has finite processing
resources. Typically, M1 representation of the trained body
parts has been shown to enlarge at the expense of the representations of less trained body parts (e.g. 1). According to this
competitive model of cortical resources, it might be possible
to surmount training limits by “weakening” the adjacent fingers’ representations. In this study, we tested the hypothesis
that the combination of training and concomitant immobilization of the adjacent fingers would effectively enhance neural changes and boost motor learning.
Methods: Participants (planned n=60) had to train at a visuomotor tracking task that requires abduction-adduction movements of one finger for one week (3 x 10 min/day). They used
either their index (Group 1) or their little finger (Group 2) and
half of them had their three non-trained fingers immobilized
(Groups 3 & 4). The two control groups consisted in wearing
the same splints without training (Groups 5 & 6). We recorded
their performances before and after the intervention and examined the neurobiological underpinnings of motor skill learning
through local changes in corticospinal excitability (CSE) profiles of hand muscles using neuronavigated Transcranial Magnetic Stimulation (nTMS) and changes in connectivity patterns
using functional Magnetic Resonance Imaging.
Results: Our preliminary data (n = 45) show that immobilization-only deteriorates performance of the deprived finger but
this “de-learning” can be prevented by training the adjacent
finger. This was associated with preserved CSE profiles of the
deprived muscle while immobilization-only induces a drastic
down-regulation. Concomitant immobilization of the adjacent
fingers did not facilitate tracking performance with the trained
finger despite of a stronger enlargement of the trained muscle’s CSE profile and different patterns of neural connectivity
changes affected by the training task.
Conclusion: The data show that concomitant immobilization
of the adjacent fingers does not boost motor learning, at least
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Research Symposia
for continuous visuomotor tracking. Although it needs to be
confirmed in larger trials, the results rather show that it might
be possible to avoid a decay in performance after limb immobilization by concurrent training of the adjacent body part.
Nudo RJ et al. (1996). J Neurosci 16, 785-807
requirements.
SA072
ZIEMANN, U., ILIC, T. V., PAULI, C., MEINTZSCHEL, F. & RUGE, D. (2004).
Learning modifies subsequent induction of LTP-like and LTD-like plasticity in human motor cortex. J Neurosci 24, 1666-1672.
requirements.
SA073
Potential role of the phosphoprotein PEA-15 in vascular
disease
Modification of plasticity and learning in the human motor
system by transcranial brain stimulation
G.F. Nixon
U. Ziemann
School of Medical Sciences, University of Aberdeen, Aberdeen,
UK
Neurology, University Tübingen, Tübingen, Germany
We have employed paired associative transcranial magnetic
stimulation (PAS), or theta-burst transcranial magnetic stimulation (TBS) for induction of long-term potentiation (LTP)-like
and long-term depression (LTD)-like plasticity in human motor
cortex, as indicated by lasting increases/decreases in motor
evoked potential amplitudes. In a first set of experiments we
demonstrated that practice of simple ballistic finger movements, leading to improved peak acceleration (i.e. kinematic
motor learning) of the trained movement, resulted in abolition of subsequent PAS-induced LTP-like plasticity but enhancement of LTD-like plasticity (Ziemann et al., 2004). These interactions strongly suggested that PAS-induced plasticity and
kinematic motor learning share common mechanisms. In a
next step, using PAS-induced plasticity as a surrogate marker
for motor learning, we demonstrated that priming with
another PAS protocol resulted in homeostatic or non-homeostatic regulation of the subsequent PAS-induced plasticity,
depending on the interval between the two protocols (MüllerDahlhaus et al., in preparation). In additional TBS experiments,
we showed that plasticity of inhibitory circuits, as tested by
paired-pulse transcranial magnetic stimulation are regulated
by homeostatic metaplasticity (Murakami et al., 2012). In a
third step, we demonstrated that kinematic motor learning is
regulated by homeostatic or non-homeostatic metaplasticity
when primed by PAS-induced LTP- or LTD-like plasticity, depending on the interval between priming PAS and kinematic motor
learning (Jung & Ziemann, 2009). These data are rather important because they suggest that priming of motor learning by
non-invasive brain stimulation may be utilized to enhance learning success. Finally, we showed absent regulation of kinematic
motor learning by preceding PAS in patients with task-dependent hand dystonia (writer’s cramp), with the magnitude of this
abnormality correlating to the clinical severity of dystonia
(Kang et al., 2011). Therefore, testing the interaction between
brain stimulation and motor learning may contribute to our
understanding of the pathophysiology of movement disorders
and may also be used for diagnostic purposes.
JUNG, P. & ZIEMANN, U. (2009). Homeostatic and non-homeostatic
modulation of learning in human motor cortex. J Neurosci 29, 55975604.
KANG, J.-S., TERRANOVA, C., HILKER, R., QUARTARONE, A. & ZIEMANN,
U. (2011). Deficient homeostatic regulation of practice-dependent
plasticity in writer’s cramp. Cereb Cortex 21, 1203-1212.
MURAKAMI, T., MÜLLER-DAHLHAUS, F., LU, M. K. & ZIEMANN, U.
(2012). Homeostatic Metaplasticity of Corticospinal Excitatory and
intracortical Inhibitory Neural Circuits in Human Motor Cortex. J Physiol 590, 5765-5781.
36P
Cardiovascular disease most commonly presents as an arterial
occlusion due to the presence of atherosclerotic lipid plaques.
Typically, balloon angioplasty is subsequently performed to
remove the plaque and restore blood supply to ischaemic tissue. Despite the increased use of metal stents to maintain
lumen diameter, re-occlusion still occurs in approximately 30%
of cases. This re-occlusion is the result of arterial hyperplasia
predominantly due to increased vascular smooth muscle (VSM)
cell proliferation. The primary mechanisms which initiate this
proliferation are not clear but probably involve regulation at
the transcriptional level. An important event in the modulation of VSM cells to a proliferative phenotype is the active
repression of smooth muscle (SM) marker proteins associated
with contractility, such as calponin and SM α-actin. This
decrease in SM marker proteins is controlled by growth factors via activation of the mitogen-activated protein kinases,
extracellular signal-regulated kinase (ERK)1/2. When an appropriate growth signal is received by the VSM cell, this leads to
phosphorylation of ERK1/2 which in turn regulates specific
transcription factors including Elk-1. Elk-1 has a critical role in
gene expression and in VSM cells is responsible for repressing
SM marker genes. We recently revealed a novel mechanism
regulating ERK1/2 and subsequently Elk-1 in VSM cells which
is likely to be important in regulating VSM cell proliferation.
An essential step in ERK1/2-induced changes in gene expression is the translocation of ERK1/2 from the cytoplasm to the
nucleus. The ability of growth factors to repress SM marker
genes is inhibited (and proliferation is decreased) if ERK1/2
nuclear translocation is prevented in VSM cells. Our experimental evidence indicates that a protein previously uncharacterized in VSM cells, phosphoprotein enriched in astrocytes15 (PEA-15), has an essential role in regulating ERK1/2 nuclear
localization. PEA-15 has a cytoplasmic distribution (courtesy
of a nuclear export sequence) and one of its primary roles is
to function as an ERK1/2-binding protein (Greig and Nixon,
2014). We have confirmed that PEA-15 binds and sequesters
ERK1/2 in the cytoplasm in VSM cells. Furthermore, in cultured
human coronary artery VSM cells, knockdown of PEA-15 results
in ERK1/2 nuclear localization, which can be reversed by PEA15 overexpression (Hunter et al, 2011). During physiological
stimulation by growth factors in VSM cells, ERK1/2 can only
be released from PEA-15 when PEA-15 is phosphorylated. This
occurs via growth factor-induced activation of protein kinase
C and Ca2+/calmodulin-dependent protein kinase II. This PEA15 phosphorylation is required for growth factor-induced VSM
cell proliferation. PEA-15 therefore has a critical role in VSM
cell physiology.
As PEA-15 has an important role in regulating VSM cell proliferation, changes in PEA-15 expression could have profound
effects on blood vessel structure and function. Specifically in
Research Symposia
vascular disease, such as arterial hyperplasia, decreased PEA15 expression would result in increased ERK1/2 nuclear localization and potentially increased proliferation. We have now
examined PEA-15 expression using an in vivo vascular injury
mouse model. Following wire injury of the mouse carotid
artery, PEA-15 expression was determined at different timepoints post-surgery. Our results reveal a significantly decreased
PEA-15 expression at 3 days post-injury compared to control
arteries. Importantly this occurs before hyperplasia is observed.
PEA-15 expression in arteries returns to normal at day 7 postinjury. Similar results for PEA-15 expression were also obtained
in an ex vivo model of vascular injury using human saphenous
vein.
In conclusion, the phosphoprotein PEA-15 has an important
role in VSM cell biology. It acts as a switch to regulate cell
phenotype and may also be involved in the pathogenesis of
vascular disease.
Greig FH & Nixon GF (2014). Pharmacol & Ther (in press).
Hunter I et al.(2011). Cardiovasc Res 90, 557-564.
This work was funded by the Medical Research Council
requirements.
SA074
Role of smooth muscle microRNAs in the regulation of
calcium signaling and vascular tone
S. Albinsson, A. Bhattachariya, D. Dahan, K. Turczynska,
K. Swärd and P. Hellstrand
Department of Experimental Medical Science, Lund University,
Lund, Sweden
The myogenic response is the intrinsic ability of small arteries
to contract in response to increased internal pressure. Although
microRNAs have been shown to play an important role in vascular smooth muscle function their importance for the regulation of the myogenic response has not been investigated previously. In this study we used smooth muscle-specific and
inducible Dicer KO mice, which are deficient of most microRNAs, and found that the myogenic response was ablated in the
small mesenteric arteries of these mice. This was associated
with an increased PTEN expression, possibly due to deletion
of miR-26a. Increased expression of PTEN, a negative regulator of PI3K/Akt pathway may reduce pressure-induced Aktphosphorylation and pressure induced calcium influx through
voltage gated L-type calcium channels. Furthermore, myogenic
tone was rescued by the L-type channel agonist BayK 8644 or
by transient stimulation with Angiotensin II (Ang II). The effect
of Ang II was dependent on AT1-receptor stimulation and activation of the PI3K/Akt pathway. These results suggest a novel
mechanism for regulation of myogenic tone in vascular smooth
muscle by microRNAs.
This work was supported by the Swedish Research Council; the
Swedish Heart and Lung Foundation, the Crafoord Foundation;
the Royal Physiographic Society; the Ake Wiberg Foundation;
the Jeansson Foundation; the
Tore Nilson Foundation; the Greta and Johan Kock Foundation;
the Magnus Bergvall Foundation and the Lars Hierta Memorial
Foundation. A.B and K.M.T. were supported by the European
Union FP7 Marie Curie
Initial Training Network Small Artery Remodeling (SmArt).
SMMHC-CreERT2 and Dicerfl/fl mice were kindly provided by
Prof. Stefan Offermanns and Prof. Matthias Merkenschlager,
respectively.
requirements.
SA075
Stem cells in vascular remodeling
S. Li
Department of Bioengineering, University of California, Berkeley,
Berkeley, CA, USA
Vascular stem cells (VSCs) play an important role in vascular
remodeling such as neointima formation. However, the relative contribution of VSCs and smooth muscle cells (SMCs) to
the proliferative SMCs remains controversial. Here we use lineage tracing models to compare the expansion and differentiation potential of VSCs (Sox10+) and SMCs (MHC+). Single
cell analysis showed that VSCs proliferate at a much higher
rate than SMCs, and the derived cells demonstrated the phenotype of myofibroblasts and proliferative SMCs, suggesting
that VSCs are a major contributor of traditionally defined proliferative SMC population. In addition, VSCs have the potential to differentiate into osteoblastic and adipogenic cells. In
vivo lineage tracing showed that neointima might contain
either a major population of VSC-derived cells or a large population of SMCs, suggesting different mechanisms of neointima formation. Furthermore, we demonstrated that VSCs contribute to the formation of microvessels during tissue
regeneration. These results highlight the important roles of
VSCs in vascular remodeling, and may lead to the development
of new therapy strategies to treat vascular diseases and to promote tissue regeneration.
requirements.
SA076
Neuropilins play a role in the development of neointimal
hyperplasia following arterial injury by modulating vascular
smooth muscle cell migration and proliferation
C. Pellet-Many, V. Mehta, I. Evans, L. Fields, J. Ruivo and
I. Zachary
Medicine, University College London, London, UK
With about two million procedures performed worldwide
every year (www.ptca.org), percutaneous transluminal coronary angioplasty (PTCA) remains the preferred method of coronary revascularisation following heart attack. The development of drug-eluting stents has greatly improved the outcome
following vascular intervention but restenosis remains an
important reason for the failure of revascularisation. The abnormal accumulation of neointimal vascular smooth muscle cells
(VSMC) resulting from both media-to-intima migration and
proliferation is the major underlying cause of restenosis. These
processes, together with deposition of extracellular matrix,
lead to neointimal hyperplasia, causing luminal narrowing, limiting the beneficial effects of the intervention.
Neuropilin 1 (NRP1) and 2 (NRP2) are versatile membrane coreceptors playing crucial roles in the developing cardiovascu-
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Research Symposia
lar and neuronal systems, but also in adult physiological and
pathophysiological settings such as the primary immune
response and cancer 1. NRP1 and 2 share an overall amino acid
homology of 44% and a similar domain structure. NRP1 null
mice die in utero between E12 and E13.5 with a spectrum of
cardiovascular and neuronal defects. NRP2-deficient mice survive to adulthood with some abnormality in the fasciculation
of certain cranial nerves and fewer and smaller lymphatic vessels, but no other obvious vascular defects 2,3. Neuropilins
(NRPs) complex with VEGF receptors in the endothelium where
they convey VEGF signaling 2 and therefore NRPs have been
shown to be crucially important for endothelial cell migration
and angiogenesis in several in vitro and in vivo models.
Moreover, NRPs are also able to mediate cellular functions via
non-canonical ligands involving signaling and the formation
of complexes with other growth factor receptors, including
PDGF/PDGFRs and transforming growth factor β (TGFβ) and
its receptors 4. Neuropilins are highly expressed in VSMC, mesenchymal stem cells (MSC) and hepatic stellate cells where
they have been shown to play a role in cell migration in
response to platelet–derived growth factor (PDGF) by associating with PDGF receptors α and β (PDGFRα and PDGFRβ) and
regulating their phosphorylation state and downstream
chemotactic signaling 5. So far, the role of NRPs in VSMC migration in vivo has not been explored. Since PDGF signaling is a
key regulator of VSMC migration and proliferation following
endovascular injury, we examined whether NRPs play a role
in the neointimal remodelling induced by balloon angioplasty
in the rat carotid artery, a well-characterised model of arterial
remodeling following endothelial denudation.
We found that endogenous NRP1 and NRP2 were up-regulated
both at mRNA and protein levels following arterial injury. The
down-regulation of NRP1 and NRP2 expression via adventitial
adenovirus delivery using shRNA constructs resulted in a clear
reduction of neointimal hyperplasia following arterial injury,
we then over-expressed wild-type (WT) NRP1 and a NRP1
mutant lacking the cytoplasmic domain (ΔC) and observed a
decreased neointimal response with the NRP1 ΔC infection,
whereas the NRP1 WT construct had no apparent effect. Bromodeoxyuridine (BrdU) staining revealed that in vivo proliferation was reduced in arteries transduced with the adenoviral
shNRP2 construct only. In vitro studies carried on rat aortic
smooth muscle cells (RAoSMC) supported this finding and
shown limited proliferation ability for the cells in which NRP2
was knocked-down, whereas cell infected with the other adenoviral constructs (NRP1 WT, NRP1 ΔC, shNRP1) displayed no
impaired proliferation in response to PDGF-BB. We used the
transwell assay to assess RAoSMC chemotaxis and found that
cells infected with NRP1 ΔC, shNRP1 and shNRP2 all displayed
a reduced migration towards the PDGF-BB gradient, and this
was in accordance with previous results in Human coronary
artery smooth muscle cells (HCASMC) 5. Moreover, NRP1 and
NRP2 are able to modulate PDGFR phosphorylation, which
could critically affect SMC function and ultimately the extent
of the neointima. Therefore, targeting NRPs expression in
human VSMCs may represent an attractive approach for the
treatment of proliferative vascular diseases.
Pellet-Many C, Frankel P, Jia H, Zachary I. Neuropilins: structure, function and role in disease. Biochem J. Apr 2008;411(2):211-226.
Kawasaki T, Kitsukawa T, Bekku Y, et al. A requirement for neuropilin1 in embryonic vessel formation. Development. November
1999;126(21):4895-4902.
Yuan L, Moyon D, Pardanaud L, et al. Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development. October
2002;129(20):4797-4806.
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Glinka Y, Stoilova S, Mohammed N, Prud’homme GJ. Neuropilin-1
exerts co-receptor function for TGF-beta-1 on the membrane of cancer cells and enhances responses to both latent and active TGF-beta.
Carcinogenesis. Apr 2011;32(4):613-621.
Pellet-Many C, Frankel P, Evans IM, Herzog B, Junemann-Ramirez M,
Zachary IC. Neuropilin-1 mediates PDGF stimulation of vascular smooth
muscle cell migration and signalling via p130Cas. Biochem J. May 1
2011;435(3):609-618.
This work is funded by the British Heart Foundation, grant no
PG/12/65/29840.
requirements.
SA077
Wnt signaling in vascular smooth muscle cells
S.J. George
School of Clinical Sciences, University of Bristol, BRISTOL, UK
Altered behavior of vascular smooth muscle cells (VSMCs) plays
an important role in cardiovascular diseases including atherosclerosis and restenosis. Accelerated VSMC proliferation and
migration lead to thickening of the intima and enhanced VSMC
apoptosis leads to atherosclerotic plaque rupture and clinical
symptoms of atherosclerosis. Consequently, the underlying
mechanisms for this altered VSMC behaviour is of great interest. It is well established that the Wnt signaling pathways are
critical for embryogenesis and development. In recent years
an important role of the Wnt signalling pathways in vascular
disease has emerged and is supported by observations of essential Wnt signalling in vascular development. In our laboratory
we have investigated the role of Wnt proteins in the regulation of VSMC behaviour. We have demonstrated that Wnt proteins enhance intimal thickening. Interestingly, Wnts have differing effects on VSMCs; Wnt4 promotes proliferation, whilst
Wnt2 augments migration. Moreover, we have identified that
Wnt proteins act as survival factors for VSMCs and retard VSMC
apoptosis at least in part by the up-regulation of WISP-1 (Wntinducible secreted protein-1). In summary, it is apparent that
Wnts have divergent effects on VSMC behaviour and a greater
understanding of Wnt pathways may reveal new therapeutic
targets for vascular disease.
This work is funded by grants from British Heart Foundation,
Heart Research UK and NIHR BRU Bristol Cardiovascular.
requirements.
SA078
Skeletal muscle AMPK: Role in regulating insulin sensitivity
and energy metabolism
G.R. Steinberg, K. Marcinko and J. Lally
Department of Medicine, McMaster University, Hamilton, ON,
Canada
Since the initial discovery that AMP-activated protein kinase
(AMPK) is increased in accordance with exercise intensity, there
has been significant interest in the proposed role of this ancient
stress-sensing kinase to act as a critical integrative switch controlling metabolic responses. AMPK exists as a heterotrimer
Research Symposia
that consists of an α catalytic subunit (α1,α2), a scaffolding β
subunit (β1, β2) and a nucleotide-binding γ subunit (γ1,γ2,γ3).
The C-terminal of the β subunit contains a highly conserved α
and γ subunit-binding sequence that is required for the formation of a stable, active AMPK αβγ complex. AMPK is stimulated by metabolic stresses (muscle contraction) or stresses
resulting in inhibition of ATP synthesis (ischaemia, hypoxia,
glucose/nutrient deprivation). The regulation of AMPK activity depends on AMP and ADP mediated phosphorylation of
the α catalytic subunit at T172 by upstream kinases (which in
skeletal muscle and liver is LKB1). Once activated, AMPK phosphorylates key metabolic enzymes that decrease ATP consumption (fatty acid & triglyceride synthesis) or generate ATP
(glucose uptake, fatty acid oxidation) (reviewed in Ref 1).
We have recently characterized the metabolic phenotype of
mice lacking both AMPK β1 and β2 isoforms in muscle (Ref
2). These were the first mice completely lacking AMPK activity in skeletal muscle. The most striking phenotype of the AMPK
Double Muscle Knock-Out mice (AMPK DMKO) is that these
mice are nearly completely exercise intolerant. This is evidenced by our observations that when given an exercise wheel
to run AMPK DMKO mice are completely inactive compared
to their wildtype littermates. Similarly, during treadmill exercise AMPK DMKO mice become fatigued immediately when
they start running compared to wildtype littermates that can
run for several hours at the same exercise intensity. Importantly, this much greater rate of fatigue is also observed when
muscles are contracted in vitro indicating that it is not due to
potential issues related to blood-flow or perfusion. In addition
this effect is not due to a dramatic fibre type-shift or reductions in maximal force production. As our studies have shown
that there is no substitute for skeletal muscle AMPK during
exercise/muscle contractions we hypothesized that AMPK may
be the molecular switch communicates exercise intensity to
other parts of the body. In this presentation we will discuss
recent findings linking skeletal muscle AMPK with the control
of insulin sensitivity and systemic energy metabolism.
O’Neill HM, Holloway GP and Steinberg GR. AMPK regulation of fatty
acid metabolism and mitochondrial biogenesis: implications for obesity. Molecular and Cellular Endocrinology 366(2):135-51, 2013.
O’Neill H, Maarbjerg S, Crane J, Jeppesen J, Jørgensen SB, Shyroka O,
Schertzer J.D., vanDenderen BJ, Kiens, B., Tarnopolsky MA., Kemp BE,
Richter EA, Steinberg GR. AMPK β1β2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose
uptake during exercise. Proceedings of the National Academy of Sciences. 108(38):16092-7, 2011.
These studies are supported by grants from the Canadian
Institutes of Health Research (CIHR) and the Natural Science
and Engineering Research Council (NSERC) of Canada. GRS is
supported by a Canada Research Chair in Metabolism and
Obesity.
requirements.
SA079
Does Ca2+ increase glucose uptake in skeletal muscle?
T.E. Jensen1, L. Sylow1, A.J. Rose2, S.J. Maarbjerg1 and
E.A. Richter1
1Nutrition, Exercise and Sports, University of Copenhagen,
Copenhagen, Denmark and 2German Cancer Research Center
(DKFZ), University of Heidelberg, Heidelberg, Germany
Skeletal muscle contraction rapidly increases the translocation
of glucose transporters (GLUTs) to facilitate the transport of
glucose across the cell membrane. The prevailing concept holds
that sarcoplasmatic reticulum (SR) Ca2+ release is neccesary
and sufficient to cause a substantial feed-forward increase in
glucose transport, with subsequent fine-tuning by feed-back
signals relating to the muscle work-load.
In the work presented here, a series of experiments were performed in incubated mouse muscles to scrutinize this model.
Our data challenge the current Ca2+-centric paradigm by showing that increased cytosolic Ca2+ coming from the SR was
unable to stimulate glucose transport in the absence of signals relating to ATP turnover and mechanical stress. Conversely,
artificial activation of AMPK and mechanical stress-signaling
elicited a full contraction-mimicking glucose transportresponse without increasing Ca2+ signaling.
These results call for a major reconsideration of the current
working model describing glucose uptake in contracting skeletal muscle and may provide a useful framework for future investigations.
requirements.
SA080
Novel regulatory mechanisms in muscle metabolism during
exercise: Role of nitric oxide in exercise induced glucose
uptake
G. McConell
ISEAL, Victoria University, Footscray Park, VIC, Australia
People with type 2 diabetes (T2D) have elevated blood glucose levels due, at least in part, to a reduction in skeletal muscle glucose uptake in response to insulin (insulin resistance).
The major site of whole body insulin-stimulated glucose uptake
is skeletal muscle (80-90%). Importantly, although skeletal muscle insulin-stimulated glucose uptake is impaired in people
with type 2 diabetes, glucose uptake during exercise is normal in these individuals. Contraction-stimulated glucose uptake
during exercise is so effective in people with T2D that as little
as 45 minutes of intense exercise can decrease blood glucose
levels to within normal levels.
The factors regulating skeletal muscle glucose uptake during
contraction are not entirely clear; however, there is evidence
that nitric oxide (NO) is involved (1). We have shown that NO
regulates leg glucose uptake during exercise independently of
leg blood flow. Importantly, we found that people with T2D
are especially reliant on NO to achieve normal glucose uptake
during exercise, since their glucose uptake was attenuated to
a greater extent by NO synthase (NOS) inhibition than controls
(2).
We have also shown that NOS inhibition in rats (ex vivo and in
situ) and mice (ex vivo) attenuates the normal increase in mus-
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Research Symposia
cle glucose uptake during contractions/exercise (1). However,
there are conflicting results from other laboratories concerning the role of NO in contraction-stimulated glucose uptake
(3, 4). Although the cGMP/PKG (PKG: cGMP-dependent protein kinase) signalling pathway is generally considered to be
the major downstream target of NO, we have preliminary evidence for cGMP/PKG-independent NO signalling of glucose
uptake during contraction via S-nitrosylation, S-glutathionylation and tyrosine nitration (5). More work is required including in mice that do not express nNOSμ, the isoform of NOS in
skeletal muscle which appears to be most involved in NO production during contraction/ exercise.
Once there is a better understanding of the mechanisms
though which NO signals glucose uptake during exercise, specific therapeutics can be designed for people with type 2 diabetes to mimic the contraction signalling pathway. Such an
agent could assist with blood glucose control of people with
type 2 diabetes who are either unable or unwilling to exercise
regularly.
McConell GK, Rattigan S, Lee-Young RS, Wadley GD, Merry TL. Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism. Am J Physiol Endocrinol Metab. 2012 Aug 1;303(3):E301-7.
vated by both insulin and muscle contraction in mouse and
human skeletal muscle. We identify Rac1 as an important regulator of muscle glucose uptake because pharmacological inhibition as well as knockout of Rac1 reduces insulin- and contraction-stimulated glucose uptake. Rac1 knockout mice
display significantly reduced glucose transport in response to
ex vivo electrically-induced contraction in isolated skeletal muscle. In vivo treadmill running-induced glucose uptake was also
significantly reduced in Rac1 knockout mice despite similar
exercise performance and similar activation of AMPK. We also
have found that Rac1 is activated by passive stretching (a major
component of the contraction stimuli) and that stretch-stimulated glucose transport is reduced in muscles of Rac1 knockout mice. These findings suggest that Rac1 may signal to glucose transport during contractions via stretch-activated
pathways. Our findings thus identify Rac1 as a novel convergence point between insulin and contraction-signaling to glucose uptake in skeletal muscle.
requirements.
Kingwell B, Formosa M, Muhlmann M, Bradley S, McConell G. Nitric
oxide synthase inhibition reduces glucose uptake during exercise in
individuals with type 2 diabetes more than in control subjects. Diabetes. 2002;51(8):2572-80.
Higaki Y, Hirshman MF, Fujii N, Goodyear LJ. Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and
contraction pathways in rat skeletal muscle. Diabetes. 2001;50(2):2417.
Roberts CK, Barnard RJ, Scheck SH, Balon TW. Exercise-stimulated glucose transport in skeletal muscle is nitric oxide dependent. Am J Physiol. 1997;273(1 Pt 1):E220-5.
Merry TL, Lynch GS, McConell GK. Downstream mechanisms of nitric
oxide-mediated skeletal muscle glucose uptake during contraction.
Am J Physiol Regul Integr Comp Physiol. 2010 Dec;299(6):R1656-65
National Health and Medical Research Council of Australia
(NHMRC)
requirements.
SA081
Rac1 in regulation of muscle glucose uptake
L. Sylow, I.L. Nielsen, M. Kleinert, T.E. Jensen, L.L. Møller and
E.A. Richter
Department of Nutrition, Exercise and Sports, University of
Copenhagen, Copenhagen, Denmark
Insulin resistance in skeletal muscle is established as an early
and key event in the pathogenesis of Type 2 Diabetes and contributes to whole body hyperinsulinemia and hyperglycemia.
During normal conditions, glucose is taken up into skeletal
muscle when glucose transporter (GLUT) 4 translocates to the
plasma membrane in response to stimuli such as insulin and
muscle contraction. Contraction-stimulated glucose uptake is
not affected by insulin resistance, likely because the intracellular events that regulate GLUT4 translocation by insulin and
muscle contraction are distinct. Because of this, activation of
glucose uptake by exercise is an important alternative way to
maintain whole body glucose homeostasis in insulin resistant
states such as Type 2 Diabetes.
Rac1 is a small GTPase that regulates the actin cytoskeleton
in cultured muscle cells. We have found that Rac1 can be acti-
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SA082
Regulation of lipid metabolism in exercise
B. Kiens
Molecular Physiology, Dept of Nutrition, Exercise and Sports
(NEXS), University of Copenhagen, Copenhagen, Denmark
Long-chain fatty acids (LCFA) from plasma and from lipolysis
of intramyocellular triacylglycerol (IMTG) both serve as fuel for
energy production. It is evident that the control of LCFA utilization in skeletal muscle is complex and is subject to a
plethora of regulatory mechanisms that have different degrees
of importance at rest and during exercise. From studies using
genetic manipulation of FAT/CD36, it seems that FAT/CD36 is
an important component in the control of plasma LCFA uptake
and utilization in skeletal muscle during exercise and the molecular mechanisms behind FAT/CD36 trafficking is central in
understanding how LCFA utilization is regulated.
The mobilization of fatty acids from the IMTG pool is catalyzed
by three lipases:
adipose triglyceride lipase (ATGL), hormone sensitive lipase
(HSL), and monoacylglycerol
lipase (MAGL) which sequentially degrade TG, diacylglycerol
(DAG) and monoacylglycerol (MAG), respectively.
In skeletal muscle, HSL has for long time been considered to
be the primary lipase activated by contractions. This notion
was based on in vitro activity measurements, where the contraction induced increase in TG-lipase activity was completely
blocked when adding an HSL-antibody to the assay medium.
However, the in vitro activity assay does not include changes
in important regulatory events such as translocation of lipases
to the lipid droplets and interaction with lipid droplet associated proteins, and therefore may not entirely reflect the acute
activation of muscle TG-lipases in vivo. In addition, in several
human studies dissociations between in vitro HSL activity and
net change in IMTG content during exercise have been
observed. This may reflect that lipases other than HSL are at
play. Knowledge about ATGL in skeletal muscle is limited, but
ATGL protein expression and activity have been demonstrated
in both rodent and human skeletal muscle. The functional
importance of ATGL for basal TG-hydrolysis in skeletal muscle
is highlighted by the finding of massive IMTG-accumulation
in ATGL-KO mice. These data strongly suggest that ATGL has
Research Symposia
an important role for skeletal muscle TG-hydrolysis. Furthermore, recent data suggest that ATGL is also activated in skeletal muscle during contractions and plays a major role in breakdown of IMTG.
requirements.
SA083
Post-translational regulation of renal epithelial transport
proteins
R.A. Fenton
Dept. of Biomedicine, InterPrET Center, Aarhus University, Aarhus,
Denmark
The function and activity of membrane-associated proteins
are often acutely regulated via various signalling molecules
and hormones via alterations in protein post-translational modification (PTM). The overall effects of their regulated function
are to maintain and modulate the homeostasis of the epithelial cell the proteins are expressed in. Our research focuses on
examining how various PTMs exert their effects on membrane
proteins to modulate their function. In particular, we are interested in the interplay between PTMs and the role of the membrane lipid bilayer for modulating the overall effects. In this
talk I will focus on our studies of a water channel (AQP2) and
a sodium-chloride cotransporter (NCC). The vasopressin-regulated water channel AQP2 is expressed in kidney collecting
duct principal cells where its apical plasma membrane abundance is the rate-limiting step for controlling the reabsorption
of water. A regulated balance of exocytosis and endocytosis
of AQP2 determines the levels of apical membrane AQP2 and
thus water reabsorption. PTMs of AQP2, in particular phosphorylation and ubiquitylation, are essential mechanisms for
modulating AQP2 subcellular sorting and distribution, degradation, and protein:protein interactions. Site-specific phosphorylation of AQP2 can determine the rate of its exocytosis
and endocytosis, whereas ubiquitylation of AQP2 can mediate its endocytic retrieval from the plasma membrane. Phosphorylation of AQP2 does not appear to affect the transport
capacity of the channel. Our recent data suggests that site specific phosphorylation of AQP2 is able to over-ride the internalization signal mediated by K63-linked polyubiquitylation,
providing a novel cell biological concept for membrane protein regulation. Lipid-rich domains within the plasma membrane play a pivotal role in determining the phosphorylation/ubiquitylation ratio. In contrast phosphorylation of NCC
does not alter its exocytic trafficking, but modulates NCC activity in the plasma membrane. We recently determined that
phosphorylation of NCC modulates NCC plasma membrane
abundance by limiting NCC ubiquitylation and thus endocytic
retrieval. In conclusion, PTM of membrane-associated proteins
is a diverse mechanism for modulation of protein function.
Interplay or ‘cross-talk’ between the PTMs increase the complexity of the system – with one particular type of PTM often
influencing the likelihood that neighboring amino acids
undergo further PTM to modulate protein function.
Fenton RA, Moeller HB, Hoffert JD, Yu MJ, Nielsen S, Knepper MA. Acute
regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin.
Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):3134-9.
Moeller HB, Praetorius J, Rützler MR, Fenton RA. Phosphorylation of
aquaporin-2 regulates its endocytosis and protein-protein interactions.
Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):424-9.
Pedersen NB, Hofmeister MV, Rosenbaek LL, Nielsen J, Fenton RA.
Vasopressin induces phosphorylation of the thiazide-sensitive sodium
chloride cotransporter in the distal convoluted tubule. Kidney Int. 2010
Jul;78(2):160-9.
Moeller HB, Olesen ET, Fenton RA. Regulation of the water channel
aquaporin-2 by posttranslational modification. Am J Physiol Renal Physiol. 2011 May;300(5):F1062-73.
Rosenbaek LL, Assentoft M, Pedersen NB, MacAulay N, Fenton RA.
Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC. J Physiol. 2012 Dec 1;590(Pt 23):6121-39.
Picard N, Trompf K, Yang CL, Miller RL, Carrel M, Loffing-Cueni D, Fenton RA, Ellison DH, Loffing J. Protein phosphatase 1 inhibitor-1 deficiency reduces phosphorylation of renal NaCl cotransporter and causes
arterial hypotension. J Am Soc Nephrol. 2014 Mar;25(3):511-22.
Rosenbaek LL, Kortenoeven ML, Aroankins TS, Fenton RA. Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Co-transporter NCC and Subsequent Clathrin-mediated Endocytosis. J Biol
Chem. 2014 Mar 25.
Funding was provided by the Danish Medical Research Council,
The Lundbeck Foundation, the Novo Nordisk Foundation, the
Carlsberg Foundation, and the Aarhus University Research
Foundation.
requirements.
SA084
miRNA in regulation of airway epithelial CFTR expression
C. Greene
Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
MicroRNAs (miRNAs) are small non-coding RNAs that have
emerged as major regulators of the protein content of a cell.
In the most part, miRNAs negatively regulate target mRNA
expression, with sets of miRNAs predicted to regulate certain
signaling pathways. We have demonstrated that the non-coding RNA expression profiles of endobronchial brushings is
altered in people with cystic fibrosis (CF) compared to those
without CF (1, 2). How this impacts on CF has important implications for our growing understanding of the pathophysiology of CF lung disease and the development of new therapeutics to treat its pulmonary manifestations (3-5). Expression
of the cystic fibrosis transmembrane conductance regulator
(CFTR) is altered in individuals with the F508del CFTR mutation. Here the role of miRNAs that are up regulated in vivo in
bronchial brushings from individuals carrying one or two
F508del CFTR alleles will be discussed (6). In particular studies that investigate miRNA-mediated regulation of CFTR expression and function will be presented including in vitro validation studies in the CFBE41o- cell line and assessment of the
impact of defective chloride ion conductance, genotype and
Pseudomonas colonization status on miRNA expression. The
results from overexpression and inhibition studies performed
using premiRs or antimiRs, respectively, and a CFTR 3’UTR
luciferase reporter gene will be shown. Collectively the data
show that miR-145, miR-223 and miR-494 are up regulated in
CF versus non-CF bronchial brushings and cell lines; in F508del
CFTR homozygotoes vs. heterozygotes; in subjects positive
for P. aeruginosa and; in cells treated with a CFTR inhibitor or
IL-1β. Reciprocal down or up regulation of CFTR gene and/or
protein expression was observed following miRNA manipulation and direct miRNA/target relationships demonstrated via
a reporter system containing a wild type or mutated full length
CFTR 3’UTR. The conclusions from this work are that increased
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Research Symposia
expression of miR-145, miR-223 and miR-494 in vivo in
bronchial epithelium of individuals carrying the Fdel508 CFTR
mutation correlates with decreased CFTR expression and that
defective CFTR function, Pseudomonas colonization and
inflammation may affect miRNA expression and contribute
to the regulation of Fdel508 CFTR..
Oglesby IK, Bray IM, Chotirmall SH, Stallings RL, O’Neill SJ, McElvaney
NG,Greene CM. miR-126 is downregulated in cystic fibrosis airway
epithelial cells and regulates TOM1 expression. J Immunol. 2010 Feb
15;184(4):1702-9. doi: 10.4049/jimmunol.0902669. Epub 2010 Jan
18. PubMed PMID: 20083669.
McKiernan PJ, Molloy K, Cryan SA, McElvaney NG, Greene CM. Long
noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis
bronchial epithelium. Int J Biochem Cell Biol. 2014 Mar 12. pii: S13572725(14)00074-0. doi:10.1016/j.biocel.2014.02.022. [Epub ahead of
print] PubMed PMID: 24631641.
Oglesby IK, McElvaney NG, Greene CM. MicroRNAs in inflammatory
lung disease—master regulators or target practice? Respir Res. 2010
Oct 28;11:148. doi: 10.1186/1465-9921-11-148. Review. PubMed
PMID: 21029443; PubMed Central PMCID:PMC2984500.
Hassan T, McKiernan PJ, McElvaney NG, Cryan SA, Greene CM. Therapeutic modulation of miRNA for the treatment of proinflammatory
lung diseases. Expert Rev Anti Infect Ther. 2012 Mar;10(3):359-68. doi:
10.1586/eri.11.175. Review. PubMed PMID: 22397568.
McKiernan PJ, Cunningham O, Greene CM, Cryan SA. Targeting miRNAbased medicines to cystic fibrosis airway epithelial cells using nanotechnology. Int J Nanomedicine. 2013;8:3907-15. doi:
10.2147/IJN.S47551. Epub 2013 Oct 11. PubMed PMID: 24143095;
PubMed Central PMCID: PMC3798151.
Oglesby IK, Chotirmall SH, McElvaney NG, Greene CM. Regulation of
cystic fibrosis transmembrane conductance regulator by microRNA145, -223, and -494 is altered in ΔF508 cystic fibrosis airway epithelium. J Immunol. 2013 Apr 1;190(7):3354-62. doi: 10.4049/jimmunol.1202960. Epub 2013 Feb 22. PubMed PMID:23436935.
Funding for this work is gratefully acknowledged from Science
Foundation Ireland, GlaxoSmithKline, the European Respiratory
Society and the National Children’s Research Centre.
requirements.
SA085
Proteolytic regulation of the epithelial sodium channel
(ENaC) by trypsin IV and cathepsin S
S. Haerteis and C. Korbmacher
Institut für Zelluläre und Molekulare Physiologie, FriedrichAlexander-Universität Erlangen-Nürnberg, Erlangen, Germany
Introduction: A unique feature of the epithelial sodium channel (ENaC) is its proteolytic activation (1, 2) which involves specific cleavage sites and the release of inhibitory peptide fragments (3). Proteolytic cleavage occurs in the extracellular
domains of α- and γENaC and is essential for channel activation under (patho-)physiological conditions. In humans, cleavage of δENaC may also contribute to channel activation (4).
Inappropriate ENaC activation by proteases may be involved
in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease (e.g. in nephrotic syndrome) (5). The pivotal final step in proteolytic channel activation probably happens at the plasma membrane, where
γENaC is cleaved by membrane-bound proteases and/or extracellular proteases (6).
The availability of several cleavage sites within a defined region
of the γ-subunit with preferences for different types of proteases may provide a basis for tissue-specific proteolytic ENaC
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activation. However, at present, the (patho-)physiologically
relevant proteases and molecular mechanisms involved in proteolytic channel activation remain to be determined. The prototypical serine protease trypsin I can activate ENaC in vitro
but is unlikely to be the physiologically relevant ENaC activating protease in vivo. In contrast, trypsin IV (mesotrypsin), a
form of trypsin known to be present in several extrapancreatic epithelial cells, may be co-expressed with ENaC. Moreover,
under pathophysiological conditions the cysteine protease
cathepsin-S (Cat-S) may reach ENaC in the apical membrane
of epithelial cells. Therefore, trypsin IV and Cat-S are candidate
proteases for proteolytic ENaC activation. The aim of the present study was to determine whether trypsin IV and Cat-S (7)
can indeed activate human ENaC expressed in the oocyte
expression system.
Methods: Site-directed mutagenesis was used to identify functionally relevant cleavage sites in the γ-subunit of the channel. Human wild-type or mutant αβγENaC was expressed in
Xenopus laevis oocytes. Amiloride-sensitive whole-cell currents
(ΔIami) were determined by two-electrode voltage-clamp
before and after 30 min incubation of the oocytes in human
trypsin IV (10 μg/ml) or Cat-S (1 μM). Biotinylated cell surface
γENaC cleavage products were detected by western blot analysis using a γENaC antibody.
Results: In Xenopus laevis oocytes, we monitored the proteolytic activation of ENaC currents and the appearance of γENaC
cleavage products at the cell surface. We demonstrated that
trypsin IV can stimulate ENaC. ENaC cleavage and activation
by trypsin IV requires a critical cleavage site (K189) in the extracellular domain of the γ-subunit. To confirm that the observed
ENaC activation is caused by the proteolytic activity of trypsin
IV, we examined the effect of the serine protease inhibitor
melagatran. Melagatran completely prevented stimulation of
ENaC currents by trypsin IV. In contrast, activation of ENaC by
trypsin IV was not prevented by the polypeptide inhibitor SBTI
(soybean trypsin inhibitor) confirming that trypsin IV, unlike
trypsin, is resistant to SBTI.
Furthermore, we demonstrated that Cat-S stimulates ΔIami in
ENaC expressing oocytes. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at
the plasma membrane indicating proteolytic channel activation. Interestingly, mutating two valine residues (V182;V193)
in the critical region of γENaC prevented proteolytic activation
of ENaC by Cat-S.
Conclusion: Our results show, for the first time, an activation
of ENaC by trypsin IV and by Cat-S, a member of the family of
cysteine proteases. Interestingly, both proteases use specific
cleavage sites to achieve channel activation. Hence, preferential cleavage sites may provide a mechanism for differential
ENaC regulation by tissue-specific proteases under (patho)physiological conditions.
Rossier BC & Stutts MJ (2009). Annu Rev Physiol 71, 361-79.
Kleyman TR et al. (2009). J Biol Chem 284, 20447-51.
Haerteis S et al. (2012) Cell Physiol Biochem 29, 761-74.
Haerteis S et al. (2009) J Biol Chem 284, 29024-40.
Svenningsen P et al. (2009). J Am Soc Nephrol 20, 299-310.
Haerteis S et al. (2012). J Gen Physiol 140, 375-89.
Haerteis S et al. (2012). Pflügers Arch 464,353-65.
This work was supported by the Interdisziplinäres Zentrum für
Klinische Forschung (IZKF) and the ELAN program of the
University of Erlangen-Nürnberg. We thank Erik Lindström,
Vera Baraznenok and Ian Henderson (Medivir AB) for providing
recombinant human cathepsin-S enzyme and cathepsin-S
inhibitor LHVS.
Research Symposia
requirements.
SA086
Protein-protein interactions and signalling complexes in
regulation of the brush border Na/H antiporter, NHE3
M. Donowitz
Johns Hopkins University School of Medicine, Baltimore, USA Minor
Outlying Islands
The epithelial brush border Na/H exchanger NHE3 accounts
for the majority of intestinal and renal Na absorption. In the
small intestine it is highly regulated related to eating; under
fasting conditions it is active, becomes quickly inhibited after
eating and then it is stimulated in the post-prandial state. The
inhibitory aspect of this regulation is duplicated as part of the
pathophysiology of diarrhea. Both stimulation and inhibition
of NHE3 involve large multi-protein signaling complexes that
form on the NHE3 C-terminus, which makes up its 377aa long
intracellular regulatory domain. The size of the NHE3 complexes are estimated as high as 1.7 mDa by density gradient
centfiguation, with changes in signaling leading to changes
in the size of the complexes and the associating proteins.
Insights in the specific complexes include: Domain 1: Close to
the NHE3 N- terminus: a) By homology with NHE1, calcineurin
homologous protein binds to the beginning of the NHE3 C-terminus (NHE3 aa 474-500). A peptide mimicking this domain
and presumably altering CHP binding stimulates NHE3 activity ( Babich, Vadnagara and Di Sole. FASEB J 27: 4646-4658,
2013). b) Just downstream there is an alpha-helical domain at
which ezrin directly binds (aa 504-528). Ezrin binding is
required for basal NHE3 activity (the PI 3-kinase dependent
component) and for stimulation of NHE3 by exocytosis by agonists such as EGF and LPA. Ezrin binding here is a regulated
process. On one side of this alpha-helix are three clustered Ser;
two of these are phosphorylated under basal conditions by
sequential activation of two kinase,s GSK-3B and Akt. Without
these phosphorylations, ezrin does not bind to this domain
of NHE3. Thus this signaling complex takes part in basal and
stimulated NHE3 activity by regulating direct ezrin binding to
NHE3. Domain 2: Middle of the NHE3 C-terminus: multiple
proteins associate with NHE3 between aa 585-606. This part
of the protein is involved with regulation of basal NHE3 activity controlling both stimulatory and inhibitory contributions
to basal activity. The identified associating proteins include
two kinases CK2 and CaMKII, NHERF 1,2,and 3; PLCγ, CaM and
slightly further downstream IRBIT. CK2 stimulates basal NHE3
activity. CaM KII inhibits basal NHE3 activity. Both kinases phosphorylate NHE3 further downstream in the C-terminus, CK2
at 719 and CaMKII at three sites. There are two other examples of three phosphorylation sites that are clustered in the
NHE3 C-teminus being required for regulation of NHE3 by a
single kinase (cGKII, RSK as well as CaMKII). This domain of
NHE3 is also involved in regulated NHE3 activity, particularly
by agonists that mimick the decrease that occurs after eating. Inhibition of NHE3 by elevated intracellular Ca2+ requires
CK2 phosphorylation of NHE3, presence of NHERF2 and
NHERF3 but not the presence of NHERF1. The NHERF2 and
NHERF3 dependence requires hetero-dimerization of NHERF2
and NHERF3. A peptide which mimicks this domain of NHE3
stimulates basal NHE3 activity at low concentrations in multiple cell types, including human enteroids, and should be considered as a model for drug development to treat diarrhea.
Conclusions: NHE3 is one of the most highly regulated transport proteins. The regulation requires two large, multi-protein
containing signaling complexes that form on its C-terminus.
One complex in domain 1 is involved in setting basal NHE3
activity and is necessary for acute stimulation of NHE3. This is
regulated by control of direct binding of NHE3 to ezrin. The
second complex in domain 2 sets basal NHE3 activity by both
increasing and decreasing NHE3 activity by altering its phosphorylation state. This complex takes part in inhibition of NHE3
activity which mimics the NHE3 inhibition that occurs immediately after eating, which spreads digestive enzymes over the
absorptive/digestive surface of the intestine and represents
the major function of NHE3 in digestion. Peptide mimicks of
the parts of NHE3 which form the large signaling complexes
stimulate NHE3 activity and thus identify NHE3 as a drug target for treating diarrhea.
requirements.
SA087
The hearts “little brain” in controlling cardiac function in
the rabbit
K. Brack
Cardiovascular Sciences, University of Leicester, Leicester, UK
Introduction: Until recently, interpretation of neural effects on
the heart was confined to the effects of centrally derived extrinsic sympathetic and parasympathetic inputs. However, it is
now known that neurocardiac control is more complex due
to an extensive independent network of neurones constituting a ‘little brain’, that are capable of modulating different cardiac functions. 1 These intrinsic neurons occur in discrete
regions on the surface of the heart and within epicardial fat
pads where they form “ganglionic plexuses” and function as
integrative circuits acting throughout the heart. 2 A recent
study in the rabbit shows that the locations of these ganglionic
plexuses are similar to other species and are primarily located
on the heart hilum, around the pulmonary arteries and right
caudal vena cava and on the conus arteriosus. 3 In the rabbit,
information pertaining to the functional effects of these ganglionic plexuses and their neurotransmitter content is absent.
The aims of this study were to 1) assess the neurotransmitter
phenotype, and 2) investigate the functional role of the ganglionic plexuses in the rabbit heart.
Methods: New Zealand White rabbit hearts (1-3kg) were used.
After sedation (ketamine, 10mg/kg [Ketaset]; medetomidine
hydrochloride [Sedator], 0.2mg/kg; butorphanol, 0.05mg/kg
[Torbugesic]; im), animals were heparinised (1000 IU) and killed
with sodium pentobarbitone (160mg/kg, iv). Aim 1: The left
and right side of the heart was cannulated and the heart
flushed from blood using 0.1M phosphate buffered saline (4oC)
and inflated with 20% gelatine. Following removal of material
overlaying the heart hilum, hearts were fixed with 4%
paraformaldehyde, and treated with hyaluronidase (0.5%) then
briefly stained for aceylcholinesterase (AChE) to confirm ganglia location. The whole mounts were then micro-dissected
from the heart and treated with appropriate blocking buffers
and incubated with primary antibodies raised against choline
acteyltransferase (ChAT), tyrosine hydroxylase (TH), neuronal
nitric oxide synthase (nNOS) and protein gene product 9.5
(PGP) for 48 hours (4oC). Visualisation of each neurotransmitter target was achieved following incubation with appropriate secondary antibodies tagged to specific Alexa Fluor flu-
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Research Symposia
orescent dyes. Aim 2: Hearts were perfused in constant flow
Langendorff mode and instrumented to record left ventricular pressure, perfusion pressure, atrial and ventricular electrograms. Nicotine was applied to different regions of the heart
that contained ganglionic plexuses (0.2 to 4 mg in 10 to 100
μL saline) and heart rate and atrio-ventricular conduction measured. Tests were carried out in sinus rhythm and during atrial
pacing in the absence and presence of muscarinic, nicotinic
and beta-adrenoreceptor blockade.
Results: Aim 1) Cell bodies and epicardial nerves were positively labelled for AChE throughout the heart hilum, around
the pulmonary arteries and right caudal vena cava and on the
conus arteriosus. ChAT was the primary neurotransmitter
found in the intrinsic cardiac nerve plexus with lesser amounts
of cell bodies and nerves immuno-reactive for TH and nNOS.
Co-location with PGP confirms the structures positively stained
were neuronal in nature.
Aim 2) Nicotine applied locally to regions containing ganglia
elicited either a bradycardia, tachycardia or brady/tachycardia
that was either dependent on cholinergic or adrenergic nerves.
In addition to these heart rate effects, nicotine primarily produced a delay in atrio-ventricular conduction. Ganglionic plexus
with specific cardiac actions were found.
Conclusion: These are the first data that describe the neurotransmitter content and functional effects of activation of different ganglionic plexuses in the rabbit heart.
Armour JA, 2008. Exp Physiol 93(2): 165-176.
Ardell JL, 1994. Neurocardiology. New York; Oxford University Press,
1994, pp95-114.
Saburkina et al, 2014. J Anat DOI: 10.1111/joa.12166.
Acknowledgment: KEB is supported on a British Heart
Foundation Intermediated Basic Science Fellowship (FS
12/2/29300).
requirements.
SA088
Renal sympathetic nerves – what have they got to do with
cardiovascular disease?
C. Barrett
Physiology, University of Auckland, Auckland, New Zealand
A hallmark of cardiovascular diseases such as hypertension and
heart failure is an elevation in sympathetic nerve activity (SNA),
especially to the heart and kidneys. In the case of heart failure, levels of cardiac and renal norepinephrine spillover have
been reported to be as much as 50 times above normal (Esler
& Kay, 1998). Understanding the significance of the elevation
in renal SNA would seem essential given sympathetic activation to the kidney is associated with poor prognosis (Petersson et al. 2005).
Elevations in renal SNA result in renal vasoconstriction,
increased renal sodium retention and increased renin release,
and consequently elevated angiotensin II and aldosterone levels (DiBona & Kopp, 1997). Sensory information from baroreceptors, chemoreceptors and osmoreceptors all modulate
SNA. In our laboratory we have employed the use of telemetry to continuously monitor arterial pressure and renal sympathetic nerve activity in conscious, freely moving animals.
We have demonstrated that, in the case of hypertension, the
elevation in renal SNA is dependent on circulating levels of
44P
angiotensin II, salt intake and a function of time (Guild et al,
2012). Removing the renal sympathetic nerves shifts the renal
pressure-natriuresis curve to the left, promoting urinary
sodium excretion and lowering of blood pressure. As a consequence renal nerve ablation, using radio frequency energy
to denervate the renal nerves, is rapidly gaining popularity as
a potential treatment for a plethora of cardiovascular diseases.
While in general it would seem that decreasing renal SNA is
the key to the clinical management of cardiovascular disease,
it is clear that this approach is simplistic. There are situations
where hypertension is not necessarily accompanied by an
increase in renal SNA. In our own animal models we have
observed a decrease in renal SNA in response to a pressor dose
of angiotensin II. This decrease in SNA is mediated by the arterial baroreceptors and sustained for at least 7 days (Barrett et
al, 2005) illustrating the potential importance in establishing
whether or not an elevation in renal SNA does contribute to
the hypertension prior to considering renal denervation.
By continuously recording renal SNA in rats following coronary
ligation to induced heart failure we have observed a gradual
increase in renal SNA, with nerve activity doubling within 14
days of the ligation. The exact cause of this increase in SNA
remains contentious with blunted baroreceptor reflex, sensitization of the chemoreflexes, increased levels of circulating
angiotensin II, oxidative stress and reduced levels of nitric oxide
among the potential contributing factors. To further complicate matters in chloralose-urethane anaesthetised rats we have
demonstrated that the change in renal sympathetic nerve activity immediately following coronary ligation is sex dependent,
with greater increases in male versus female rats. The sex differences in the response to coronary ligation can, at least in
part, be explained by the sex-differences observed in the arterial baroreflex control of renal SNA. The benefit of renal nerve
denervation in heart failure remains unclear, but there is at
least preliminary evidence to suggest renal nerve denervation
can improve fluid balance and cardiac function in heart failure. Proof of concept clinical studies suggest that arterial pressure in patients with heart failure is maintained post renal
nerve ablation (Davies et al. 2013), although whether such
patients can then cope with additional stressors such as sepsis or haemorrhage remains to be determined.
Clinical and experimental evidence generally suggests reducing renal sympathetic nerve activity should be a major aim in
the clinical management of hypertension and heart failure.
However, understanding the exact nature of the changes in
renal SNA that occurring during the progression of the disease
is essential if treatment is to be effective.
Esler M, Kaye D (1998). J Auton Nerv Sys. 72:210-219.
Petersson M, Friberg P, Eisenhofer G, Lambert G and Rundqvist B
(2005). European Heart Journal 26: 906-913.
DiBona GF, Kopp UC (1997). Physiol Rev 77: 75-197.
Guild SJ, McBryde FD, Malpas SC and Barrett CJ (2012). Hypertension
59: 614-620.
Barrett CJ, Guild SJ, Ramchandra R and Malpas SC (2005). Hypertension 46: 168-172.
Davies JE, Manisty CH, Petraco R, Barron AJ, Unsworth B, Mayet J,
Hamady M, Hughes AD, Sever PS, Sobotka PA and Francis DP (2013).
Int J Cardiol 162: 189-192.
Funding sources: Health Reseach Council of New Zealand,
Lotteries Health and University of Auckland Faculty
Development Fund
requirements.
Research Symposia
SA089
Autonomic control of the heart - going beyond the classical
neurotransmitters
N. Herring
NH is a British Heart Foundation Centre of Research Excellence
Intermediate Fellow at the University of Oxford. This work is
supported by projects grants from the BHF and an Oxford
Health Services research grant.
requirements.
Physiology, Anatomy and Genetics, University of Oxford, Oxford,
Oxon, UK
Beta-blockers are the only antiarrhythmic drug proven to
improve mortality post myocardial infarction and in chronic
congestive heart failure where sympathetic drive to the heart
is high. We have previously demonstrated that high-level sympathetic stimulation causes the release of additional sympathetic neurotransmitters in addition to norepinephrine, such
as neuropeptide-y and galanin (1) (2). In this presentation I
will present preliminary data suggesting that even in the presence of beta-blockers, the release of additional sympathetic
co-transmitters, can have deleterious consequences for cardiac function.
In the isolated Langendorf perused rat heart with intact innervation from both stellate ganglia, the susceptibility of the heart
to ventricular fibrillation is increased following direct stimulation of either the right or left stellate ganglia, even in the
presence of high dose beta-blockade. However, combining
beta-blockade with an antagonist of the neuropeptide-Y Y1
receptor can abolish the antiarrhythmic effect of sympathetic
stimulation. Exogenous neuropeptide-Y also directly lowers
ventricular fibrillation threshold and steepens the action potential restitution curve measured using optical mapping techniques.
Exogenous neuropeptide Y also acts as a potent vasoconstrictor increasing coronary vascular resistance in a Y1 receptor dependent manor in the Langendorf rat heart. Immunohistochemical staining of human coronary arterioles obtained
from appendage samples in patients undergoing coronary
artery by-pass, demonstrates dense Y1 receptor staining in
vascular smooth muscle cells in the media of these vessels. We
have also recently demonstrated a strong correlation between
plasma neuropeptide-Y levels and coronary microvascular function in patients with ST-elevation myocardial infarctions being
treated by primary percutaneous coronary intervention (3).
Targeting neuropeptide-Y receptors pharmacologically may
therefore be a useful therapeutic strategy both acutely during
myocardial infarction to improve microvascular function and
prevent coronary “no reflow” after stenting and also during
chronic heart failure as an antiarrhythmic agent. Such medications would be expected to act synergistically with betablockers, ACE inhibitors, and implantable cardiac devices such
as defibrillators and vagus nerve stimulators.
Herring N, Lokale MN, Danson EJ, Heaton DA, Paterson DJ. Neuropeptide Y reduces acetylcholine release and vagal bradycardia via a
Y2 receptor-mediated, protein kinase c-dependent pathway. J Mol Cell
Cardiol 2008;44:477-485.
Herring N, Cranley J, Lokale MN, Li D, Shanks J, Alston EN, Girard BM,
Carter E, Parsons RL, Habecker BA, Paterson DJ. The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal
bradycardia: Implications for neural control of cardiac excitability. J
Mol Cell Cardiol 2012;52:667-676.
Cuculi F*, Herring N*, De Caterina AR, Banning AP, Prendergast BD,
Forfar JC, Choudhury RP, Channon KM, Kharbanda RK. Relationship of
plasma neuropeptide Y with angiographic, electrocardiographic and
coronary physiology indices of reperfusion during ST elevation myocardial infarction. Heart 2013, 99(16), 1198-203 (*joint first author)
SA090
How sympathetic are your spinal cord circuits?
S. Deuchars
School of Biomedical Sciences, University of Leeds, Leeds, UK
All sympathetic control of end organs in the body relies on the
activity of the sympathetic preganglionic neurones (SPNs)
within the spinal cord that form the final pathway from the
central nervous system. These SPNs exhibit heterogeneity with
respect to their neurochemistry, location, descending inputs
and patterns of activity. Part of this heterogeneity is bestowed
by the local spinal cord circuitry, comprising interneurones
located in a number of regions of the spinal cord. Our understanding of the role of these local circuits and indeed the significance of connections between the SPNs themselves, through
specialised gap junctions, is patchy, but it is clear that the spinal
cord itself is capable of generating activity that is physiologically relevant.
Transneuronal tracing studies first identified presympathetic
interneurones that were located in laminae V, VII, X and the
intermediolateral cell column (IML) and these IML presympathetic interneurones are fast firing due to the presence of the
Kv3.1b channel subunit (1) while those in lamina X are GABAergic and directly inhibit SPNs (2;3). Some of the interneurones
in lamina X may play a more integrative role than just the control of sympathetic outflow since axons of single filled interneurones directly synapse onto both SPNs and motoneurones in
a 500 μm spinal cord slice. There may be a further subset of
these interneurones that colocalise acetylcholine and GABA
but their role is yet to be fully investigated. GABAergic inputs
onto SPNs exert their effects via a number of mechanisms,
through activation of GABAA and GABAB receptors (4) and also
through extrasynaptic receptors which serve to stabilise the
membrane at more negative potentials (5). These GABAergic
interneurones contribute to the rhythmic patterns of activity
that can be generated in the spinal cord either spontaneously
or through activation of 5-HT2 receptors since bicuculline
reduces the network oscillatory activity (6).
Gap junctions also play a significant role in sympathetic control; they are located on SPNS in the IML where they enable
rapid communication between these neurones. Blockade of
these gap junctions reduces local rhythmic activity in the spinal
cord slice (6) and also disrupts autonomic control in the working heart brainstem preparation (7). In mice lacking the connexin 36 subunits (Cx36 -/-) that are the main component of
SPN gap junctions, spikelet occurrence is almost abolished in
single SPNs and is severely diminished even after 5-HT application, while in Cx 36 +/- mice, there is a significant reduction
in ongoing and evoked spikelet frequency compared to the
wild-type. In the working heart brainstem preparation of Cx36
-/- mice, respiratory-related sympathetic activity is diminished
and responses to chemoreceptor activation are also reduced.
Using telemetry recordings in the awake Cx36 -/- mouse, the
variance in blood pressure and heart rate is much greater than
the wild type animals indicating that these gap junctions are
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Research Symposia
critical to enable the rapid changes in sympathetic activity that
underlie homeostasis.
These studies together indicate that local spinal cord circuitry
is critical in enabling appropriate levels and patterning of activity in sympathetic outflow. It is of interest to understand how
these circuits may be harnessed in the situation of spinal cord
injury. Another avenue of exploration into recovery of functionality after spinal cord injury is to elucidate whether there
is neurogenic capacity within the spinal cord, similar to that
observed in other CNS regions such as the subventricular zone.
Our very recent research suggests that it is possible to manipulate this plasticity, which may in future facilitate restoration
normal sympathetic function after injury.
nels (TRPM6 and TRPM7) form prime targets for hormonal control of the active Ca2+ and Mg2+ flux from the urine space or
intestinal lumen to the blood compartment. The characteristics of the newly identified transporters will be discussed and
in particular the distinctive molecular regulation of these new
epithelial Ca2+ and Mg2+ channels in (patho)physiological situations will be highlighted.
requirements.
SA092
Deuchars SA, Brooke RE, Frater B, Deuchars J. Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role
of the potassium channel subunit Kv3.1. Neuroscience
2001;106(2):433-46.
Vascularizing the tumor: emerging role for TRP channels
Deuchars SA, Milligan CJ, Stornetta RL, Deuchars J. GABAergic neurons
in the central region of the spinal cord: a novel substrate for sympathetic inhibition. J Neurosci 2005 Feb 2;25(5):1063-70.
Dept. Life Science and Systems Biology, University of Torino, Torino,
Italy
Deuchars SA. Multi-tasking in the spinal cord—do ‘sympathetic’
interneurones work harder than we give them credit for? J Physiol 2007
May 1;580(Pt.3):723-9.
Wang L, Bruce G, Spary E, Deuchars J, Deuchars SA. GABA(B) Mediated Regulation of Sympathetic Preganglionic Neurons: Pre- and Postsynaptic Sites of Action. Front Neurol 2010;1:142.
Wang L, Spary E, Deuchars J, Deuchars SA. Tonic GABAergic inhibition of sympathetic preganglionic neurons: a novel substrate for sympathetic control. J Neurosci 2008 Nov 19;28(47):12445-52.
Pierce ML, Deuchars J, Deuchars SA. Spontaneous rhythmogenic capabilities of sympathetic neuronal assemblies in the rat spinal cord slice.
Neuroscience 2010 Oct 27;170(3):827-38.
Lall VK, Dutschmann M, Deuchars J, Deuchars SA. The anti-malarial
drug Mefloquine disrupts central autonomic and respiratory control
in the working heart brainstem preparation of the rat. J Biomed Sci
2012;19:103.
We are very grateful for the generous support of The British
Heart Foundation and The Wellcome Trust for this work.
requirements.
SA091
The role of TRP channels in calcium & magnesium transport
in renal tubules
R. Bindels
Radboud university medical center, Nijmegen, Netherlands
Ca2+ and Mg2+ are of great physiological importance in their
function in neural excitability, muscle contraction, blood coagulation, bone formation, hormone secretion and cell adhesion.
The human body is equipped with an efficient negative feedback system counteracting variations of the Ca2+ and Mg2+
balance. These divalents are maintained within a narrow range
by the small intestine and kidney which both increase their
fractional (re)absorption under conditions of deprivation. Rapid
progress has recently been made in identification and characterization of the Ca2+ and Mg2+ transport proteins contributing to the delicate balance of divalent cations. Expression cloning approaches in combination with knockout mice
models and genetic studies in families with a disturbed Mg2+
balance revealed novel gatekeeper proteins that belong to the
super family of the transient receptor potential (TRP) channels. These epithelial Ca2+ (TRPV5 and TRPV6) and Mg2+ chan-
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A. Fiorio Pla
Transient Receptor Potential (TRP) channels modulate intracellular Ca2+ concentrations, controlling critical cytosolic and
nuclear events that are involved in the initiation and progression of cancer. It is not, therefore, surprising that the expression of some TRP channels is altered during tumor growth and
metastasis which are strictly dependent on tumor vascularization.
On the other hand, although the expression and role of ion
channels and transporters in the vascular endothelium is well
recognized and subject of recent reviews, only recently TRP
channels involvement in tumor vascularization have been recognized.
In the last years we investigated in detail the Ca2+ machinery
and TRP channels roles in Tumor derived endothelial cells (TEC)
compared with normal endothelial cells (EC) and evaluated
the differential involvement of key-intracellular messengers
triggered by proangiogenic factors (VEGF, bFGF) in vascular
ECs, such as AA, nitric oxide (NO) and hydrogen sulfide (H2S).
We correlated cell migration, proliferation and tubulogenesis
in vitro to intracellular Ca2+ signaling 1–3.
Our data suggest that TRPV4 is likely to be a key player in tumor
vascularization and in particular tumor-derived endothelial cell
(TEC) migration induced by arachidonic acid (AA), which
endogenously activate the channel. TEC display a significant
increase in TRPV4 expression, which is correlated with greater
Ca2+ entry. Wound healing assays revealed a key role of TRPV4
in regulating cell migration of TEC but not “normal” EC
(HMVEC). Knockdown of TRPV4 expression completely abolished AA-induced TEC migration, suggesting that TRPV4 mediates the pro-angiogenic effects promoted by AA. Furthermore,
pre-incubation of TEC with AA induced actin remodeling and
a subsequent increase in the surface expression of TRPV4. This
was consistent with the increased plasma membrane localization of TRPV4 and higher AA-stimulated Ca2+ entry in the
migrating cell 4.
On the other hand recent advancement in the characterization of the TRPM8 differential expression in normal versus TEC
and its role in cell migration inhibition suggest that the balance between TRPV4 and TRPM8 could be critical to control
such mechanism.
The possible implication of this balancing in tumor vascularization and the cellular and molecular mechanisms implicated
will be discussed.
Munaron, L., Genova, T., Avanzato, D., Antoniotti, S. & Fiorio Pla, A.
Targeting calcium channels to block tumor vascularization. Recent Pat.
Anticancer. Drug Discov. 8, 27–37 (2013).
Research Symposia
Fiorio Pla, A. & Gkika, D. Emerging role of TRP channels in cell migration: from tumor vascularization to metastasis. Front. Physiol. 4, 311
(2013).
Fiorio Pla, A., Avanzato, D., Munaron, L. & Ambudkar, I. S. Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRP channels as molecular targets. Am. J. Physiol. Cell Physiol. 302, C9–15
(2012).
Fiorio Pla, A. et al. TRPV4 mediates tumor-derived endothelial cell
migration via arachidonic acid-activated actin remodeling. Oncogene
31, 200–12 (2012).
requirements.
SA093
Remodeling of channel-forming TRP and ORAI proteins
determines an oncogenic switch in prostate cancer
N. Prevarskaya, C. Dubois, V. Fabien, L. V’yacheslav and
R. Maylis
Laboratoire de physiologie cellulaire inserm u1003, Villeneuve
d’Ascq, France
Normal cell progression to their malignant derivatives is associated with remodeling of the proteins controlling such major
cellular functions as apoptosis and proliferation. Here, we show
that prostate cancer cells use ORAI and TRP protein redistribution as an oncogenic switch mechanism. In particular, ORAI3
and TRPV6 remodeling results from genomic and microenvironment perturbations that disrupt the equilibrium of channels and favors the formation of novel Ca2+ channels activated
in a store-independent manner. This remodeling of Ca2+ signaling in turn induces cell progression to a more aggressive
pro-proliferative phenotype. Our study specifically positions
these channels at the center of molecular machinery linking
dysregulated arachidonic acid metabolism, calcium homeostasis, and oncogenesis.
requirements.
SA094
Voltage-gated sodium channels and metastatic disease
W.J. Brackenbury
Biology, University of York, York, UK
Since the 1970s, cancer incidence in the UK has steadily
increased and although survival rates continue to improve,
cancer is responsible for more than one in four deaths. Metastasis, the spreading of cancer cells from primary sites to form
secondary tumours in distant organs, is a major cause of physical and psychological morbidity, and is the leading cause of
cancer-related deaths. Metastasis is estimated to be responsible for around 90% of deaths from solid tumours. Treatment
options for patients with metastatic disease are largely limited
to palliation. Thus, there is an urgent unmet need to identify
new molecular targets and cost-effective adjuvant therapies
with curative intent.
Voltage-gated Na+ channels (VGSCs) are heteromeric proteins
composed of pore-forming α subunits and smaller β subunits.
The β subunits are multifunctional channel modulators and
are members of the immunoglobulin superfamily of cell adhe-
sion molecules. VGSC α and β subunits are best characterised
in the central nervous system (CNS), where they play a critical role in regulating electrical excitability, neurite outgrowth,
fasciculation, pathfinding and migration during development
(Brackenbury & Isom, 2011). VGSCs are also present in metastatic cells from a number of cancers (Brackenbury, 2012). In particular, expression of α subunits has been shown to favour an
invasive/metastatic phenotype. For example, Nav1.5 (encoded
by SCN5A) is expressed in metastatic breast cancer cells, and
potentiates their invasion and migration (Fraser et al., 2005).
Using the Oncomine expression array database of clinical cancer specimens, we have found that SCN5A is up-regulated in
breast cancer and associates with disease recurrence, metastasis and reduced survival. The VGSC-blocking anticonvulsant
phenytoin inhibits Na+ current in metastatic breast cancer cells,
particularly at depolarised holding voltages. It may thus be
an effective VGSC blocker in cancer cells, which typically have
depolarised membrane potentials (Yang & Brackenbury, 2013).
At a concentration within the therapeutic range used to treat
epilepsy, phenytoin inhibits migration and invasion of breast
cancer cells in vitro, suggesting that pharmacological targeting of Na+ current may have therapeutic utility (Yang et al.,
2012).
β subunits are also expressed in tumour cells. For example,
β1 (encoded by SCN1B) is expressed in breast cancer cells,
where it functions as a cell adhesion molecule (Chioni et al.,
2009). SCN1B mRNA and β1 protein are up-regulated in clinical breast cancer specimens, compared with normal tissue.
Further, β1 up-regulation substantially increases tumour
growth and metastasis in an orthotopic xenograft mouse
model of breast cancer. β1 over-expression also increases vascularisation and reduces apoptosis in the primary tumours,
and β1-expressing tumour cells have an elongate mesenchymal-like morphology. β1 potentiates outgrowth of elongated
processes from breast cancer cells co-cultured with fibroblasts,
via trans-homophilic adhesion. β1-mediated process outgrowth
in breast cancer cells requires both Na+ current and the presence and activity of fyn kinase, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons.
We conclude that when present in breast tumours, β1
enhances pathological growth and cellular dissemination by
recapitulating its well-defined role in CNS ontogeny. This is the
first demonstration of a functional role for β1 in cancer in vivo.
Moreover, β1 expression in breast cancer supports the notion
that carcinomas can acquire neuronal-like characteristics in
order to progress.
Together, our data support the hypothesis that VGSC α and β
subunits are up-regulated in breast cancer, favouring an invasive/metastatic phenotype, and may thus be promising targets for therapeutic intervention. We propose that repurposing existing VGSC-blocking drugs (e.g. antiepileptics,
antiarrhythmics) may have the potential to improve patient
outcomes in the metastatic setting. In particular, phenytoin,
a clinically approved oral anticonvulsant, should be further
studied as a potentially cost-effective treatment. In addition,
targeting β1-mediated adhesion interactions may have potential as a novel anti-cancer therapy.
Brackenbury WJ. (2012). Voltage-gated sodium channels and metastatic disease. Channels (Austin) 6, 352-361.
Brackenbury WJ & Isom LL. (2011). Na Channel beta Subunits: Overachievers of the Ion Channel Family. Frontiers in pharmacology 2, 53.
Chioni AM, Brackenbury WJ, Calhoun JD, Isom LL & Djamgoz MB.
(2009). A novel adhesion molecule in human breast cancer cells: Voltage-gated Na+ channel β1 subunit. Int J Biochem Cell Biol 41, 12161227.
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Research Symposia
Fraser SP, Diss JK, Chioni AM, Mycielska M, Pan H, Yamaci RF, Pani F,
Siwy Z, Krasowska M, Grzywna Z, Brackenbury WJ, Theodorou D, Koyuturk M, Kaya H, Battaloglu E, Tamburo De Bella M, Slade MJ, Tolhurst
R, Palmieri C, Jiang J, Latchman DS, Coombes RC & Djamgoz MB.
(2005). Voltage-gated sodium channel expression and potentiation
of human breast cancer metastasis. Clin Cancer Res 11, 5381-5389.
Yang M & Brackenbury WJ. (2013). Membrane potential and cancer
progression. Frontiers in Physiology 4, 185.
Yang M, Kozminski DJ, Wold LA, Modak R, Calhoun JD, Isom LL & Brackenbury WJ. (2012). Therapeutic potential for phenytoin: targeting
Na(v)1.5 sodium channels to reduce migration and invasion in metastatic breast cancer. Breast Cancer Res Treat 134, 603-615.
This work was supported by the Medical Research Council
[Fellowship number G1000508(95657)] and was part-funded
by the Wellcome Trust [ref: 097829] through the Centre for
Chronic Diseases and Disorders (C2D2) at the University of
York. The Breast Cancer Campaign Tissue Bank collected and
made available the samples used in this study.
requirements.
SA095
Voltage-gated potassium channels in tumour biology
L. Pardo
Max-Planck-Institute of Experimental Medicine, Göttingen,
Germany
The crucial role of voltage-gated channels in the pathophysiology of cancer has become a generally accepted concept. The
molecular nature of this implication is however obscure, and
appears to be specific for each channel. KV10.1 is a brain-specific voltage-gated potassium channel ectopically expressed
in a majority of extra-cranial solid tumors and a significant fraction of acute myeloid leukemia. Expression of KV10.1 is tightly
controlled by E2F1, p53 and miR34a; alterations on any of these
factors lead to aberrant expression of the channel. Once
expressed, KV10.1 confers a selective advantage to the cells,
at least partly mediated by increased angiogenesis (through
higher HIF1 activity and VEGF secretion), but it also directly
modifies cell cycle through at least two independent mechanisms, direct activation of cyclin-dependent kinases and altered
coordination of ciliogenesis. Its specificity for tumor tissue
makes KV10.1 an attractive target for tumor management.
Channel expression correlates with bad prognosis in several
tumor types, and pharmacological or genetic inhibition of
KV10.1 is able to reduce tumor progression. Channel inhibition alone does not induce regression in immunocompromised
mice, possibly because the role of KV10.1 in tumor cells is partly
independent of ion permeation. Immuno-targeted tools taking advantage of the tumor specificity of channel expression
(a single-chain antibody against KV10.1 fused to the human
soluble TNF-related apoptosis-inducing ligand (TRAIL) induced
apoptosis in KV10.1 positive cancer cells, but not in non-tumor
or KV10.1-negative tumor cells; in vivo, this resulted in efficacious tumor reduction in combination with doxorubicin.
Supported by the Max-Planck Society,
requirements.
48P
SA096
If only we were dolphins: Effects of obstructive sleep apnea
on chronic lung disease
M. Teodorescu
University of Wisconsin-Madison, Madison, WI, USA
Accumulating literature supports a bidirectional relationship
of obstructive sleep apnea (OSA) with chronic lung disease
(CLD), such as asthma, chronic obstructive pulmonary disease
(COPD) and pulmonary fibrosis. OSA is more common in CLD
and apart from traditional risks, a unique set of characteristics
related to CLD may predispose. In asthma, for example, these
appear related to complex dynamic physiology between upper
and lower airway during sleep, asthma-related inflammation,
and additional effects of corticosteroids. Once established,
OSA aggravates CLD. Patients with overlap of OSA and COPD
or pulmonary fibrosis have higher morbidity and mortality.
Likewise, in asthma, OSA relates to worse control, quality of
life and more health care use. Mechanistically, OSA features
appear intricately linked with modulating the lower airway caliber, inflammatory milieu and remodeling. For example, in
rodents with allergen-induced inflammation, chronic intermittent hypoxia (CIH) caused airflow limitation, a monocytepredominant Th-1 inflammation, and collagen deposition in
more proximal airways, coincident with matrix degradation in
the distal airways and parenchyma. Furthermore, following
bleomycin-induced lung injury, CIH augmented the total lung
collagen content and led to impaired gas exchange. These
changes are concerning, since none are responsive to current
standard therapies for example, for asthma. Although the Sleep
field has been dominated by pulmonologists, much work is
needed to understand this interaction. This talk will review the
pathophysiology of OSA in the context of lung disease, describing available clinical data on the effects of OSA on CLD, and
mechanisms emerging from human and animal studies. Further research directions will also be outlined.
requirements.
SA097
Interaction between intermittent hypoxia and high fat diet
to generate oxidative stress, sympathetic hyperactivity,
insulin resistance, and systemic hypertension
T. Gallego-Martin1,2, E. Olea1,2, C. Gonzalez1,2 and S. Yubero1,2
1University of Valladolid School of Medicine, Valladolid, Spain and
2CIBERES,
ISC III, Madrid, Spain
Obstructive sleep apnea (OSA) constitutes a common pathological process consisting in sleep-related repetitive obstructions of upper airways with consequent episodes of recurrent
or intermittent hypoxia (IH). Oxygen desaturation in each
episode of IH is the obvious element of altered respiration and,
as a consequence, experimental models of OSA are based on
the exposure of animals to atmospheres which composition
oscillates from room air to oxygen-poor gas mixtures; frequency of oscillations and of levels of oxygen can be adjusted
to mimic different severities of IH encountered in patients suffering OSA. As time progresses, OSA commonly generates a
pathologies affecting the cardiovascular system, in the form
of hypertension and increased frequency of heart and brain
Research Symposia
strokes, and the glucose and lipid metabolism in the form of
type II diabetes. Altogether define the obstructive sleep apnea
syndrome (OSAS). Clinical and experimental evidence supports
the notion that the pathogenesis of OSA-associated pathologies is linked to the oxidative status generated by the repetitive hypoxic episodes and to sympathetic hyperactivity mediated by carotid body overdrive of brainstem cardioregulatory
center. Obese subjects commonly develop cardiovascular and
metabolic pathologies which define the metabolic syndrome
(MS) and are analogous to those encountered in OSAS. This
fact has lead to propose a bidirectional interrelationship
between both processes, with obesity generating or aggravating OSA and OSA aggravating obesity and the associated
pathologies. From this perspective, we have aimed: a) to define
the effects of IH and obesity on redox status and sympathetic
activity assessed as plasma and renal artery catecholamine levels; b) to define the effects of IH and obesity on glycemic
metabolism and arterial pressure, and; c) to define the effects
of experimentally associating obesity and IH on all those
parameters. To achieve those aims we have used male rats
which at the age of 12 weeks continued to be fed with standard rodent chow (3.8 kcal/g with 10% Kcal as fat; control, C)
or started to be feed with fat-rich diet (5.2 kcal/g with 60% kcal
as fat, obese, O) for an additional 12 weeks period. These two
groups were further divided: half C rats were exposed to IH
(40s, 5% O2/80s, air; 8h/day; for 2 weeks; CIH) and half O rats
were equally exposed to IH (OIH). Experiments were performed
when the rats reached 24 weeks of age. Breathing parameters
were monitored by plethysmography in freely moving animals.
Arterial blood pressure (AP) was recorded in ketamine (100
mg/Kg, i.p) and, blood and tissue samples were removed from
pentobarbital anaesthetised rats (60 mg/Kg; i.p) and conveniently prepared for analysis. Major findings include: a) IH and
O cause an oxidative status (increased liver lipid peroxides and
diminished activities of mitochondrial and cytoplasmic superoxide dismutase), an inflammatory status (augmented C-reactive protein and nuclear factor kappa-B activation) and sympathetic hyperactivity (augmented plasma and renal artery
catecholamine levels and rate of synthesis); combined treatments in OIH rats worsened those alterations. b) Both IH and
O augmented lipid content in liver, plasma cholesterol, triglycerides, and leptin levels, also augmenting glycaemia, insulin
levels and HOMA index; IH and O animals were hypertensive
and most of the parameters aggravated when IH and O were
combined in OIH rats. c) CIH rats had a diminished ventilatory response to hypoxia and hypercapnia and O animals, while
maintaining minute ventilation, exhibited a restrictive ventilatory pattern; combination of treatments maintained the
restrictive pattern and hypoventilation. As a whole, data indicate that IH and O cause metabolic and cardiovascular pathologies associated to a misregulation of the redox status and sympathetic activity. In our experimental paradigm, which
intended to mimic the clinical situation, i.e., the appearance
of OSA in O patients, the deviations from normality were more
intense in O than in CIH, and the exposure of O animals to IH
as in OIH rats worsened the greatest part of the parameters
measured. Mechanisms generating the oxidative status and
the sympathetic augmented tone and their participation in
the genesis of hypertension and metabolic alterations will be
discussed.
Supported by Grants BFU2012-37459 and CIBER CB06/06/0050
to C.G.
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SA098
Protective mechanisms in acute myocardial infarction and
sleep disordered breathing
L. Lavie
Technion-Israel Institute of Technology, Haifa, Israel
Rationale: Mobilization and functions of endothelial progenitor
cells (EPCs) are increased in acute myocardial infarction (AMI)
patients. Yet, sleep disordered breathing (SDB) is highly prevalent in AMI patients.
Objective: To compare EPCs numbers and functions in AMI
patients with SDB (AMI-SDB) and without SDB (AMI-only) and
to determine the effects of intermittent hypoxia (IH) in-vitro
on EPCs proliferative and angiogenic properties.
Methods: Forty male AMI patients underwent a whole-night
sleep study using ambulatory monitoring. Nineteen had SDB
(oxygen desaturation index >5 events/hour). AMI-SDB and AMIonly patients were matched by age, BMI, blood chemistry and
co-morbidities. Blood samples were analyzed by flow cytometry, endothelial cell colony-forming-units (EC-CFUs), paracrine
measures, blood chemistry, and oxidative stress, inflammatory and angiogenic markers. Effects of IH in-vitro were studied in twelve healthy subjects.
Measurements and main results: Circulating EPCs (CD34+/
KDR+), angiogenic T cells (CD3+/CD31+/CXCR4+) and vascular endothelial growth factor (VEGF) in monocytes were significantly higher in AMI-SDB patients. Also, EC-CFUs numbers
and EC-CFUs paracrine effects on endothelial tube formation
were significantly higher in AMI-SDB then in AMI-only patients.
Similarly, in cell cultures from healthy subjects, EC-CFUs numbers and their paracrine effects on endothelial tube formation
were increased after exposure to IH in-vitro, compared to normoxia.
Conclusions: Co-existent mild-moderate SDB in AMI patients
increased the mobilization, proliferative and angiogenic capacities of EPCs, angiogenic T cells numbers and VEGF expression in monocytes, compared to AMI patients without SDB. IH
in-vitro had similar effects on healthy EPCs functions.
requirements.
SA099
Basic mechanisms of cardiovascular diseases in obstructive
sleep apnoea syndrome
S. Ryan
St. Vincent’s University Hospital and University College Dublin,
Dublin, Ireland
Obstructive sleep apnoea (OSA) is a highly prevalent disease
and associated with the development of various cardiovascular diseases which represents a significant public health burden. OSA comprises various pathophysiological triggers for
cardiovascular diseases, which include sleep fragmentation,
intrathoracic pressure swings and recurrent hypercapnia. However, there is now strong evidence that the unique form of
intermittent hypoxia (IH) observed in OSA, with repetitive short
cycles of desaturation followed by rapid reoxygenation, plays
a pivotal role in the development of cardiovascular co-morbidities. The pathogenesis is likely multifactorial and our current concept involves sympathetic nervous system overactiv-
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Research Symposia
ity, systemic inflammation and oxidative stress leading to
endothelial dysfunction, and possibly metabolic dysfunction
as the most important pathways.
Inflammatory processes are central in this pathogenesis and
there is ample evidence – arising from both cell culture and in
vivo models - that IH preferentially activates the transcription
factor nuclear factor-kappa B (NF-κB). NF-κB is a key player in
inflammatory and innate immune responses and when chronically activated contributes to atherosclerosis through driving production of inflammatory mediators such as tumour
necrosis factor alpha (TNF-α), interleukin (IL)- 8 and IL-6 and
these mediators have been found to be upregulated in OSA
patients. OSA and obesity are closely linked and there is increasing evidence that white adipose tissue is a potent source organ
of inflammatory mediators in OSA.
A greater understanding of the basic mechanisms of cardiovascular complications in OSA should lead to the identification
of therapeutic targets and therefore, further translational studies involving cell, animal and human models are strongly
required.
requirements.
in patients with OSA. However, further extensive research is
required to substantiate the potential clinical impact of the
association between OSA (via intermittent hypoxia) and cancer.
Almendros I et al. (2012). Eur Respir J 30, 215-217.
Almendros I et al. (2012). Sleep Medicine 13, 1254-1260.
Almendros I et al. (2013). Respir Physiol Neurobiol 186, 303-307.
Almendros I et al. (2014). Am J Respir Crit Care Med 189, 593-601.
Nieto FJ et al. (2012). Am J Respir Crit Care Med 186, 190-194.
Campos-Rodriguez F et al. (2013). Am J Respir Crit Care Med 187, 99105.
Martinez-Garcia MA et al. (2014). Eur Respir J in press.
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SA101
Exercise capacity and hemoglobin concentration in Tibetan
high altitude natives
P.D. Wagner1, T.S. Simonson1, G. Wei2, H. Wagner1, T. Wuren2,
M. Yan2, G. Qin2 and R.L. Ge2
SA100
Obstructive Sleep Apnea and Cancer: An Emerging
Relationship
R. Farre
University of Barcelona-CIBERES, Barcelona, Catalonia, Spain
Chronic intermittent is a challenge experienced by patients
with obstructive sleep apnea (OSA). It was well documented
that continuous or very low-frequency intermittent hypoxia
increase tumor growth. However, whether the high frequency
intermittent hypoxia characterizing OSA (up to 60 event/h)
enhances tumor progression and metastasis remained
unknown. Very recent data in animal models and in patient
populations have provided solid evidence on a positive relationship between OSA and cancer. Mice with injected subcutaneous melanoma cells showed a two-fold tumor growth rate
when subjected to intermittent hypoxia mimicking OSA as
compared with control animals breathing normoxic air (1).
This enhanced tumor progression was associated with
increased tumor vasculatisation and correlated with increased
circulating levels of vascular endothelial growth factor (2). This
mice model of OSA also showed that metastasis from the primary tumor to the lung was increased in the animals subjected
to intermittent hypoxia simulating OSA as compared with normoxic controls (3). Enhancement of cancer progression by
intermittent hypoxia has been observed in mouse tumors other
than melanoma (4). Very recent experimental data strongly
suggest that the increase in tumor progression caused by intermittent hypoxia is boosted through immune activation (4).
Moreover, data in humans confirm the observed cancer-OSA
association revealed by research in animal models. Increased
cancer mortality was observed in two different cohorts: in
1546 subjects of the general population followed-up for 22
years (5) and in 5320 patients with suspected OSA followedup for 4.5 years (6). In both cases, cancer mortality was significantly associated with the severity of nocturnal hypoxemia.
A more recent pilot study has shown that human melanoma
progression is associated with the severity of OSA (7). In conclusion, the data obtained in animal models and in patient
studies strongly suggest that there is an increased cancer risk
50P
1Dept of Medicine, Division of Physiology, University of California,
San Diego, La Jolla, CA, USA and 2Research Center for High-Altitude
Medicine, Qinghai Medical College, Xining, Qinghai, China
It has been known for some time that healthy Tibetans born
and raised on the high plateau (about 4300m) display a range
of hemoglobin values that vary from those of normal Caucasians at sea level (~15-17g/dl) to frankly polycythemic
(greater than 20g/dl). Genetic studies of this population have
revealed that the apparent loss of altitude-related polycythemic
response in those Tibetans without polycythemia is associated
with mutations causing down-regulation of hypoxia-sensitive
genes, in particular those of the HIF family. Moreover, genetic
analysis suggests this is of adaptive benefit, but if so, and how
from a physiological standpoint, has not been tested. In a
group of 21 healthy Tibetan males with similar daily activities
and [Hb] ranging from 16 to 23 g/dl, those with lower [Hb]
were able to reach a higher VO2peak/kg (at 4300m) than those
with high [Hb]. Multivariate analysis showed that this was
almost fully explained by a combination of higher cardiac output (QT) and greater muscle diffusing capacity for O2 (DMO2).
On the other hand, indices reflecting lung structure and function, such as arterial PO2, PCO2 and AaPO2 difference during
peak exercise, and lung volumes and flow rates at rest, did not
vary as a function of [Hb] and did not contribute statistically
to variance in peak VO2. That those with lower [Hb] not just
matched but exceeded their higher [Hb] counterparts in exercise capacity suggests that QT and DMO2 enhancement may
be the primary adaptive physiological change, and not compensatory to the lower [Hb]. Both the physiological and genetic
underpinnings of these observations remain to be investigated.
requirements.
Research Symposia
SA102
SA103
Short-term adaptation and chronic cardiac remodelling to
high altitude in lowlanders and native highland populations
Metabolic insight into mechanisms of high-altitude
adaptation in Tibetans
M. Stembridge1, P.N. Ainslie2 and R. Shave1
D.A. McClain
1Cardiff
University of Utah School of Medicine, Utah, UT, USA
School of Sport, Cardiff Metropolitan University, Cardiff,
UK and 2School of Health and Exercise Sciences, University of
British Columbia, Okanagan, BC, Canada
Both short-term and life-long high altitude exposure challenge
the cardiovascular system to meet the metabolic demand for
oxygen (O2) in a hypoxic environment. As the demand for O2
delivery increases during exercise, the circulatory component
of oxygen transport is placed under additional stress. Acute
adaptation and chronic remodelling of cardiac structure and
function may occur to facilitate O2 delivery in lowlanders during sojourn to high altitude and permanent highland residents.
However, our understanding of cardiac adaptation in highlanders remains limited. The Sherpa, one of the most successful
high altitude populations, are considered to be of Tibetan lineage and have resided at high altitude for over 25,000 years.
Previous research suggests Sherpa are able to achieve higher
maximal heart rates than acclimatised lowlanders and report
a lower severity of hypoxic pulmonary vasoconstriction compared to other ethnic groups native to high altitude. Despite
the potential for lower pulmonary pressures, neither the interaction of right ventricular (RV) afterload with cardiac structure
and function, nor the relationship between RV function and
left ventricular (LV) filling have been assessed. Ventricular form
and function are intrinsically linked through the unique
arrangement of myofibres that facilitate efficient ejection, minimize myofibre stress during contraction and aid diastolic recoil.
The myocardial mechanics (twist, rotation and strain) that
underpin ventricular function reflect this relationship and are
known to acutely change and chronically remodel in response
to altered physiological demand, as seen with hypoxia. Therefore, examination of myocardial mechanics allows detailed
insight into cardiac adaptation in high altitude populations.
Data from recent studies examining cardiac structure, function and mechanics in adolescent and adult Sherpa at high altitude and lowlanders before and after ascent to 5050 m will be
presented. Collectively, our findings indicate chronic structural
ventricular adaptation, with adult Sherpa having smaller
absolute and relative ventricular size. In line with structural
remodelling, cardiac mechanics also differ in Sherpa when
compared to lowlanders at high altitude. These changes may
reflect a functional cardiac adaptation that affords Sherpa
the same mechanical reserve seen in lowlanders at sea level
but not when they ascend to high altitude.
These studies were carried out within the framework of the
Ev-K2-CNR Project in collaboration with the Nepal Academy of
Science and Technology as foreseen by the Memorandum of
Understanding between Nepal and Italy, and thanks to
contributions from the Italian National Research Council.
requirements.
Recent studies have identified genes involved in high-altitude
adaptation in Tibetans. Three of these genes (EPAS1, EGLN1,
and PPARA) are associated with relatively decreased hemoglobin (Hb) levels. Consistent with the phenotype, EGLN1 in
Tibetans has a gain of function mutation that confers a higher
affinity for oxygen, hence less sensitivity to hypoxia. Considering the strong metabolic demands imposed by hypoxia, we
hypothesized that other selected genes might alter metabolism to allow adaptation to altitude despite the desensitization of the upstream hypoxia sensing caused by the EGLN1
mutation. A shift in fuel preference to glucose oxidation and
glycolysis at the expense of fatty acid oxidation would provide
adaptation to decreased oxygen availability. Measurements of
serum metabolites from Tibetans living at high altitude are
consistent with this hypothesis: The EPAS1 haplotype is significantly associated with increased lactate levels (suggesting
increased anaerobic metabolism), and the PPARAhaplotype
and serum free fatty acids are positively related (suggesting
decreased fat oxidation). These data, combined with preliminary calorimetry data in a Tibetan population living at low altitude, suggest that the high altitude adaptations may offer protection from diabetes at high altitude but increase diabetes
risk at lower elevations and/or with adoption of a nontraditional diet. We also hypothesize that because iron is a cofactor for EGLN1, there will be significant associations of phenotypes with the degrees of variation seen in tissue iron among
human populations, many of which are iron deficient. We have
begun to investigate the interactions of iron and hypoxia in
animal models in order to allow us to make predictions that
we can test in human populations. We have discovered that
low iron is synergistic with hypoxia in triggering cellular
autophagy, a process by which cellular organelles including
mitochondria are digested to provide metabolites that can
be used to provide energy, for example, providing amino acids
for gluconeogenesis in the liver.
requirements.
SA104
Adaptive changes related to hemoglobin levels in native
high-altitude Tibetans
T. Simonson1, T. Wuren2, G. Qin2, G. Wei2, H. Wagner1,
M. Yan2, D.J. Witherspoon3, C.D. Huff4, J. Xing5, L.B. Jorde3,
R. Ge2 and P.D. Wagner1
1Division of Physiology, University of California San Diego, La Jolla,
CA, USA, 2Research Center for High-Altitude Medicine, Qinghai
Medical College, Xining, Qinghai, China, 3Department of Human
Genetics, University of Utah, Salt Lake City, UT, USA, 4MD Anderson
Cancer Center, The University of Texas, Houston, TX, USA and
5Department of Genetics, Rutgers, Piscataway, NJ, USA
Studies of native high-altitude residents, who have been challenged by hypoxia for hundreds of generations, provide the
unique opportunity to identify and link genetic adaptations
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Research Symposia
to physiological changes in an extreme environment. Previous
research has shown that highland populations from different
continents and non-native highlanders respond to altitude in
different ways, yet a great deal remains to be learned about
the mechanisms underlying this variation. Most humans living at altitudes above 4000 meters develop polycythemia, classically considered an adaptive response to enhance oxygen
transport at altitude. It is therefore surprising to find that many
healthy Tibetan highland natives are not polycythemic. Recent
genomic analyses show that Tibetans have several genetic
adaptations to high altitude and haplotypes harboring a few
of these genes, including those involved in the hypoxia
inducible factor pathway (EPAS1, EGLN1, and PPARA), are further associated with loss of polycythemia (Simonson et al.
2010; Beall et al. 2010; Yi et al. 2010). Several genes reported
in more than one genomic analysis of Tibetan adaptation (e.g.
HMOX2, CYP17A1, PKLR, HFE, and HBB/HBG2 gene regions
[Wuren et al. 2014]) have not as of yet been associated with
traits exhibited by this population but warrant further analysis in a physiological genomics context. In order to determine
the physiological relevance, genetic underpinnings, and
sequence of adaptive events in Tibetans, we are examining
exercise capacity and oxygen transport, the precise genetic
targets that afford evolutionary advantages, and the relationships among these factors and relatively lower hemoglobin levels (loss of polycythemia) in adapted Tibetans. This natural experiment in human adaptation has broad implications
for understanding evolutionary processes and provides important insight into hypoxia sensing and response in humans at
altitude and in oxygen-limited disease states.
provides important physical support for cells and tissue, but
also has a crucial role in regulating cell behaviour mediating
survival, proliferation, differentiation and migration via interaction with specific cell adhesion receptors such as the integrins. Matrix metalloproteinases (MMPs) are a large family of
zinc-dependent proteolytic enzymes. They are best known
for their role in degradation of ECM molecules, release of ECMsequestered cytokines and growth factors, and cleavage and
activation of membrane-bound cytokines. We have previously
shown that advanced glycation endproducts (AGEs) accumulate in ECM proteins in the endoneurium of rats with streptozotocin-induced diabetes and that glycation of ECM proteins
with methylgloxal causes a reduction in both neurotrophinstimulated and preconditioned neurite-outgrowth from sensory neurons (Duran-Jimenez et al, 2009). Furthermore,
reduced MMP-2 expression in the sciatic nerve in diabetes may
also contribute to the failure of regeneration in experimental
diabetic neuropathy. These changes may provide a potential
mechanism for the failure of axonal regeneration and collateral sprouting observed in diabetic neuropathy and represent
a target for therapeutic intervention.
Duran-Jimenez, B, Dobler D, Moffatt S, Rabbani N, Streuli CH, Thornalley PJ, Tomlinson DR and Gardiner NJ (2009). Advanced glycation
end products in extracellular matrix proteins contribute to the failure
of sensory nerve regeneration in diabetes. Diabetes 58(12):2893-903
We thank the JDRF (2-2009-226) and Diabetes UK for support.
requirements.
Simonson et al. (2010). Science 329:5987, 72-75.
Yi et al. (2010). Science 329:5987, 75-78.
SA107
Beall et al. (2010) PNAS 107:25, 11459-11464.
Wuren et al. (2014). PLoS One 9(3):e88252.
requirements.
SA106
Diabetic neuropathy: nerve regeneration and the
extracellular matrix
N. Gardiner
Faculty of Life Sciences, University of Manchester, Manchester, UK
Diabetic neuropathy is a common secondary complication of
diabetes mellitus with no effective treatment. It has a complex
aetiology, associated with biochemical and structural changes
in the nervous system, nerve conduction velocity deficits and
altered mechanical and thermal sensitivity. A common feature
of both clinical and experimental diabetic neuropathy is degeneration of distal nerve fibres, and a reduced capacity for regeneration of injured axons and/or attenuated collateral branching from undamaged axons. This means that the rate of axonal
degeneration eventually exceeds that of regeneration leading
to numbness, loss of protective sensation and an increased
risk of amputation. In addition, diabetic neuropathy can be
accompanied by paresthesias, allodynia and ongoing pain and
can therefore impact on patient’s quality of life. We are therefore interested in elucidating the changes at both the cellular
and extracellular level that underlie the development of experimental diabetic neuropathy.
The main focus of this talk will be the effects of modification
of the extracellular matrix (ECM) in diabetes. The ECM not only
52P
Manipulating growth factors as potential therapeutics for
diabetic neuropathy
R. Hulse
Cancer Biology, School of Medicine, University of Nottingham,
Nottingham, UK
Painful neuropathy is a major complaint in many disease states,
particularly in diabetic mellitus patients. The impact of disease
or physical injury on neuronal systems leads to an array of neuronal alterations. These principally result in neuronal cell death
and neural degeneration highlighted by nerve regression and
neuronal apoptosis, events that affect neuronal signalling and
neurotransmission and are highly correlated to pain. A large
proportion of patients with painful neuropathy are diabetics,
and this neuropathy significantly affects their quality of life.
Damage to the peripheral nerves is not only associated with
pain, numbness or altered sensation, but also with a lack of
motor control and coordination (when motor neurons are damaged, or proprioception is impaired), and impact on autonomic
functions (e.g. salivary secretion), all of which also profoundly
affect quality of life.
In neurodegenerative disease peripheral nerve fibres are compromised, resulting eventually in the standard clinical hallmarks of neuropathy - regression of epidermal nerve fibre terminals and pain. The sensory nerve terminal regression found
in type I diabetes is as a result of a poor neuroprotective/regenerative ability. This is likely to be attributable to loss of necessary growth/support factors such as NGF, vascular endothelial growth factor-A (VEGF) and brain derived neuroptrophic
factor (BDNF). Therefore administration of the specific isoforms of growth factors such as VEGF165b, which has neuroprotective actions in vitro and in vivo, maybe beneficial. Sys-
Research Symposia
temic treatment of diabetic rats with VEGF165b prevented the
development of diabetic neuropathy, which is typified by
enhanced nociception to mechanical, thermal and chemical
stimuli, as well as neuronal loss of cutaneous nerve terminals
and sensory neuronal apoptosis in vivo.
When damage to peripheral nerve trunks occurs, the degeneration of damaged fibres results in changes in the sensory
properties of intact nerve fibres, often resulting in pain. Some
key molecules have been particularly implicated in diabetic
neuropathy, such as TRPA1, a channel fundamental to the sensitisation of neurones in chronic pain states. TRPA1 activity is
increased in diabetic neuropathy. This can contribute to both
sensitisation and death of nociceptors when it activated by
mitochondrial dysfunction, oxidative stress and other endogenous diabetic metabolites. VEGF165b treatment inhibits the
hyperglycaemic induced potentiation of TRPA1 activity. These
findings demonstrate that manipulation of VEGF expression
maybe a useful tool in treating diabetic complications, and in
particular neuropathy.
requirements.
SA108
Endoplasmic reticulum stress in diabetic retinopathy
S.X. Zhang
Ophthalmology, The State University of New York at Buffalo,
Buffalo, NY, USA
The endoplasmic reticulum (ER) is the primary cell machinery
responsible for lipid and protein biosynthesis, protein folding
and intracellular calcium storage. Maintaining a homeostatic
environment in the ER is vital for normal cellular activities and
survival. Undesired changes in cell metabolism disturb the balance between the protein synthesis and folding processes
resulting in ER stress. The ability to eliminate ER stress by an
intricate set of adaptive signaling pathways, known as unfolded
protein response (UPR), is closely correlated with cell function
and cell fate. Impaired and/or dysregulated UPR signaling has
been implicated in the pathogenesis of various human diseases. The presentation will discuss the impact and mechanisms of ER stress and the UPR signaling in diabetes-related
neurovascular injury of the retina and highlight the potential
of targeting UPR in the prevention and treatment of diabetic
retinopathy.
NIH grant EY019949, a Research Award from ADA, and
Unrestricted Grants from Research to Prevent Blindness to the
Department of Ophthalmology of University at Buffalo.
requirements.
SA109
Vascular Endothelial Growth Factor (VEGF-A)165b is
protective in diabetic nephropathy
Y. Qiu1, S. Oltean1, J.K. Ferguson1, M. Stevens1, C. Neal1,
S.C. Satchell2, S.J. Harper1, D.O. Bates3 and A.J. Salmon1,2
1School
of Physiology and Pharmacology, University of Bristol,
Bristol, UK, 2Academic Renal Unit, School of Clinical Science,
University of Bristol, Bristol, UK and 3Cancer Biology, Division of
Oncology, School of Medicine, University of Nottingham,
Nottingham, UK
Diabetic nephropathy is the leading cause of end-stage kidney
disease worldwide. VEGF-A is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, and both
VEGF-A knockout and overexpression of angiogenic VEGF-A
isoforms worsen diabetic nephropathy. We examined the vasculoprotective effects of the anti-angiogenic VEGF-A isoform
VEGF-A165b in diabetic nephropathy. VEGF-A165b was upregulated in diabetic individuals with well-preserved kidney function, but not in those with progressive disease. VEGF-A165b
overexpression in mouse podocytes in vivo prevented functional and histological abnormalities in diabetic nephropathy. Systemic VEGF-A165b injections, before and after the onset
of kidney disease, reduced features of diabetic nephropathy
in models of both type 1 and type 2 diabetes. VEGF-A165b
normalised glomerular permeability through phosphorylation
of VEGFR2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of
diabetic human glomeruli. These results show that VEGF-A165b
acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.
requirements.
SA110
Endothelial dysfunction in diabetes
A.W. Stitt
Queen’s University Belfast, Belfast, UK
Diabetes is marked by degenerative pathology in blood vessels. The macro and microvascular complications arising from
diabetes impose an ever-increasing burden on healthcare
authorities globally. The precise molecular defects that lead
to diabetes-related vascular cell dysfunction, and endothelial
cells in particular, remains ill-defined. This lack of understanding has seriously limited therapeutic options for conditions such as retinopathy, neuropathy, nephropathy and accelerated atherosclerosis in patients with diabetes. There is a
pressing requirement to identify definitive pathways that initiate cell damage and drive progression to overt micro and
macrovasculopathy. This presentation will review the molecular pathogenesis of diabetic microvascular complications,
with particular emphasis on restoring function to the endothelium, especially in the context of diabetic retinopathy (DR).
Patients with DR may irreversibly lose sight as a result of the
development of diabetic macular oedema (DMO) and/or proliferative diabetic retinopathy (PDR). Endothelial dysfunction
and degeneration plays an essential role in their pathogenesis. For example, with prolonged exposure to the diabetic
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milieu the replicative potential of the endothelium can become
exhausted leading to a premature senescent phenotype. It has
been proposed that the primary defect in DR (and other
microvasculopathies) lies with the vascular endothelium and
that these complications could be considered to be, at least
in part, “endotheliopathies”. Indeed, damage to these cells
impacts on the normal function of other cells in the capillary
complex.
The precise basis of endothelial, dysfunction in the diabetic
microvasculature in vivo remains obscure but is most likely
related to an array of cumulative biochemical insults coupled
with impaired ability of the cells to repair and renew themselves. Our group has focused on the reparative potential of
endothelial progenitor cells (EPCs). EPCs are a low-frequency
population of circulating cells known to be recruited to sites
of vessel damage and tissue ischemia where they promote vascular healing and re-perfusion. A growing body of evidence
suggests the number and function of EPCs is altered in patients
with varying degrees of diabetes duration, metabolic control
and in the presence of absence of complications. There is
mounting evidence that some EPC sub-types may be involved
in the pathogenesis of DR and may also serve as biomarkers
for disease progression and stratification. Moreover, some EPC
sub-types have considerable potential as therapeutic modalities in the context of cell therapy.
This review presents basic clinical concepts of endothelial dysfunction and highlights the potential for EPCs to be used to
repair damaged vasculature during diabetes.
requirements.
SA111
Serotonin spillover onto the axon initial segment of
motoneurons induces central fatigue by inhibiting action
potential initiation
F. Cotel1, R. Exley2, S. Cragg2 and J. Perrier1
1Neuroscience
and Pharmacology, University of Copenhagen,
Copenhagen, Denmark and 2Department of Physiology, Anatomy
and Genetics, University of Oxford, Oxford, UK
Motor fatigue induced by physical activity is an everyday experience characterized by a decreased capacity to generate motor
force. Factors in both muscles and the central nervous system are involved. The central component of fatigue modulates
the ability of motoneurons to activate muscle adequately independently of the muscle physiology. Indirect evidence indicates that central fatigue is caused by serotonin (5-HT), but
the cellular mechanisms are unknown. In a slice preparation
from the spinal cord of the adult turtle, we found that prolonged stimulation of the raphe-spinal pathway-as during
motor exercise-activated 5-HT1A receptors that decreased
motoneuronal excitability. Electrophysiological tests combined
with pharmacology showed that focal activation of 5-HT1A
receptors at the axon initial segment (AIS), but not on other
motoneuronal compartments, inhibited the action potential
initiation by modulating a Na+ current. Immunohistochemical staining against 5-HT revealed a high-density innervation
of 5-HT terminals on the somatodendritic membrane and a
complete absence on the AIS. This observation raised the
hypothesis that a 5-HT spillover activates receptors at this latter compartment. We tested it by measuring the level of extracellular 5-HT with cyclic voltammetry and found that prolonged
stimulations of the raphe-spinal pathway increased the level
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of 5-HT to a concentration sufficient to activate 5-HT1A receptors. Together our results demonstrate that prolonged release
of 5-HT during motor activity spills over from its release sites
to the AIS of motoneurons. Here, activated 5-HT1A receptors
inhibit firing and, thereby, muscle contraction. Hence, this is
a cellular mechanism for central fatigue.
requirements.
SA112
Activity-dependent plasticity of the axon initial segment
and its synapses
W. Wefelmeyer and J. Burrone
MRC Centre for Developmental Neurobiology, King’s College
London, London, UK
The axon initial segment (AIS) is a structure at the proximal
end of the axon with a high density of sodium and potassium
channels that defines the site of action potential generation.
It has recently been shown that this structure is plastic and can
change its position along the axon as well as its length in a
homeostatic manner (Grubb & Burrone 2010; Kuba et al.
2010). Here, we investigated AIS plasticity in CA1 neurons of
rat hippocampal organotypic cultures, using optogenetics to
excite individual pyramidal neurons expressing channelrhodopsin-2 (ChR2). 48 hour photostimulation (20 ms blue
light flashes delivered in bursts of 5 at 20 Hz, mean of 1 Hz)
led to an outward shift of the AIS by around 7 μm. This effect
was cell-autonomous and independent of network activity.
The structural plasticity of the AIS was accompanied by an
increase in current threshold for eliciting an action potential
and a shift in the input/output curve towards higher input currents resulting in an overall decrease in excitability.
In parallel to this, we also mapped and characterised the axoaxonic synapses that Chandelier interneurons form exclusively
onto the AIS of pyramidal cells. We find that these synapses,
labelled with either presynaptic (vGAT) or postsynaptic
(gephyrin, GABA-A alpha2 receptor) markers, did not translocate with the AIS after chronic stimulation. As a result, the
synapses formed onto the proximal end of the AIS were left
behind, creating a partial mismatch between axo-axonic
synapses and the AIS. We are currently exploring the functional
consequences of this intriguing form of plasticity.
Grubb, MS & Burrone, J. (2010). Nature 465,1070-1074.
Kuba, H et al. (2010). Nature 465,1075-1078.
This work was funded by a Wellcome Trust investigator award
to JB.
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Research Symposia
SA113
tion of axonal excitability can alter the temporal features of
spike trains.
Supported by NIH grants R00DC011080 and R01DA035913.
Molecular processes involved in the plasticity of the axon
initial segment
B. Dargent
requirements.
School of Medecine, CNRS-Aix Marseille University, Marseille,
France
SA115
The axonal initial segment (AIS) is a unique sub-domain that
plays a central role in the physiology of the neuron, as it orchestrates both electrogenesis and the maintenance of neuronal
polarity. The voltage-gated ion channels responsible for generating action potentials are concentrated at the plasma membrane of the AIS through interactions with the scaffolding protein ankyrin G (ankG). AnkG also binds to adhesion protein
Neurofascin-186 and NrCAM, and links membrane proteins to
the actin cytoskeleton through β4-spectrin, and to microtubules through End Binding proteins EB3 and EB1. The precise location and length of the AIS can vary from neuron to
neuron and thus influence neuronal function. Moreover, recent
studies revealed that the entire AIS is a dynamic structure,
adapting its length and its position along development and in
response to electrical activity.
Since the cellular morphology of the AIS regulates neuronal
excitability, it is crucial to understand the detailed molecular
mechanisms accounting for AIS stabilization and positioning
along the proximal axon. AIS maintenance depends on the stabilization of membrane and cytoskeletal protein complexes,
but also on distal axon properties. We have recently discovered new AIS components and I will present their role in the
proper AIS positioning and morphology.
requirements.
SA114
Neuromodulation of action potential initiation
K. Bender
UCSF, San Francisco, CA, USA
The axon initial segment is a specialized compartment that is
the site of action potential initiation in central neurons, and is
enriched with sodium and potassium channels that mediate
the depolarizing and repolarizing phases of spikes. In addition to these channel classes, we recently found that the initial segment is further enriched with low-voltage activated calcium channels (T- and R-type). Since these channels can be
active over long durations at hyperpolarized potentials, they
play a critical role in the generation of high-frequency volleys
of action potentials, termed bursts, and blocking initial segment calcium influx can suppress both spontaneous and
evoked bursting. Moreover, initial segment T-type channels
are regulated by dopamine in cells expressing Gi-coupled, type
3 dopamine receptors (D3). Upon activation, D3 receptors suppress calcium influx through these initial segment-localized
channels, without affecting the function of similar channels
expressed in the dendrites, leading to a suppression of burst
initiation. Here, I will discuss our recent efforts to understand
the molecular signaling cascade that supports dopaminergic
modulation of axonal channels, and how calcium channel localization and kinetics contribute to neuronal excitability. These
results will advance our understanding of how neuromodula-
Tuning electrotonic coupling of axonal and dendritic trees
M. Kole and K. Helmes
Axonal Signalling, Netherlands Institute of Neuroscience,
Amsterdam, Netherlands
High-densities of ion channels in the axon initial segment (AIS)
and nodes enable temporally precise action potential generation and conduction in vertebrate neurons (1). Computational
models of large cortical pyramidal neurons show that the
dimensions of the dendritic tree and the associated cable properties can impose a conductance and capacitive load on the
initial segment causing axial lateral current loss into soma during the onset of the action potential. As a consequence of the
reduced efficacy of sodium current to initiate action potentials it was recently hypothesized that the AIS ion channel density scales proportionally with the degree of dendritic conductance load (2). The passive cable properties of the AIS and
its relationship to the dendritic tree are, however, not well
understood. Here, we used a novel approach to obtain functional and structural information of single pyramidal neurons
to examine whether features of the AIS scale with the dendritic
tree. Wistar rats (n = 12) were anaesthetized (3% v/v isoflurane) and parasagittal brain slices (300 μm thick) prepared
using standard methods. Whole-cell patch-clamp recordings
were obtained from thick-tufted layer 5 pyramidal neurons
filled with biocytin and quantified for action potential properties and the subthreshold transients (n = 23). Slices were
subsequently fixed in 4% paraformaldehyde and stained for
AIS-specific anchoring protein markers and biocytin-streptavidin allowing examination and identification of the entire
axonal and dendritic tree in the slice. High-resolution confocal
laser-scanning microscopy (Leica TCS SP8) was used to collect
detailed information on the AIS and nodal domains together
with structural information of dendrites and axons. The results
indicated that the soma area positively correlated with the AIS
area (r = 0.85, Pearson’s bivariate correlation coefficient, p <
0.001, n = 24). Structure-function correlation showed furthermore that the location, but no other aspects of the AIS
correlated with the somatic action potential rate-of-rise (r =
–0.72, Pearson’s p < 0.001, n = 24). To systematically explore
the causal relationships we integrated immunofluorescence
data into three-dimensional reconstructions allowing a detailed
quantitative representation of the axonal domains into a morphologically realistic conductance-based computer model, constrained by the experimental data. Preliminary analyses indicate
that
both
geometry-dependent
and
geometry-independent rules exist for the size and location of
the axon initial segment. These data suggest that the geometry of the axon initial segment in part compensates for the
capacitive and conductance load of the cell, leading to both
normalization and tuning of the axo-somatic firing properties.
Kole MHP & Stuart GJ (2012). Neuron, 73(2), 235–247.
Hay E et al. (2013). J Neurophysiol, 109(12), 2972–2981.
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The investigation was in part supported by a European Research
Council Starting Grant (ERC-StG) #261114 to M.H.P. Kole. The
authors thank Sharon de Vries for the technical support.
requirements.
SA116
Control of vascular tone by endothelium
J. Pearson
King’s College London, London, UK
The potent in vivo vasodilator actions of acetylcholine were
first described by Henry Dale exactly 100 years ago. However,
isolated rings from blood vessels usually constrict to acetylcholine, a conundrum that exercised Robert Furchgott as early
as the 1950s, when he wrote an extensive review on the pharmacology of vascular smooth muscle. It was not until 1980
that Furchgott solved the conundrum, with the publication
of his classic paper demonstrating the obligatory role of
endothelium in the relaxation of vascular smooth muscle
caused by acetylcholine. It then took a further 7 years for Salvador Moncada’s group to show that NO, enzymatically formed
from arginine, was the endothelium-derived relaxing factor
(EDRF) described by Furchgott. At the same time Masashi Yanigasawa and colleagues discovered the potent endotheliumderived constriction factor (EDCF) endothelin-1 (ET 1). However, unlike NO, it is unlikely that ET-1 plays a highly dynamic
role in the control of vascular tone. Several other candidate
EDRFs (not least prostacyclin and H2O2) and EDCFs (e.g. other
prostanoids) exist. Amongst EDRFs, some cause vasorelaxation
by inducing hyperpolarisation of smooth muscle, and were
thus named endothelium-derived hyperpolarising factors
(EDHFs). The best characterised of these are epoxyeicosatrienoic acids (EETs). Particularly in resistance arteries,
there is evidence of myo-endothelial gap junctions directly linking electrical activity between endothelial and smooth muscle cells. Tudor Griffith in the 1990s pioneered studies to
demonstrate that these gap junctions were an important route
by which endothelium could directly hyperpolarise smooth
muscle and cause vasorelaxation without the need for secreted
factors. Subsequently others (particularly Steven Segal, Kim
Dora and Chris Garland) have shown that this route of electrical coupling between endothelium and smooth muscle is
also required to enable efficient conducted vasodilatation –
the physiological process whereby local vasodilation induced
at one site is conducted rapidly and co-ordinately upstream.
While there is now a wealth of data indicating that lack of NO
bioavailability (often due to uncoupling of NO synthase due
to loss of its required cofactor tetrahydrobopterin) underlies
impaired endothelium-dependent vasodilatation in disease
states such as diabetes, more recent data increasingly suggest
that the relative importance of other individually characterised
EDRFs and EDCFs in the control of vascular tone depends critically on the vascular bed being studied and its state of health
or disease.
requirements.
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SA117
The urothelium as a therapeutic target
B. Vahabi
Department of Biological, Biomedical and Analytical Sciences,
University of the West of England, Bristol, UK and Bristol Urological
Institute, Bristol, UK
The urothelium, which lines the inner surface of the urinary
bladder, is composed of at least three layers of cells: a basal
cell layer attached to a basement membrane, an intermediate
layer and a superficial apical layer with large hexagonal cells
also known as umbrella cells. The region between the urothelium and the underlying smooth muscle layers is called the
lamina propria and is richly supplied by blood vessels, nerves
and myofibroblasts (or interstitial cells (IC)). In addition to
being a barrier to urine, toxic metabolites and pathogens, the
urothelium exhibits specialised sensory properties and functions as primary transducer of some mechanical and nociceptive stimuli. Three lines of evidence suggest that the urothelial cells participates in the detection of both physical and
chemical stimuli: 1) close proximity to bladder nerves (efferent and afferent) 2) expression of numerous receptors/ion
channels by urothelial cells such as purinergic, adrenergic and
muscarinic receptors and transient receptor potential (TRP)
channels and 3) secretion of chemical mediators such as neurotrophins, ATP, acetylcholine, prostaglandins etc. By receiving, amplifying, and transmitting information, the urothelium
can convey information about the luminal pressure and urine
composition to the nervous and muscular systems and help
coordinate bladder function during filling and voiding.
Defects in urothelial-cell signalling/barrier function are likely
to contribute to the pathophysiology of bladder diseases. Alteration in tissue pH, mechanical or chemical trauma, bacterial
infection, interstitial cystitis or spinal cord injury can result in
altered expression/sensitivity of urothelial receptors/channels
and enhanced release of chemical mediators such as nitric
oxide (NO) and ATP from the urothelial cells. This may contribute to symptoms of urgency, frequency and pain during
bladder filling and voiding.
In addition, recent studies have shown that bladder urothelium expresses transmembrane channels called aquaporins
(AQPs). AQPs regulate transepithelial water movement in various tissues e.g. kidney and are regulated by circulating vasopressin. We have also found the expression of AQPs in pig urinary bladder, a valuable alternative to human bladder for
elucidating physiological principles. The discovery of AQPs in
bladder urothelium supports a more controversial hypothesis that urothelium is able to modify composition and volume
of urine in the lower urinary tract and may play an unappreciated but important role in water, salt and solute homeostasis. These observations open new exciting avenues for research
in urothelial biology and could lead to new strategies for the
clinical management of bladder-associated diseases.
requirements.
Research Symposia
SA118
Layers of complexity: Urothelium, interstitial cells and
smooth muscle interaction in urinary bladder
K.D. McCloskey
Queen’s University, Belfast, Belfast, Northern Ireland, UK
The unique physiological properties of the diverse array of cells
within the bladder wall enable its normal functions of filling
and emptying. Studies of diseased bladders by our group and
others have shown that lesions tend to occur in multiple cell
types (urothelium, nerves, interstitial cells (IC) or smooth muscle) underpinning dysfunction. The spinal cord injured (SCI)
rat bladder exhibits early areflexia followed by chronic overactivity and late decompensation. Loss of the urothelium in
the first few days after SCI is reversed by week 2 (1) and our
recent (unpublished) work has shown that this is accompanied
by acute damage to the interstitial cells of the lamina propria
(IC-LP). Restoration of the IC-LP occurs by day 7 during urothe-
lial regeneration. Patchy denervation after SCI is associated
with loss of a population of detrusor IC indicating that the
survival of some IC is dependent on normal nerve interactions
(2). In acute, areflexic SCI, many of the IC proliferate and
undergo phenotype change through myofibroblastic to de
novo smooth muscle formation, both in the detrusor and in
the lamina propria. This at least partly accounts for characteristic SCI bladder smooth muscle hypertrophy which is a
pathophysiological response to urinary retention.
Other insults to bladder function include diabetes and pelvic
radiation therapy, both of which cause changes to the physiology of urothelial cells, IC and smooth muscle. The available
experimental evidence will be presented with emphasis on disruption of cell-cell signaling with key areas of current and future
research highlighted.
Birder LA. (2011) Handb Exp Pharmacol. 202:207-31.
Johnston L et al, (2012) J Cell Mol Med.
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Oral Communications
C01
requirements.
Does the hypodynamic phase of sepsis provoke changes in
the excitability of cardiac myocytes?
L.P. Silva1, J.F. Pinho da Silva2, L.C. Rezende3, R.S. Poncianno2,
D.G. Souza3 and J.S. Cruz2
1Physiology and Biophysics, Universidade Federal de Minas Gerais,
Belo Horizonte, Minas Gerais, Brazil, 2Biochemical and
Immunology, Universidade Federal de Minas Gerais, Belo
Horizonte, Minas Gerais, Brazil and 3Microbiology, Universidade
Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Sepsis is a systemic inflammatory syndrome associated to infection with high mortality. Recent data show that sepsis mortality rates can reach up to 80% depending on the severity
(1). Cardiovascular dysfunction is associated with high mortality varying from 70% to 90% compared with 20% in patients
without any cardiovascular damage (2). Despite the high number of cases, sepsis does not have an effective therapeutic
option for mortality control, largely due to the still limited
knowledge about cellular mechanisms and sub-cellular components involved in cardiovascular changes during the development of sepsis (3). This work proposes to investigate if there
are changes in excitability and/or contractility at the cellular
level that could be correlated with the heart dysfunction
observed during sepsis. Balb C mice (8-12 weeks of age) were
anesthetized (xylasine/ketamine 10/50 mg/kg) and submitted
to cecal ligation and puncture (CLP) based on Baker et al.
(1983). After, 6 and 24 hours (h) of CLP left ventricular cardiomyocytes were isolated from Sham and CLP operated mice
as described by Shioya (2007). The cells were submitted to
electrophysiological recordings to measure action potentials
(AP). The same batch of isolated cells was used to determine
contractile parameters. Experiments were performed in accordance with Ethics Committee in Animal Experimentation
(CEUA) from Universidade Federal de Minas Gerais, Protocol
Nr: 137/2012. In a non-lethal sepsis experimental set, we could
not detect important differences between AP from the studied groups neither after 6 nor 24 h post CLP procedure. There
were no significant differences among the experimental groups
in the cardiac AP overshoot, time to peak, depolarization rate
(dV/dt) and time to 10%, 50% and 90% of repolarization. Considering an experimental set of lethal sepsis, 6 h after CLP, it
was observed changes in AP that suggests modification in Na+
channel function and/or expression, once we observed that
the depolarization rate was significantly increased in AP measured from septic cells (263±12 V/s, SHAM, n=16 and 578±64
V/s, SEPTIC, n=5, *One way ANOVA p<0.05). Likewise, contractile parameters showed no significant changes in fractional
shortening, shortening rate and diastolic length. Interestingly,
the relaxation rate was significantly different (156±2 μm/s,
SHAM and 108±19 μm/s, SEPTIC. One Way ANOVA *p<0.05,
n=20). Based in our results, we can conclude that the heart
failure developed during sepsis may be not related to a reduction in cardiac excitability nor contractility in general. These
observations points to the notion that the circulatory disorder
that occurs after 6 h of sepsis is not due to a global cardiac dysfunction but, instead, the heart acts in a compensatory way
to balance the hypodynamic vascular dysfunction. Clearly further studies are needed.
Jawad I et al. (2012). J Glob Health 2, 10404.
Celes MR et al. (2010). Lab Invest 90, 53-54.
Hochstadt A et al. (2011) J Cardiothorac Vasc Anesth 25, 526-535.
CNPq, FAPEMIG, Capes and CAPES/CASADINHO
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Heterogeneity in transverse (t)-tubules in pig atria
H.C. Gadeberg1, R.C. Bond1, C.H. Kong1, M.B. Cannell1,
R. Ascione2, G. Chanoit3 and A.F. James1
1School of Physiology & Pharmacology, University of Bristol, Bristol,
UK, 2School of Clinical Sciences, University of Bristol, Bristol, UK
and 3School of Clinical Veterinary Sciences, University of Bristol,
Bristol, UK
Transverse (t)-tubules are invaginations of the sarcolemma
that play a key role in excitation-contraction coupling and associated Ca2+ fluxes in mammalian cardiac myocytes. T-tubules
were considered to be effectively absent from atrial myocytes.
However, recent studies have demonstrated t-tubules in the
atria of larger mammalian species (1–4). To date there have
been no reports of the presence of t-tubules in pig atrial
myocytes. The aim of the present study was to investigate
the t-tubule network of pig atrial myocytes in comparison with
ventricular cells.
Animal procedures were approved by local ethics committee
and conducted according to UK legislation. Atrial and ventricular tissue was obtained from 5-6 month old female Landrace White pigs (45-75 kg). Atrial cells were isolated by arterial perfusion with a collagenase-containing Tyrode’s solution.
Membranes of isolated cells were visualised using di-8-ANEPPS
or AlexaFluor 488-conjugated wheat germ agglutinin (WGA).
The spatio-temporal properties of Ca2+ transients were examined in field-stimulated (1 Hz) isolated cells loaded with fluo4-AM (5 μM) and superfused with Tyrode’s solution (pH 7.4,
22 °C). T-tubules were visualised in fixed, frozen tissue sections
stained with AlexaFluor 488-conjugated WGA and AlexaFluor
633-conjugated phalloidin. Binary images were obtained by
application of a threshold and t-tubule density (TTD) calculated as the percentage of bright pixels. A Gaussian function
was fitted to the distribution of TTD. A distance mapping
approach was used to calculate the percentage of pixels within
0.5 μm of t-tubule-like staining (%TT0.5) (2, 3). Data were
analysed by Student’s unpaired t-test or one or two-way
ANOVA with Bonferroni post-tests. P<0.05 were considered
significant.
Isolated ventricular cells showed a well-developed t-tubule network whereas atrial myocytes did not. The spatio-temporal
properties of the atrial Ca2+ transient were consistent with the
absence of t-tubules. In tissue sections, TTD was significantly
lower in atrial (2.69±0.378, n=33) compared to ventricular
(7.52±0.379, n=32; P<0.0001) myocytes. However, t-tubules
could be identified in 9 of 33 atrial cells. The probability distribution of TTD in atrial cells was consistent with the existence
of a sub-population of cells with t-tubules (P<0.001). The %TT0.5
of atrial myocytes with t-tubules (24.0±2.50 %, n=9) was significantly greater than atrial myocytes without t-tubules
(7.04±0.679 %, n=19; P<0.0001) but not different from ventricular cells (23.7±1.19 %, n=31).
These data demonstrate heterogeneity in the extent of the ttubule network in pig atrial myocytes, consistent with a previous report from the dog (4). Heterogeneity in the extent of
the t-tubule network is likely to reflect heterogeneity in Ca
transport and electrical activity within the atria and may
thereby play a role in the origin of ectopic activity.
Oral Communications
Lenaerts I, Bito V, Heinzel FR, Driesen RB, Holemans P, D’hooge J, et
al. Ultrastructural and functional remodeling of the coupling between
Ca influx and sarcoplasmic reticulum Ca release in right atrial myocytes
from experimental persistent atrial fibrillation. Circ Res. 2009 Oct
23;105(9):876–85.
Dibb KM, Clarke JD, Horn MA, Richards MA, Graham HK, Eisner DA, et
al. Characterization of an Extensive Transverse Tubular Network in
Sheep Atrial Myocytes and its Depletion in Heart Failure. Circ - Heart
Fail. 2009;2(5):482–9.
Richards MA, Clarke JD, Saravanan P, Voigt N, Dobrev D, Eisner DA, et
al. Transverse tubules are a common feature in large mammalian atrial
myocytes including human. Am J Physiol - Heart Circ Physiol. 2011 Nov
1;301(5):H1996–2005.
λ in WT (8.69±0.72 to 6.72±0.86 mm, n=8, p<0.05) but
increased it in RyR2s/s (6.27±0.78 to 7.38±0.83 mm, n=10,
p<0.05). Activation and inactivation studies revealed smaller
Na+ currents in RyR2s/s than WT. Flec increased Na+ currents
in RyR2s/s yet reduced them in WT. This suggests that the
expected inhibition of Na+ channels by flec was opposed by
concurrent inhibition of RyR2-mediated release of SR Ca2+, previously shown to reduce Na+ channel function (3,5).
These findings demonstrate that flec decreases atrial arrhythmia with increased λ and Na+current in RyR2s/s, yet increases
atrial arrhythmia with decreased λ and Na+ current in WT.
Dolber PC, Bauman RP, Rembert JC, Greenfield JC. Regional changes
in myocyte structure in model of canine right atrial hypertrophy. Am
J Physiol - Heart Circ Physiol. 1994 Oct 1;267(4):H1279–1287.
requirements.
C03
Contrasting effects of flecainide on atrial arrhythmia in wildtype and RyR2-P2328S mice
S.C. Salvage1, J.H. King1, K.H. Chandrasekharan1,
H.R. Matthews1, L. Guzadhur2, C.L. Huang1,2 and J.A. Fraser1
1Physiology,
Development and Neuroscience, University of
Cambridge, Cambridge, UK and 2Biochemistry, University of
Cambridge, Cambridge, UK
Atrial fibrillation (AF), is the commonest cardiac arrhythmia
and is associated with increased morbidity and mortality (1).
Yet its underlying mechanisms are incompletely understood.
Cardiac ryanodine receptor (RyR2) gain-of-function mutations
predispose to catecholaminergic polymorphic ventricular
tachycardia (CPVT) (2) and in some cases, including RyR2P2328S (RyR2s/s), atrial arrhythmia (3). Recently, an anti-arrhythmic effect of flecainide (flec) was reported in CPVT (4). However, the effect of flec on RyR2-related atrial arrhythmias has
not been studied.
We explored the effects of flec (1 μM) on arrhythmic incidence,
action potential conduction velocity (CV), refractory period
(RP), wavelength (λ = CV x RP) and Na+ current activation and
inactivation, in wild-type (WT) and RyR2s/s hearts. WT and
RyR2s/s mice (3–11 months) were killed in accordance with the
UK Animals (Scientific Procedures) Act (1986). Hearts were
Langendorff-perfused and paced at 8 Hz (S1) with interposed
S2 stimuli at progressively shorter S1-S2 intervals (3). Left atrial
epicardial excitation was recorded by a 32 pin multi-electrode
array (MEA). CV was determined using velocity vector analysis, while RP was quantified as the last non-refractory S1-S2
interval. Loose patch-clamp experiments determined Na+ current properties in superfused atrial preparations as previously
described (5). Data are expressed as means ± SEM. Statistical
significance (to p<0.05) was tested using two-way ANOVA or
paired t-tests as appropriate.
Arrhythmic score in RyR2s/s (2.65±0.38, n=17) was higher than
that of WT (1.10±0.22, n=10) (Fig. 1). Flec increased arrhythmic score in WT (2.65±0.38, n=9) but decreased it in RyR2s/s
(1.59±0.39, n=17). Flec slowed CV in WT from 0.38±0.024 m
s-1, n=14 to 0.22±0.02 m s-1, n=10, p<0.001, and did not influence RP (control: 24.6±1.5 ms; flec: 27.7±3 ms, n=9). In contrast, flec did not alter CV in RyR2s/s (control: 0.28±0.02 m s-1,
n=14; flec: 0.29±0.02 m s-1, n=10), but increased RP from
23±1.8 ms to 34±3.9 ms, n=17, p<0.001. Thus, flec decreased
Fig. 1. 1 μM flec increases arrhythmic events in WT hearts, but is antiarrhythmic in RyR2s/s hearts. Paired symbols denote p<0.05.
Kannel WB et al. (1998). Am J Cardiol 82(8A), 2N–9N.
Laitinen PJ et al. (2001). Circulation 103, 485–90.
King JH et al. (2012). Acta Physiol 207, 308–23.
Watanabe H et al. (2009). Nat Med 15, 380–3.
King JH et al. (2013). Cardiovasc Res 99, 751–9.
This research was supported by a BBSRC David Phillips
fellowship awarded to JAF.
requirements.
C04
Enhanced neuronal calcium conductance in cardiac stellate
neurons from young pro-hypertensive rats: A neuronal
channelopathy?
G. Hao, H.E. Larsen, K. Liu, D. Li, E. Mann and D.J. Paterson
Physiology, Anatomy and Genetics, University of Oxford, Oxford,
UK
Background Hypertension is associated with heightened activation of the sympathetic nervous system where a significant
component of the over-activity occurs at the end organ. In particular post-ganglionic sympathetic neurones (PGSN) show
enhanced intrinsic excitability resulting in greater transmitter
release1. Furthermore intracellular Ca2+ transients are significantly enhanced in the SHR stellate neurons compared to age
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Oral Communications
matched neurons from normotensive rats. Interestingly, this
Ca2+ phenotype is also seen in the stellate neurons from the
pre-hypertensive SHR, suggesting it is an early hallmark of the
disease. The increased Ca2+ transients have been shown to be
due in part to altered mitochondrial Ca2+ handling2, but this
does not explain the total event. In this study we investigated
the whole cell Ca2+ current in cardiac sympathetic stellate ganglions of the SHR and WKY in order to elucidate whether the
neuronal Ca2+ channels are involved in the neuronal phenotype seen in animals with a pre-disposition to high blood pressure.
Methods & Results Cardiac stellate ganglia were excised from
4 week old male pro-hypertensive SHR and WKY rats in accordance with the Home Office Animals (Scientific Procedures)
Act 1986 (UK). The ganglia were digested with collagenase (1
mg/ml) and trypsin (2 mg/ml) and mechanically separated to
generate a single cell suspension. Whole cell voltage clamp
recordings were performed by holding the cell at -90 mV before
a set of voltage steps from -60 to +50 mV were applied at 10
mV intervals. Access to the cell was obtained in normal Tyrode
solution before the desired current was isolated using a modified Tyrode solution containing 2 mM Ba2+ and 1 μM
Tetrodotoxin. Greater than 75% series resistance compensation was applied to all cells and a cellular resting membrane
potential of > -40 mV was required for inclusion in the study.
Experiments were conducted at 37°C. We show that the whole
cell Ca2+ current in the stellate neurons was significantly larger
in the SHR (-27.7 ± 1.8 pA/pF; n=7) when compared to the
WKY (-22.2 ± 1.3 pA/pF; n=8 p<0.05. Unpaired T-test).
Conclusions These data suggest that a part of the abnormally
high intracellular Ca2+ levels observed in stellate neurons from
the SHR arise from dysregulation of the neuronal Ca2+ channel. This feature is seen at an early stage in the development
of the disease itself, and also suggests a neuronal Ca2+ channelopathy is present in this genetic based model of hypertension.
Key words: Hypertension, channelopathy, stellate neurons,
electrophysiology
Shanks, J. & Herring, N. Peripheral cardiac sympathetic hyperactivity
in cardiovascular disease: role of neuropeptides. American journal of
physiology. Regulatory, integrative and comparative physiology 305,
R1411-1420 (2013).
Li, D., et al. Abnormal intracellular calcium homeostasis in sympathetic
neurons from young prehypertensive rats. Hypertension 59, 642-649
(2012).
Acknowledgements: OXION Wellcome Trust Centre & British
Heart Foundation Centre for Research Excellence, Oxford, UK
requirements.
C05
Acute intermittent hypoxia evokes sympathetic long-term
facilitation (LTF) via activation of the renin-angiotensin
system (RAS)
A.Y. Fong1, A.M. Hammond1, T. Xing2 and P.M. Pilowsky3
1Physiology,
University of Melbourne, Parkville, VIC, Australia,
School of Advanced Medicine, Macquarie University,
North Ryde, VIC, Australia and 3Heart Research Institute, University
of Sydney, Newtown, NSW, Australia
2Australian
Sleep apnoea is commonly associated with co-morbidities
including hypertension with elevated sympathetic nerve activ-
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ity (SNA). Activation of the renin-angiotensin system (RAS) is
thought to be a mechanism involved in the pathogenesis of
hypertension is sleep apnoeics. The intermittent hypoxia experienced in sleep apnoea is thought to contribute to the activation of the SNA, contributing to the development of hypertension. We have reported that acute intermittent hypoxia
(AIH, 10x45s of 10% O2, separated by 5mins hyperoxia) elicits a sustained elevation of splanchnic SNA in the anaesthetized
rat (Xing & Pilowsky, 2010). The aim of the present study was
to examine the role of the renin-angiotensin system in the sustained elevation of SNA, termed sympathetic long-term facilitation (LTF) in anaesthetised (urethane, 1.5g/kg, i.p.), vagotomised, paralysed (pancuronium, induction dose 0.8mg/kg,
continuous infusion 0.4mg/hr) and ventilated Sprague-Dawley rats (Xing & Pilowsky, 2010). The greater splanchnic nerve,
the renal sympathetic nerve, and the phrenic nerve were continuously recorded during and for at least an hour after AIH.
AIH evoked LTF in the splanchnic (+63±8.5% above baseline,
n=6) and renal sympathetic nerves (+38±9.7% above baseline,
n=6). Pretreatment before AIH with an angiotensin 1A receptor blocker, valsartan (0.1mg/kg, i.v.) abolished the sympathetic LTF in the splanchnic nerve (+2±9.5% above baseline,
n=7). Intermittent injections of angiotensin II (10x35pmol in
0.1ml, i.v., 5mins intervals) also elicited sympathetic LTF (+42.4
±11.5% above baseline, n=5). Intermittent phenylephrine injections (10x 25μg in 0.1mL, i.v.), resulting in intermittent renal
hypoperfusion also produced sympathetic LTF (+72.4±21.6%
above baseline, n=5), that was abolished by pretreatment with
the renin inhibitor, aliskiren (1mg/kg, i.v., n=4). Continuous
infusion of the equivalent dose of angiotensin II (350pmol in
1mL) or phenylephrine (250μg in 1mL) over 10 mins, did not
cause sympathetic LTF. This data supports the idea that activation of the RAS is sufficient for the development of sympathetic LTF following AIH. In addition, the temporal nature of
intermittent activation of RAS is required for sympathetic LTF.
This data is in agreement with data in human studies that AIH
activates angiotensin type I receptors to increase arterial blood
pressure (Foster et al., 2010), and suggests that activation of
RAS may be relevant to sympathetic overactivity and hypertension in sleep apnoea patients.
Foster GE, Hanly PJ, Ahmed SB, Beaudin AE, Pialoux V, Poulin MJ. 2010.
Intermittent hypoxia increases arterial blood pressure in humans
through a Renin-Angiotensin system-dependent mechanism. Hypertension. 56:369-377.
Xing T, Pilowsky PM. 2010. Acute intermittent hypoxia in rat in vivo
elicits a robust increase in tonic sympathetic nerve activity that is independent of respiratory drive. J. Physiol. 588:3075-3088.
Funding by NHRMC (Australia), ARC (Australia).
requirements.
Oral Communications
C06
requirements.
Ventricular conduction system remodelling in rabbits with
congestive heart failure
S.R. Logantha1, X. Cai1, J. Yanni1, C.B. Jones2, J. Li1, A. D’Souza1,
A. Vohra3,1, H. Dobrzynski1, G. Hart1 and M.R. Boyett1
1Institute
of Cardiovascular Sciences, University of Manchester,
Manchester, UK, 2Alder Hey Children’s Hospital, Liverpool, UK and
3Manchester Academic Health Sciences Centre, Manchester Royal
Infirmary, Manchester, UK
In the heart, the ventricular conduction system comprises an
intricate network of Purkinje fibres (PFs) that conducts electrical activity from the atrioventricular node to the ventricular myocardium and activates coordinated ventricular contraction. Conduction abnormalities associated with the
ventricular conduction system are common in heart failure (1)
and such patients often require cardiac resynchronisation therapy. In a rabbit model of congestive heart failure (CHF), we
have investigated electrical and molecular remodelling in PFs.
Three month old male New Zealand white rabbits (~3 kg) were
anaesthetised with ketamine intramuscular injection and isoflurane inhalation. CHF was induced (n=10) by combined volume
and pressure overload caused by surgical destruction of aortic valve (week 0) and banding of abdominal aorta (week 3)
respectively. CHF characteristics were confirmed by echocardiography prior to termination at week 8. Ten sham operated
animals were used as controls. In left ventricular (LV) preparations, intracellular action potentials were recorded using
microelectrodes from LV myocardium and three regions of the
ventricular conduction system: left bundle branch, free-running PFs and junctional PFs (at Purkinje-ventricular junction).
In free-running PFs and LV myocardium, mRNA expression of
~30 ion channels was measured using qPCR. This work was
carried out in accordance with UK Home Office regulations.
In CHF rabbits, echocardiography showed significant LV hypertrophy, reduced LV fractional shortening (25.5±3.2 vs. 41.3±1.4
% in control; P<0.05) and increased diastolic LV internal diameter (2.2±0.1 vs. 1.5±0.03 cm in control; P<0.05). In anaesthetized CHF rabbits, the ECG RR-interval was 228±18 ms, similar to control. In the CHF group, the left bundle branch action
potentials showed a slightly increased maximum upstroke
velocity (dV/dtmax), amplitude and action potential duration,
but the differences were not significant. Action potential duration was prolonged in free-running PFs and junctional PFs by
84% and 81%, respectively, in the CHF cohort. In agreement
with this observation, in free-running PFs, we found downregulation of mRNA responsible for major repolarizing K+ channels: Kv1.5 (responsible for IK,ur), KChIP2 (responsible for Ito),
ERG (responsible for IK,r) and KvLQT1 (responsible for IK,s).
Action potentials recorded in the LV myocardium showed a
43% increase in dV/dtmax in the CHF group, consistent with
an upregulation of Nav1.5 mRNA that is responsible for INa and
the action potential upstroke.
In CHF, we observe ion channel remodelling and action potential changes that are heterogeneous within the ventricular conduction system. LV dilatation and consequent stretching of
the PFs are likely to contribute to the marked changes observed
in the free-running PFs.
Padeletti L et al. (2010). J Card Fail 16, 320-326.
Research funded by a British Heart Foundation programme
grant (RG/11/18/29257).
C07
The properties of sodium channels in rat left atrial and
pulmonary vein cardiomyocytes
L. Hutchison1, A. Rankin2, R. Drummond1 and E. Rowan1
1SIPBS,
University of Strathclyde, Glasgow, UK and 2School of
Medicine, University of Glasgow, Glasgow, UK
Atrial fibrillation is the most common cardiac arrhythmia,
affecting up to one million people in the UK. Clinical studies
have demonstrated that ectopic activity originating from the
cardiomyocyte sleeve around the pulmonary vein is an important contributing factor in atrial fibrillation. However the underlying mechanisms of this ectopic activity remain unclear.
Recent studies have proposed variations in the properties of
ion channels, in particular sodium channels, in atrial and pulmonary vein cardiomyocytes as a potential mechanism.
Tetrodotoxin (TTX)-sensitive neuronal sodium channels are
found in atrial and ventricular cardiomyocytes; however their
presence and function in pulmonary vein cardiomyocytes is
unknown. Thus, the main aim of this study was to examine the
characteristics of cardiac sodium channels in rat pulmonary
vein and atrial cardiomyocytes and to establish whether TTXsensitive sodium channels are present in the cardiomyocytes
from these two regions.
Male Sprague-Dawley rats (250-400g) were euthanised by cervical dislocation and the heart and lungs removed en bloc.
Thereafter, the pulmonary veins and left atrium were microdissected. Using reverse transcription-polymerase chain reaction,
the TTX-sensitive Nav1.1 transcript was found to be present in
the rat pulmonary vein but absent from the left atria. The main
cardiac sodium channel, Nav1.5 and the four sodium channel
associated β-subunits were present in both tissues. Sodium
currents were recorded from isolated pulmonary vein and left
atrial cardiomyocytes using whole-cell recording techniques.
Values are means ± S.E.M. Significance (P<0.05) was calculated
by an unpaired t-test. Peak sodium current density at a holding potential of -120 mV with a step to -45 mV was -64.0 ± 4.8
pA/pF, n=10 in pulmonary veins and -64.0 ± 5.9 pA/pF, n=6 in
left atrial cardiomyocytes. Voltage of half activation (V½) were
similar in pulmonary vein (-55.7 ± 2.3 mV, n=10) and left atrial
cardiomyocytes (-58.4 ± 1.4 mV, n=6). There was no significant difference in inactivation V½ in pulmonary vein (-98.3 ±
1.3 mV, n=7) and left atrial (-100.1 ± 2.4 mV, n=6) cardiomyocytes. Application of nM concentrations of TTX, which mainly
blocks TTX-sensitive channels, indicated a TTX-sensitive component accounting for approximately 20% of the total sodium
current in pulmonary vein but not left atrial cardiomyocytes.
The TTX-sensitive current had a KD value of 5.2 nM with a peak
current density of -13.0 ± 0.5 pA/pF, n=3 compared to a KD of
650.0 nM and peak current density of -46.8 ± 2.6 pA/pF, n=3
for the TTX-resistant current.
Although there was no significant difference in the characteristics of the total sodium current in pulmonary vein and left
atrial cardiomyocytes, a variation in sodium channel subtype
expression does exist as a TTX-sensitive current was detected
in pulmonary vein but not left atrial cardiomyocytes.
requirements.
61P
Oral Communications
C08
Spontaneous Ca2+ signalling in pulmonary vein sleeve cells
increases during ageing
K. Rietdorf, S.R. Masoud and M.D. Bootman
Biomedical Research Network, The Open University, Milton Keynes,
UK
Atrial fibrillation (AF) is the most common form of a sustained
cardiac arrhythmia, with age being a significant risk factor (1).
Substantial evidence indicates that cardiomyocytes located in
the pulmonary veins (pulmonary vein sleeve cells; PVCs) cause
AF by generating ectopic electrical activity (2). Electrical ablation, to isolate PVCs from their left atrial junctions, is a major
treatment for AF (3). In small rodents, the sleeve of PVC cardiomyocytes extends along the pulmonary veins deep inside
the lungs (4). Since ageing is a major risk factor for AF development, we investigated changes in the morphology of PVCs,
and their spontaneous and electrically-paced Ca2+ signals in
aged mice.
Lung slices were prepared, as previously described (5), from
mice between 3 and 24 months of age. Briefly, lungs were
injected with 1.8% agarose and subsequently sliced into 180
μm thick sections. Lung slices were either used for live-cell Ca2+
imaging studies, or were fixed in 4% paraformaldehyde for
immunofluorescence staining or electron microscopic (EM)
studies.
For Ca2+ imaging, the slices were loaded with 5 μM fluo-4 AM,
and line scans were collected on a Zeiss LSM 510 confocal
microscope. Immunofluorescence staining was performed as
previously described (5), using an antibody against type 2 ryanodine receptor (RyR2). For EM studies, the slices were postfixed in 1% osmium tetroxide, dehydrated through a graded
series of acetone concentrations before flat-embedding in Epon
resin. Ultrathin sections ∼70 nm were cut and counter-stained
with 4% uranyl acetate and Reynolds’ lead citrate before imaging in a JEM 1400 transmission EM.
PVCs from lung slices of all ages showed spontaneous Ca2+
transients, with a higher spontaneous activity in slices from
24 compared to 3 month old mice (1.0 ± 0.19 vs. 0.71 ± 0.11
Hz, n.s., unpaired t-test, n = 3 and 4 slices, respectively). Electrical pacing with all frequencies was more successful in slices
from 3 month old mice (full pacing with 1 Hz in 40.3 ± 9.9%
of the slices, 3 Hz 37.4 ± 9.4 %, and 5 Hz 32.6 ± 10.1%, 4 slices)
compared to slices from 24 month old mice (12.5 % full pacing at all frequencies, 3 slices). However, the differences are
not statistically significant (unpaired t-test).
Immunofluorescence staining showed a striated expression of
RyR2 in lung slices from all age groups, with no difference in
the distance between the striations (1.9 ± 0.03 vs. 1.9 ± 0.06
μm, respectively; unpaired t-test). EM images confirmed the
presence of striations in PVCs.
The data presented here indicate that PVCs become increasingly spontaneously active and more difficult to electrically
pace as they age, but do not have gross morphological
changes. The reason underlying the enhanced spontaneous
Ca2+release is unknown. However, it is likely that an increased
rate of spontaneous Ca2+ signalling within the PVCs will contribute to the genesis of AF.
Chinitz, J.S., Castellano, J.M., Kovacic, J.C. & Fuster V. (2012): Atrial fibrillation, stroke, and quality of life. Ann N Y Acad Sci.;1254:140-50
Haïssaguerre, M., Jaïs, P., Shah, D.C., Takahashi, A., Hocini, M., Quiniou, G., Garrigue, S., Le Mouroux, A., Le Métayer, P. & Clémenty, J.
(1998): Spontaneous initiation of atrial fibrillation by ectopic beats
originating in the pulmonary veins. N Engl J Med.;339(10):659-66
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Calkins H. (2014): Has the time come to recommend catheter ablation of atrial fibrillation as first-line therapy? JAMA;311(7):679-80
Kracklauer, M.P., Feng, H.Z., Jiang, W., Lin, J.L., Lin, J.J. & Jin, P.P. (2013):
Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms. FEBS J 280:
880–891
Rietdorf, K., Bootman, M.D., Sanderson, M.J. (2014): Spontaneous,
pro-arrhythmic calcium signals disrupt electrical pacing in mouse pulmonary vein sleeve cells. PLoS One. 2014 Feb 20;9(2):e88649
requirements.
C09
Mechanical activity-induced changes in cardiomyocyte Ttubular shape provide a novel mechanism for maintaining
synchronous contractions
A.X. Zhang1, M.K. Morphew2, E.T. O’Toole2, C. Page2,
C. Schwartz2, A. Hoenger2, P. Kohl1 and E.A. Rog-Zielinska1
1National
Heart and Lung Institute, Imperial College London,
London, UK and 2The Boulder Laboratory for 3D Electron
Microscopy of Cells, Colorado University, Boulder, CO, USA
Rationale: In mammalian ventricular myocytes, T-tubules (Ttub) are essential for synchronous and rapid changes in cytosolic free Ca2+ concentration and, hence, efficient contraction.
T-tub form a highly complex, polymorphic network of membrane invaginations extending from the surface to the centre
of the cell. T-tub membranes contain various ion channels that
facilitate excitation-contraction coupling. Due to the complex
nature of the T-tub network they create an area of restricted
diffusion, and it has been proposed that this in turn could create a microdomain of ion concentrations that differ from bulk
extracellular space. In order to maintain synchronous contractions however there must exist a mechanism of rapid
replenishment of T-tub content in addition to passive diffusion
which might be too slow to sustain ion homeostasis, especially
at higher heart rates. Previously, transmission electron microscopic imaging has shown that T-tubules may undergo deformation as the cardiomyocytes contract which may give rise to
a novel mechanisms of partial content exchange based on convection1.
Objective: We hypothesise that the contraction and stretchinduced deformation of T-tub is associated with transitions
between circular and more elliptical cross-sectional shape,
which – in the presence for a near-constant T-tub surface area
– gives rise to changes in partial T-tub volume. This would constitute a convection-driven mechanism for ion exchange (in
addition to diffusion). We employ EM tomography (EMT) to
reconstruct and model the 3D structure of cardiomyocyte Ttub at nano-scopic resolution (4*10-9 m) and to quantify
changes in shape of the T-tubular system in as a function of
sarcomere length.
Methods: Rabbit hearts were chemically fixed and embedded
during contracture, cardioplegic arrest and balloon catheterinduced stretch. Samples were subsequently subjected to dualaxis EMT. Datasets were reconstructed and T-tub and sarcomeric Z-disc planes tracked to obtain 3D models. The
orientation of the long cross-sectional axis of the T-tub relative to the Z-disc was measured.
Results and Conclusion: Using high-resolution 3-D structural
modelling of rabbit cardiac T-tub system in different contractile states we provide evidence that mechanical activity of cardiomyocytes induces directional deformation of T-tub shape.
Oral Communications
This finding supports the suggestion that ion homeostasis
within cardiomyocyte T-tub is based on convection-assisted
diffusion, ensuring synchronous cardiac activity regulation on
a beat-to-beat basis.
Kohl P et al. (2003) Prog Biophys Mol Biol 82(1), P221-7
Mechano-Electric Feedback and Cardiac Arrhythmias
requirements.
introducing a -9 mV shift of the voltage of half-activation
(V501M+WT effect) also reduced If current amplitude and the
slope of diastolic depolarisation, resulting in a cycle length of
412 ms (+24%).
Through accelerated deactivation of the If current or a negative shift of steady-state activation each mutant may cause
bradycardia, a condition favouring atrial fibrillation. Thus, these
HCN4 mutants likely contribute to atrial fibrillation by different mechanisms.
Nof E et al. (2010). Pacing Clin Electrophysiol 33:100-106.
Severi S et al. (2012). J Physiol 590:4483-4499.
C10
Pathogenic mechanisms of two novel mutations of the
hyperpolarization activated channel type 4 gene related
to lone atrial fibrillation
Y. Vincent1, M. Chahine2, A. Mechakra1, P. Chevalier1, Y. Yang3
and G. Christé1
1EA4612
Neurocardiologie, Université Lyon 1, Lyon, France,
2Institut Universitaire en Santé Mentale, Québec City, QC, Canada
and 3Shanghai Chest Hospital, Shanghai Jiao Tong University,
Shanghai, China
The If current (funny current) is involved in the pacemaker
mechanism of cardiac cells. In the human sinus node, If is
mainly encoded by the HCN4 gene. Less than 10 pathogenic
mutations of this gene were found to date, all related to bradycardia [1].
Two novel heterozygous mutations were identified in Chinese
patients with lone atrial fibrillation and bradycardia, and were
absent in 200 ethnically matched control subjects. One,
(D600E) is located near the CNBD site, and the other (V501M)
in the pore domain. We sought for possible mutation-induced
functional changes in HCN4 channels.
Cos7 cells were transfected with either 3 μg (per 35 mm Petri
dish) wild-type (WT) HCN4 cDNA plasmid, or with 3 μg
mutated HCN4 cDNA plasmid or with a mixture of 1.5 μg of
each. Cotransfection with a CD8 surface antigen plasmid
allowed detection of transfected cells. Whole-cell currents were
recorded with the patch-clamp technique, at room temperature.
Currents measured at -130 mV in Cos7 cells expressing D600E
alone (-19.2±3.5 pA/pF, n=22) were larger (p<0.05, unpaired
Student’s t-test) than in cells expressing the WT (-11.9±2.1
pA/pF, n=30). This difference was abolished in cells expressing D600E+WT. The mutant, alone or in coexpression with the
WT, did not change the kinetics of activation. However, deactivation was accelerated 1.6 fold versus WT in cells expressing either D600E alone or D600E+WT.
Cells transfected with V501M alone did not show any HCN4
current. In cells expressing V501M+WT, the current (-11.7±5.6
pA/pF, n=5) was similar to that of WT (-11.9±2.1 pA/pF, n=30).
Neither V501M alone nor V501M+WT changed the time constants of activation and deactivation. However, V501M+WT
caused a negative shift by 9 mV of the voltage for half steadystate activation (-104.9±3.4 mV vs -96.1±1.9 mV for WT). The
reversal voltage of the HCN4 current was not changed by
mutants alone or coexpressed with the WT.
The impact of each mutant in the heterozygous condition was
explored in a computer model of sino-atrial node cell [2] run
at steady-state in spontaneous mode. When deactivation of
the If current was accelerated 1.6-fold (D600E+WT effect), the
If current amplitude and the slope of the slow diastolic depolarisation were lowered and interval between action potentials was lengthened from 332 to 352 ms (+6%). Alternatively,
requirements.
C11
Chronic sustained hypoxia induces temporal redox and
metabolic remodelling and atrophy signalling in mouse
diaphragm muscle
P. Lewis1, D. Sheehan2 and K.D. O’Halloran1
1Physiology,
2Biochemistry,
University College Cork, Cork, Ireland and
University College Cork, Cork, Ireland
Introduction: Differential structural, functional, and metabolic
adaptations have been reported in respiratory and limb muscles in animal models of chronic sustained hypoxia, similar to
COPD. We sought to elucidate temporal mechanisms that
underpin hypoxic remodelling in respiratory muscle. Altered
redox modulation is a potential mechanism that supports these
adaptations. Method: Adult male C57Bl6/J mice were exposed
to one, three and six weeks of sustained hypoxia (FiO2: 0.1)
or normoxia (n=8 at each time point). Diaphragm (main respiratory pump muscle) was excised and processed for redox
proteomics experiments using fluorescent tags to detect
changes in protein carbonyls and free thiols. Mass spectrometry was used to identify redox modified proteins. Spectrophotometric assays were used to measure activities of redox
modified metabolic proteins, antioxidants, and activity of the
redox-sensitive proteasome. ELISA was used to quantify content of phosphorylated protein synthesis/atrophy signals
including phospho(p)–mTOR, p–FOXO3a and p–Akt. Sternohyoid (upper airway dilator), EDL and soleus (hind limb)
muscles were analysed for comparative purposes. Results:
Hypoxia induces temporal and muscle specific protein oxidation despite an early antioxidant response (p<0.05). Redox
remodelling occurs to key metabolic proteins and reaches the
level of the cross-bridge. Hypoxia induces temporal and muscle specific activity changes to metabolic proteins. After six
weeks of hypoxia, GAPDH, LDH, aconitase, and creatine kinase
activities are all decreased in the diaphragm while G3PD activity is increased (P<0.05). Diaphragm and soleus proteasome
activities are increased (P<0.05) while no changes were
observed in sternohyoid or EDL. Hypoxia induces bi-phasic
changes in diaphragm atrophy signalling with p–mTOR
increased after one week and p–FOXO3a decreased after six
weeks (P<0.05). p–Akt remained unchanged. Discussion:
Hypoxia-induced molecular remodelling is muscle specific.
Hypoxia per se, contractile activity, molecular composition,
and related temporal changes in muscle demands all potentially play a role. The diaphragm, as has been observed in cardiac muscle, appears to favour fatty acids as an energy source
in hypoxia. Protein synthesis signalling in the diaphragm is
potentially a ‘training effect’ of hypoxia-induced hyperventi-
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Oral Communications
lation, but atrophy through FOXO3a signalling prevails. Bi-phasic changes in atrophy signalling are unlikely to be growth factor related on the basis of no detectable change in p–Akt content. We hypothesize that redox changes underpin respiratory
muscle remodelling in chronic sustained hypoxia.
Funding support for this work was generously provided the
Health Research Board, Ireland; The Strategic Research Fund,
University College Cork; and the Dept. of Physiology, University
College Cork.
requirements.
induced a 45.1±22.2% (donors=12) increase in CD11b (active
form) cell surface expression versus 0.6±8.4% for asthmatics
(donors=9).
In summary, human eosinophils express functional P2X1 receptors and their activity is significantly reduced in asthmatics.
P2X1 activation increases cell surface expression of CD11b
(activate form) in the healthy eosinophil, suggesting a role
for these receptors in eosinophil adhesion/migration.
requirements.
C13
C12
Impared P2X1 receptor function in eosinophils from
asthmatic patients
A. Wardlaw3, M. Smith1, F. Symon3, A. Wright3, S. Nicolas2,
M. Bafadhel3, M. Muessel3, P. Bradding3 and C. Vial1
1Cell
Physiology and Pharmacology, university of Leicester,
Leicester, UK, 2Genetics, University of Leicester, Leicester, UK and
3Institute for Lung Health, Respiratory Biomedical Unit, Hospitals
of Leicester NHS Trust, University of Leicester, Leicester, UK
Eosinophils play an important role in the pathogenesis of
asthma, and can be activated by extracellular nucleotides,
released following cell damage and inflammation. This study
aimed at identifying the P2X receptor(s) present on eosinophils
and investigating their contribution to eosinophil biology.
Human eosinophils were isolated from the blood of normal
and asthmatic volunteers. qPCR and Western blot were used
to determine respectively P2X receptor mRNA and protein
expression level in eosinophils from healthy and asthmatic
donors. Conventional whole cell patch-clamp experiments
were performed to identify the functional subtypes of P2X
receptors in eosinophils. Eosinophil CD11b (active form) cell
surface expression was measured by flow cytometry.
qPCR showed that P2X1, P2X4 and P2X5 receptor transcripts
were expressed in eosinophils from 3 healthy and 3 asthmatic
donors. The application of ATP (100 μM) elicited a rapidly activating and rapidly desensitizing inward current (75.0±18.6
pA/pF; donors=3) in healthy human eosinophils which was
abolished by 1μM of the selective P2X1 receptor antagonist
NF449 (2.7±0.5 pA/pF; donors=6, p=0.0313). The P2X1 agonist α,β-meATP (10μM) also induced a fast transient current in
eosinophils (18.4±3.2 pA/pF; donors=3) which was totally inhibited by 1 μM NF449 (2.7±1.8 pA/pF; donors=8, p=0.0078).
These P2X1-like currents were markedly reduced (66 %) in
eosinophils from asthmatic compared to healthy donors (10
μM α,β-meATP-induced currents of 8.4±1.7 pA/pF [donors=12]
and 24.4±3.6pA/pF, donors=18, p<0.0001). P2X1 transcript
and protein expression level were similar in asthmatic and normal eosinophils, suggesting that reduced P2X1 currents
observed in asthmatic eosinophils could result from increased
receptor desensitisation. Therefore, eosinophils from asthmatics were treated either with a substantial (10 IU/ml) or a
standard dose (0.32 IU/ml) of apyrase before the recordings
took place. High concentrations of apyrase rescued P2X1 activity in asthmatic eosinophils [α,β-meATP (10μM)-induced currents of 17.1±4.5 pA/pF for 10 IU/ml and 11.4±2.6 pA/pF for
0.32 IU/ml of apyrase, n=6 donors for each, p=0.0313]. We
also investigated the potential contribution of P2X1 receptor
to eosinophil integrin CD11b (the αM chain of the αMβ2 integrin) regulation. In healthy eosinophils, 10 μM α,β-meATP-
64P
Identifying a primary mechanotransducer in a model
sensory system
T. Suslak, A.P. Jarman and D. Armstrong
University of Edinburgh, Edinburgh, UK
Stretch-activated afferent neurons are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. We
have developed a novel, in vivo recording protocol to obtain
receptor potential recordings from intact dbd sensory neurons, in situ, in larval Drosophila to determine whether these
neurons are a tractable model of such receptors for investigating mechanisms of mechanotransduction.
Electrophysiological recordings from dbd neurons are shown
to be equivalent to those previously obtained from mammalian
muscle spindles. Both of these are accurately modelled by an
in silico model of stretch-activated neurons that we are developing. Subsequently, this in silico model is shown to predict
an essential role for a mechanosensory cation channel in receptor potential generation.
We identify the mechanosensory ion channel, Piezo, in this
functional role in dbd neurons, using pharmacological and
genetic techniques. Here, for the first time, DmPiezo, the only
orthologue of the Piezo family in D. melanogaster, has been
implicated in a stretch-activation response. This study shows
the utility of an in silico model for identifying components of
a mechanosensitive system. It also establishes the dbd neuron as a useful, accessible and tractable model for studying
the phenomenon of stretch-dependent mechanotransduction.
Our study represents the first direct evidence for an in vivo
role for Piezo in mechanotransduction. Whilst earlier studies
have shown effects of Piezo loss at the behavioural level, or in
cultured cells, this is the first time that the contribution of
DmPiezo to a cellular mechanism in fully differentiated neurons has been directly demonstrated.
As far as we are aware, we have developed the first model
system for patch clamp recording of mechanically activated
receptor potentials in intact, fully differentiated mechanosensory neurons in a genetically tractable model organism. Whilst
earlier studies have utilised electrophysiology of Drosophila
afferent neurons, we are unaware of any study which utilises
this approach to test in vivo responses to physiological,
mechanical stimuli. This is a unique combination of properties
in a single system. In combination with mathematical modelling of the receptor potential, with predictive capacity for candidate channel properties, this promises to be a very powerful tool for future studies dissecting the process of
mechanotransduction and identifying transducer proteins that
are activated by mechanical stimuli in the physiological range.
Suslak, TJ. et al., 2011. Network. 22(1-4), 133-42.
Oral Communications
Nair, A. et al., 2010. BMC Res. Notes, 3, 154-61.
Kim, SE. et al., 2012. Nature, 483, 209-13.
Coste, B. et al., 2010. Science, 330, 55-60.
Funding for this project came from EPSRC, MRC and BBSRC via
the DTC at the University of Edinburgh
requirements.
C14
L-DOPA induced dyskinesia is associated
neuroinflammation in an 6-OHDA rat model
with
M. Bortolanza1,2, R. Cavalcanti-Kiwiatkoski1, F.E. PadovanNeto3,2, C.A. da-Silva1,2, R. Raisman-Vozari4 and E. Del Bel1,2
reduced by NOS-inhibitor (p<0.05). There was in the dopamine
depleted lateral striatum an increase in the expression of the
iNOS (48.29± 0.56), of the GFAP/astrocytes (12850 ± 189.3)
and of the OX-42/microglia (8671±250.4), decreased by 7-NI
(iNOS=30.86±0.59; GFAP=6624 ± 172.5; OX-42=3775±126.9).
Thus, this established model of experimental diskinesia is associated with components of inflammatory cascade. These data
suggest that levodopa (chronically) to parkinsonian rats may
cause AIMS via mechanisms involving neuroinflamation.
Padovan-Neto FE, Echeverry MB, Tumas V, Del-Bel EA. Nitric oxide synthase inhibition attenuates L-DOPA-induced dyskinesias in a rodent
model of Parkinson’s disease. Neuroscience. 2009 Mar 31;159(3):92735. doi: 10.1016/j.neuroscienc
Financial support: Physoc, FAPESP, CNPq, CAPES, Catedra USPFrance; CNPQ Science without Border. .
requirements.
1Morphology,
Physiology and Basic Patology, University of Sao
Paulo- Ribeirao Preto Dental School, Ribeirao Preto, Sao Paulo,
Brazil, 2Center for Interdisciplinary Research on Applied
Neurosciences (NAPNA), University of Sao Paulo- Ribeirao Preto
Dental School, Sao Paulo, Sao Paulo, Brazil, 3NeurologiaNeurociencias, Universidade de sao Paulo- Faculdade de medicina
de Ribeirao Preto, Ribeirao Preto, Sao Paulo, Brazil and 4Institut
de Cerveau et de la Moelle Epinière, 5Sorbonne Université UPMC
UM75 INSERM U1127, CNRS UMR 7225, Paris, UK
Dyskinesia produced by long-term treatment with levodopa
in the Parkinson disease patient remains a serious clinical problem. Rodent preparation have been developed to allow studies into possible causative mechanism. One experimental
preparation is the microinjection of the neurotoxin 6-OHDA
into medial forebrain bundle (MFB), followed by a treatment
during three weeks with levodopa (30mg/kg+benserazide7.5mg/kg/21days). Nitric oxide synthase (NOS) inhibitor
abolishes levodopa-induced dyskinesia (LID). Although the
development of LID depends on the dopaminergic neuron
lesion it is not known if a neuroinflamatory reaction is engaged
in the development and/or maintenance. Dyskinesia model:
Wistar rats (male, 180-200g, n=23) anesthetized with a mixture of Ketamine (80mg/kg) xylazine (10mg/kg), i.p., were
submitted to a stereotaxic surgery (Padovan-Neto et al., 2009)
receiving a single injection of 6-OHDA (24μg/3μl/0.05% ascorbic acid-right MFB). Desipramine hydrochloride (25 mg/kg i.p),
pargyline (40 mg/kg) were administered 30 min before 6OHDA. The development of LIDS was documented using a scale
(Padovan-Neto et al., 2009). After three weeks, rats were
divided into four sub-groups, daily treated with L-DOPA
(30mg/kg) or saline for 21 days preceded by 7-NI (30mg/kg)
or vehicle (saline-DMSO 50%). Three hours after the last drug
administration, rats were anesthetized (urethane25 mg/kg),
perfused (saline 250ml + heparin (25000 IU/mL+ sodium nitrite
(1g/L solution), followed by 300ml of PFA 4%, 0.1 M PBS. Brains
were post-fixed (2 hours, 4°C), cryoprotected (sucrose 30%,
pH 7.4, 4 °C, 48 hours) frozen and processed by histology. Neuroinflamatory reaction was monitored by immunohistochemistry in the striatum of the inducible NOS (iNOS), of the
glial fibrillary acidic protein (GFAP-astrocytes), of the antiCD11b antibody/equivalent protein (clone OX-42-microglia).
Images were digitized with a video-camera (Leica DFC420),
evaluated with the Image J (http://rsb.info.nih.gov). Behavioural values are presented as means±S.E.M., compared by
Mann–Whitney U test and r-MANOVA, respectively. Rats developed abnormal involuntary movements (AIMS) (axial, limb and
orofacial=20.85±1.28; locomotor=3.78±0.76) wiche were
C15
Zebrafish: an emerging model for investigating hair cell
physiology and function
J. Olt, S. Johnson and W. Marcotti
Biomedical Science, University of Sheffield, Sheffield, UK
Hair cells convert sound information into neuronal activity with
remarkable precision, fidelity and reliability in the inner ear
and vestibular system. Although hair cells have been studied
extensively in the last few decades, and we understand a great
deal about their development and function in vitro, we still do
not know how they operate in vivo. We used the zebrafish to
investigate the biophysical properties and the development
of the hair cells in vivo (1).
Whole-cell patch clamp recordings were obtained from hair
cells in the primary neuromast of the lateral line of larval (3.05.2 dpf; days post fertilisation) and juvenile (17-37 dpf)
zebrafish (Danio rerio). Recordings were performed at room
temperature or at the temperature where fish are kept (28 °C).
Larvae were paralysed by an injection of 125 μM α-bungarotoxin into the heart and the blood flow and heart rate were
constantly monitored. Juveniles were briefly anaesthetised
with 0.04% MS-222 and then decapitated. Membrane currents
and neurotransmitter release (measured as change in membrane capacitance) were investigated from lateral line hair cells.
Values are given as mean ± SEM.
Hair cells expressed different K+-currents: IK,D, IA, Ih, IK,Ca. The
expression of those currents differed depending on the position within the neuromast (centre vs. edge) and with age (larvae vs. juvenile). Of the 41 larval hair cells investigated, 80%
showed the following current profile: IK,D, IK,Ca and a very small
IA. About 34% of hair cells expressed Ih and a large IA was only
seen in 17 % of cells (n = 41). In the juvenile zebrafish, the size
of the total outward K+ current in hair cells was found to be
similar to that of larval cells (larvae: 335 ± 18 mV, n = 41, juvenile: 400 ± 30 mV, n = 24), indicating that the overall number
of K+ channels is unlikely to increase with development. However, in juvenile zebrafish we found a larger proportion (about
53% compared to 17% in larvae) expressing the large A-type
current. Furthermore, the A-type was almost exclusively
expressed in hair cells positioned in the centre of the neuromast, which agrees with previous morphological observations
indicating that they have a more mature phenotype compared
to cells positioned at the edge of the sensory organ (2, 3).
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Oral Communications
Hair cells in juvenile zebrafish also show measurable Ca2+ currents (–10.5 ± 2.7 pA at -30 mV, n = 6) and neurotransmitter
release (6.6 ± 0.6 fF, n = 5), which reflects about 150 calcium
channels and around 178 vesicles.
This study provides crucial information on the development
and function of sensory hair cells of the zebrafish in vivo. However, it is important to consider that the majority of hair cells
only reach maturity from juvenile stages. Consequently, future
in vivo studies will require a new approach for zebrafish older
than 5.2 dpf.
Olt J, Johnson, SL, Marcotti W (2014).In vivo and in vitro biophysical
properties of hair cells from the lateral line and inner ear of developing and adult zebrafish. J Physiol, doi:10.1113/jphysiol.2013.265108.
Williams & Holder (2000). Cell turnover in neuromasts of zebrafish larvae. Hear Res, 143, 171-181.
López-Schier & Hudspeth (2006). A two-step mechanism underlies the
planar polarization of regenerating sensory hair cells. Proc Natl Acad
Sci U S A 103, 18615-20.
requirements.
C16
Probing the physiology of perception: Invariant neural
responses in ferret auditory cortex during vowel
discrimination
S. Town, K. Wood, H. Atilan and J. Bizley
Ear Institute, University College London, London, UK
Perceptual invariance is the ability to recognize an object
despite variation in sensory input. For example when listening
to a violin solo, the instrument has a constant identity despite
variations in sound waveform as different notes are played.
Likewise in speech we can recognize phonetic components,
such as the vowel “u”, across talkers with different voice
pitches. However, it is unclear how the brain supports perceptual invariance and specifically whether neurons in auditory cortex extract invariant representations of vowel identity
across pitch.
Here we study an animal model of perceptual invariance in
which ferrets (n=4) were trained in a two-alternative forced
choice task to discriminate artificial vowel sounds. On each
trial of the task, the subject was presented with two vowel
sounds and required to respond at a particular location depending on vowel identity. Across trials vowel pitch was varied and
ferrets were required to generalize discrimination across pitch
variation. During task performance, multi-unit activity was
recorded from microelectrodes positioned in auditory cortex.
We recorded 104 responsive units - units whose firing rate during vowel presentation differed by ≥ 2 standard deviations from
mean spontaneous activity. For each responsive unit, we asked
if it was possible to decode vowel identity from the spiking
responses observed across all pitches. Using a bootstrap procedure to test significance (100 iterations, p<0.05), the majority of units (n = 72) were found to encode information about
vowel identity across pitches. In a large number of units (n =
90) vowel pitch was successfully decoded across vowel identities. Many units provided information about both vowel pitch
and identity (n = 66).
Our results show that auditory cortical neurons may offer a
physiological substrate for invariant perceptual representations of sound. Our findings also indicate that information
about multiple sound features may be represented in auditory
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cortex during behavior, even when such features are irrelevant
for task performance.
requirements.
C17
CB1 receptor agonists inhibit transmitter release at mouse
neuromuscular junctions
J.M. Fogarty, L.R. Johnston, R.G. Pertwee and G.S. Bewick
School of Medical Sciences, University of Aberdeen, Aberdeen,
UK
Endocannabinoid modulation of synaptic transmission in the
mammalian CNS is well established, and generally occurs via
cannabinoid CB1 receptors1. The endocannabinoid system
(ECS) is now widely explored for its fundamental physiological interest, and its potential as a therapeutic target for treating neurodegeneration, pain and mental illness. However,
actions at neuromuscular synapses in skeletal muscle are much
less well characterised. If present, the ECS might also prove a
useful target for treating neuromuscular diseases, such as
Isaac’s disease2, congenital myasthenias and failing synapses
in motor neurone disease. Recent studies show CB1-selective
agonists, particularly arachidonylcyclopropylamide (ACPA),
inhibit neuromuscular transmission at lizard and frog neuromuscular junctions (NMJs)3,4. Here, we ask if mammalian NMJs
might have a similar system and if so, can CB1-mediated synaptic inhibition modulate nerve-evoked muscle contraction?
Diaphragm strips from adult male MF1 mice (30-45gm) were
isolated ex vivo after humane killing (Schedule 1, ASPA, 1986,
EU modification) and suspended in Krebs solution at room temperature. After ensuring healthy neurotransmission (curaresensitive nerve-evoked contraction >95% muscle evoked contraction), it was reduced by incubating in 0.5mM Ca2+ and
sufficient Mg2+ (1.19-3mM) to reduce twitch and tetanic tension to ~50% of initial values. This provided a suitable bioassay to investigate both effects on synaptic transmission
(increases or decreases in nerve-evoked contraction), and direct
stimulation-evoked muscle contractility.
0.1 - 10μM ACPA produced a dose-dependent inhibition
(P<0.0001, Two-Way ANOVA) of both nerve-evoked twitch tension, by up to 48.7 ± 5.6% (mean ± SE), and tetanic (50Hz, 0.5s)
tension, by 14.4 ± 1.3% (both n=4), vs vehicle control (DMSO,
n=8) at 10μM. It reached significance at 0.2μM (P<0.03, Bonferroni post-hoc). Inhibition by another CB1-selective agonist,
ACEA (arachidonyl-2’-chloroethylamide), was less for twitch
(maximum 32.2±6.8% at 10μM; ANOVA P<0.01 vs ACPA), but
not tetanic (maximum 14.7 ± 9.4% at 10μM, both n=6) tension over the same range. ACPA-induced inhibition of nerveevoked twitch and tetanus (both n=10) was abolished by 10μM
AM251 (CB1 antagonist/inverse agonist, n=9). Cannabinoids
had no effect on direct muscle-evoked muscle tension.
This is the first substantive evidence that CB1 receptors are
functionally important at mammalian skeletal muscle NMJs.
The data suggest that these receptors can strongly modulate
neuromuscular transmission, and hence that the CB1 receptor might be a novel therapeutic target for agonists, inverse
agonists or allosteric modulators to treat neuromuscular diseases involving such perturbed neurotransmission.
Pertwee RG (2012). Phil Trans Roy Soc B: Biol Sci 367, 3353-3363.
Meyniel C et al. (2011). Clin Neurol Neurosurg 113, 323-324.
Newman Z et al. (2007). Eur J Neurosci 25, 1619-1630.
Oral Communications
Sánchez-Pastor E et al. (2007). J Pharmacol Expt Ther 321, 439-445.
requirements.
may synchronise the activity of regionally distributed pyramidal cells.
Lapray D et al. (2012). Nat Neurosci 15, 1265-1271.
requirements.
C18
Sleep and movement segregate distinct types of
somatostatin-expressing GABAergic interneuron in
hippocampal network operations
Katona1,
Viney1,
Lapray1,2,
L.
T.
D.
B.
T. Klausberger1,3 and P. Somogyi1,3
Micklem1,
Z.
Borhegyi3,
1MRC Anatomical Neuropharmacology Unit, University of Oxford,
2Brain
Oxford, UK,
Mind Institute, EPFL, Lausanne, Switzerland
and 3Center for Brain Research, Medical University of Vienna,
Vienna, Austria
Structural and functional changes in the hippocampal neuronal network of the brain allow organisms to adapt to the
environment. Such plasticity leads to the encoding of newly
acquired information and the replacement or updating of
stored knowledge by new experience. Information is carried
by the output of active pyramidal cell assemblies during transient network oscillations. In the CA1 area, the activity of
pyramidal cells is regulated by at least 22 distinct types of
GABAergic interneuron, some expressing the neuropeptide
somatostatin (SOM) and innervating the dendrites of their
postsynaptic targets. The in vivo spike-timing of identified types
of GABAergic cell in freely moving rodents has been largely
unknown until recently. Although thousands of interneurons
have been reported from large scale extracellular recordings
their identity has remained indefinite. How do different types
of GABAergic cell regulate pyramidal cell output during behaviour? We have explored the roles of identified hippocampal
interneuron types during network rhythms. Specifically, we
have tested the hypothesis that distinct types of GABAergic
cell have different effects on pyramidal cell firing during exploration and sleep. First, we have extracellularly recorded single
interneurons in freely moving rats (male, 300-570 g, n=14) followed by neurobiotin-labelling of these neurons using the juxtacellular technique for cell type identification (Lapray et al.,
2012). Results: 1. The reported neurons are SOM- and/or NPYexpressing, dendrite-targeting cells, including O-LM (n=4), bistratified (n=5) and putative projection cells (n=5). 2. Behaviour and network states differentiate the activities of
SOM-expressing GABAergic cell types in freely moving rats, as
shown by comparing firing rates (mean±s.e.m.) per cell type
per behavioural- or network states using repeated-measures
ANOVA. 3. During sleep, O-LM cells decrease firing to 11.9±5.2
Hz from 20.9±5.2 Hz during awake states, and are mostly inhibited during sharp wave-ripples (SWRs). 4. Bistratified cells fire
at higher rates during sleep (28.7±5.0 Hz) than O-LM cells
(11.9±5.2 Hz) and projection cells (14.4±2.8 Hz), and unlike
O-LM cells, strongly increase spiking during SWRs. Projection
cells have variable SWR responses. 5. During movement, OLM (19.0±8.1 Hz) and bistratified cells (35.8±7.2 Hz) fire cooperatively at the troughs of theta oscillations but with different frequencies. Individual projection cells couple their spiking
to different phases of theta cycles. We suggest that GABA and
SOM are differentially released to distinct dendritic zones of
CA1 pyramidal cells during sleep and wakefulness to coordinate segregated glutamatergic inputs from the CA3 area and
the entorhinal cortex. Furthermore, long-range projections
C19
Mitochondrial
metabolism
regulates
neurotransmission in cerebellar granule cells
GABAergic
M.V. Accardi1, B.A. Orser2 and D. Bowie1
1Pharmacology and Therapeutics, McGill University, Montreal, QC,
Canada and 2Physiology, University of Toronto, Toronto, ON,
Canada
Reactive oxygen species (ROS) are ubiquitous signaling molecules in the CNS primarily formed as by-products of mitochondrial metabolism. Involved in both physiological and
pathophysiological processes, ROS are important in neurotransmission with recent evidence demonstrating that mitochondrial-derived ROS (mROS) regulate the strength of
GABAergic synapses in cerebellar stellate cells (Accardi et al.
2014). However, it remains unclear whether this regulation is
found at other GABAergic synapses. To test this, C57BL/6 mice
(Wild-type and δ-knock out [δ-KO], male/female, P15-28) were
anaesthetized (isoflurane, inhalation), decapitated and
parasagittal cerebellar slices were obtained, using a vibratome,
so that inhibitory events from cerebellar granule cells could
be recorded. GABAergic miniature IPSCs (mIPSCs) were pharmacologically isolated using glutamate receptor blockers and
tetrodotoxin and mIPSCs were recorded using whole-cell patchclamp electrophysiology. The mitochondrial poison, antimycinA (2 μM), was used to elevate mROS by including it in the
recording pipette solution. Antimycin-A significantly increased
the frequency of mIPSCs in granule cells in a time dependent
manner reaching a maximal effect after 20 minutes (normalized to the first minute, antimycin-A: 187.6 ± 30.7% at 20 min
vs. normalized 100% at 1 min; paired t-Test, p = 0.041; n = 7;
all values are means ± S.E.M). In the absence of antimycin-A,
mIPSC frequency was unaffected over 25 minutes demonstrating the stability and health of granule cell recordings (Control: 112.23 ± 25.7% at 25 min vs. normalized 100% at 1 min;
paired t-Test, p = 0.734; n = 7). The increase in mIPSC frequency
was blocked by the presence of 1 mM N-acetylcysteine (NAC,
n = 6), an antioxidant which acts through its thiol group to
reduce ROS by electron donation (antimycin-A: 187.6 ± 30.7%
at 20 min vs. antimycin-A + NAC: 102.9 ± 5.4% at 20 min;
unpaired t-Test, p = 0.0291). To assess the impact on tonic
GABA responses, we applied 10 μM bicuculline at the end of
each recording. Using this approach, we observed that
antimycin-A (n = 6) significantly reduced the baseline current
compared to control cells (n = 5) (Control: 20.46 ± 5.6 pA vs.
antimycin-A: 3.46 ± 0.93 pA, unpaired t-Test, p = 0.0147). The
presence of the δ-subunit, which is found in extrasynaptic
GABAA receptors, was essential for the increase in mIPSC frequency since δ-KO mice lacked an mROS-induced increase in
mIPSC frequency (δ-KO Control: 92.5 ± 10.6% at 25 min vs. δKO + antimycin-A: 85.5 ± 9.9% at 25 min; unpaired t-Test, p =
0.639; n = 6 and 6 respectively; values were normalized to their
respective first minute). Taken together, our data suggests a
more global role for mROS in regulating GABAA receptor signaling at both synaptic and extrasynaptic locations.
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Oral Communications
Accardi, MV et al.
10.1038/ncomms4168
(2014).
Nat.
Commun.
5:3168
doi:
requirements.
C20
Examining the role of interneurons in high frequency
activity in vitro
This project is funded by a research grant from Epilepsy
Research UK awarded to John Jefferys, Andrew Powell and
Premysl Jiruska.
VGAT-Venus transgenic rats were generated by Drs. Y.
Yanagawa, M. Hirabayashi and Y. Kawaguchi in National
Institute for Physiological Sciences, Okazaki, Japan, using pCS2Venus provided by Dr. A. Miyawaki.
requirements.
G. Morris1, P. Jiruska2, A.D. Powell1 and J. Jefferys1
1Neuronal Networks, University of Birmingham, Birmingham, UK
and 2Department of Developmental Epileptology, Institute of
Physiology, Prague, Czech Republic
High frequency activity (HFA) is a phenomenon where neuronal networks oscillate at frequencies faster than 100 Hz.
Physiological HFA (‘ripple band’) is implicated in learning and
memory and has a frequency of around 100-200 Hz. Network
ripples in the hippocampus in vivo are tightly phase-locked to
the firing of certain interneurons, implying a causative relationship and a pacemaker role for the interneuron within the
oscillation (Csicsvari et al 99;Ylinen et al 95). HFA that is faster
than 200 Hz (‘fast ripple band’) is unique to the epileptic brain
(Bragin et al 99). The mechanism underlying fast ripples is
unknown and could give an insight into the pathophysiology
of epilepsy, with the potential to identify new therapeutic
targets. This work examined the relationship between interneuron firing and HFA in the acute high potassium model of
epilepsy in vitro.
300 μm horizontal brain slices were prepared from adult VGATVenus A rats (Uematsu et al 08), following cardiac perfusion
with sucrose ACSF. Rats were anaesthetised with 0.24mg/kg
medotomidine and 58.2mg/kg ketamine via I.P. injection.
Epileptic-like activity, including HFA, was induced by bath perfusion of 9mM potassium. Field activity was recorded from
stratum pyramidale in hippocampal CA3b and simultaneous
single-cell recordings were made from interneurons, as identified by the fluorescent Venus transgene. The phase relationship between HFA cycles and interneuronal action potentials was determined using a custom made MatLab script; the
significance of these relationships was determined using
Rayleigh statistics (α=0.05). Averages are presented as
mean±SEM.
Perfusion of 9mM potassium caused epileptic-like discharges,
including HFA, in 83 out of 131 slices. Traces where HFA was
acquired simultaneously with successful single-cell recordings
were analysed further (n=36). Peak HFA frequency was normally distributed (Kolomogorov-Smirnov test, p=>0.05), with
a mean of 216±5 Hz, and spanned both the ripple (n=8) and
fast ripple (n=28) bands. Using interneuronal action potentials
as a reference, 11 cells showed significant phase correlation
with HFA cycles. Despite the unimodal distribution of HFA frequencies, ripples were significantly more likely than fast ripples to be correlated with interneuronal activity (Fisher’s exact
test, p=<0.001).
This suggests a fundamental mechanistic difference between
ripple and fast ripple band activity, which hints at a failure of
interneurons to modulate high frequency activity in the epileptic brain.
Csicsvari,J. et al. (1999) J.Neurosci. 19, 274-287.
Ylinen,A. et al. (1995) J.Neurosci. 15, 30-46.
Bragin,A. et al. (1999) Epilepsia 40, 127-137.
Uematsu,M. et al. (2008) Cereb.Cortex 18, 315-330.
68P
C21
Ultrafast GCaMP for tracking calcium flux in vivo
N. Helassa1, E. Esposito2, T. Carter1,3, J. Bradley2, D. Ogden2
and K. Török1
1Cardiovascular and Cell Sciences Research Institute, SGUL, London,
UK, 2Laboratoire de Physiologie Cérébrale, CNRS and Université
Paris Descartes, Paris, France and 3National Institute for Medical
Research, MRC, London, UK
Background and aims: Imaging of neuronal network activity
in free-moving animals is the aim of much research to understand brain function and the underlying mechanisms. To visualise neuronal activity in vivo at the cellular level, geneticallyencoded indicators have been developed which track calcium
flux that accompanies membrane depolarisation and action
potentials. Green Fluorescent-Calmodulin Protein (GCaMP)
type Ca2+ indicators are based on a circularly permutated EGFP
molecule (cpEGFP) flanked at the N and C termini by the
smooth muscle myosin light chain kinase derived RS20 peptide and calmodulin (CaM), respectively, which form a tight
complex upon calcium binding. This interaction stabilises the
intrinsically fluorescent deprotonated form of cpEGFP inducing a fluorescence enhancement. While the stability and brightness of GCaMP type indicators have increased significantly,
their slow response kinetics to action potentials have posed a
limitation to their application. To address the issue of slow
association and dissociation kinetics of GCaMPs, our hypothesis was that the calcium response kinetics will be accelerated if the binding affinity of Ca2+.CaM.RS20 complex is weakened. To achieve that, point mutations were introduced in the
EF-hands of CaM1 (mutants EF-1 to EF-4) and in the RS20 target peptide sequence2 (mutant RS-1) of GCaMP3 and GCaMP6.
Methods: Following site-directed mutagenesis based on our
rational design, GCaMP proteins were expressed in E.coli and
purified by a single-step affinity chromatography. All in vitro
experiments were performed at physiological ionic strength
at 20°C. Equilibrium binding constants were measured by single photon fluorescence. The calcium dissociation and association kinetics of the GCaMP mutants were obtained by
stopped-flow fluorimetry and the two-photon fluorescence
properties were measured by spectroscopy. Calcium responses
were tested in endothelial cells (HUVEC) stimulated by ionomycin.
Results: Dissociation constants (Kd) for calcium obtained from
the equilibrium calcium binding experiments were in the μM
range (0.5-5.6 μM) with Hill coefficients from 2 to 5. Calcium
dissociation rates were up to 60-fold faster than GCaMP3 with
an off-rate of 102±1 (S.E.M) s-1 (n=9). Fluorescence changes
on calcium association were highly cooperative and characterized by a rate limiting conformational change. The limiting
rates were up to 7-fold faster compared to GCaMP3, with an
observed association rate of 145±2 (S.E.M) s-1 (n=20). Two-
Oral Communications
photon cross-sections and fluorescence responses to calcium
influx in endothelial cells of mutated GCaMPs were comparable to those of GCaMP3.
Conclusion: The principles employed proved to accelerate the
calcium response kinetics of GCaMPs and can be applied to
the new generations of GCaMPs to generate low affinity
probes.
Jama A et al. JBC, 2011, 286:12308-12316
Török K and Trentham DR. Biochemistry, 1994, 33:12807-12820
Funded by the Wellcome Trust grant to KT and Royal Society
International Joint Project to KT and DO.
requirements.
C22
Serotonin controls the firing of subicular pyramidal neurons
by inhibiting a T-type calcium current
A. Petersen and J. Perrier
Department of Neuroscience and Pharmacology, Copenhagen
University, Copenhagen N, Denmark
The subiculum is the major output region of the hippocampal network. It receives inputs from CA1 and entorhinal cortex regions and sends projections to several cortical and subcortical areas. The subiculum is involved in cognitive tasks
including spatial orientation or novel object recognition memory. Under pathological conditions, the subiculum is responsible for certain forms of temporal lobe epilepsy. In this study,
we investigated how serotonin (5-HT) modulates the activity
of principal cells from the subiculum at the subcellular level.
We recorded the electrical activity of subicular pyramidal neurons with the whole-cell patch clamp technique in a slice preparation from the hippocampus of mice (P7 to P36). In agreement with previous observations, we found that subicular
neurons fired action potentials in bursts caused by the presence of a low threshold spike generated by T-type Ca2+ channels. A glass pipette filled with 5-HT was positioned near the
membrane of the recorded cell and released either by pressure or microiontophoresis. A local puff of 5-HT inhibited the
generation of action potentials. During voltage-clamp recordings performed after blocking currents mediated by Na+ and
K+ currents, puffing 5-HT decreased the amplitude of a lowthreshold voltage sensitive transient inward current sensitive
to mibefradil. These results suggest that 5-HT inhibits a current mediated by T-type Ca2+ channels. To corroborate our
findings, we monitored the variations in calcium concentrations by loading recorded cells with the calcium indicator FURA2. We observed that a burst firing evoked by depolarizing current pulses induced an increase in calcium concentration.
When 5-HT was puff-applied, the calcium signal was attenuated in all compartments of the neuron (AIS, soma, dendrites).
Puffing the 5-HT2C agonist WAY 629 instead of 5-HT had the
same inhibitory effect on firing and Ca2+ current.
Our data suggest that 5-HT modulates the activity of subicular pyramidal cells by inhibiting T-type calcium channels
through an activation of 5-HT2C receptors. We suggest that
the pathway we uncovered prevent the development of epileptic seizures for healthy subjects.
C23
Tonic signaling from O2 sensors can be reprogrammed
depending on genotype to set neural circuit activity and
behavioral state
K. Busch1, P. Laurent2 and M. de Bono2
1Centre
for Integrative Physiology, University of Edinburgh,
Edinburgh, UK and 2Cell Biology Division, MRC Laboratory of
Molecular Biology, Cambridge, UK
To maintain homeostasis of the body or avoid dangerous environments, all animals must continuously monitor key sensory
information such as oxygen levels. Tonic sensory receptors can
perceive such constant information but are not well understood. How do sensors sustain responses to external signals
for long periods of time, and how do they reconfigure neural
circuits to generate a prolonged change in behaviour?
Caenorhabditis elegans provides a unique opportunity to
explore homeostatic sensing and tonic signalling in the living
organism. It regulates oxygen homeostasis by mounting
sophisticated behavioural responses to ambient O2, preferring
concentrations substantially below atmospheric levels. Using
calcium imaging, behavioural assays and optogenetic stimulation, we studied the mechanisms underlying avoidance of
high [O2]. We found that four O2-sensing neurons continuously
respond to ambient oxygen. Tonic signalling in these neurons
is sustained by a ‘calcium relay’ that consists of cGMP-gated
channels, voltage-gated Ca2+ channels, as well as the ryanodine and IP3 receptor channels which release calcium from
intracellular stores (Busch et al., 2012). Tonic activity of these
neurons is necessary and sufficient to set the behavioural state
according to ambient [O2] for many minutes and even hours.
Tonic signalling propagates to downstream neural circuits,
including the “hub interneuron” RMG, which is thought to integrate inputs from diverse sensory neurons and is required to
mediate O2 responses.
The tuning of oxygen responses shows plasticity depending
on experience, context, or genetic background. Experiencedependent plasticity forms directly in the sensory neurons and
takes several hours to develop, showing hallmarks of longterm memory. Strikingly, O2 responses can be reprogrammed
by experience in some genetic backgrounds but not others: a
neuroglobin allele expressed in the O2 sensors and present in
many wild strains functions to potently restrict plasticity. In
this way, different alleles allow divergent behavioural strategies in reaction to sensory cues: responses can be either narrowly tuned and fixed to a certain concentration level, or
broadly tuned and flexible in a changing environment.
Busch KE, Laurent P, et al.: Tonic signaling from O2 sensors sets neural circuit activity and behavioral state (2012). Nature Neuroscience
15, 581-591.
requirements.
requirements.
69P
Oral Communications
C24
requirements.
Transcranial stimulation promotes consolidation of
imprinted memory in domestic chicks (Gallus gallus)
C25
A. Nicol and B. McCabe
Sub-department of Animal Behaviour, University of Cambridge,
Cambridge, UK
Modulation of Ca2+ entry mechanisms as a potential therapy
for acute pancreatitis
Domestic chicks learn the visual features of an object through
imprinting, subsequently approaching that stimulus in preference to others. The intermediate medial mesopallium (IMM)
stores the recognition memory of imprinting (McCabe 2013).
The responses of IMM neurons are altered by imprinting: more
respond to an imprinted stimulus than to an alternative. Behavioural and electrophysiological effects of imprinting are
impaired if sleep is disturbed shortly after imprinting, but maintained in undisturbed chicks (Jackson et al 2008). Here we
show the effect of post-training transcranial stimulation on
imprinted preferences.
Chicks (n=57) were hatched and reared in darkness to ~12h
post-hatch, then placed in running wheels and tested to establish which of 2 rotating stimuli (red box, RB, or blue cylinder,
BC) was approached preferentially before imprinting. The preference test (PT0) comprised serial exposures to the stimuli
(4x2min, RB-BC-BC-RB). Under anaesthetic (0.12ml Equithesin,
i.p.), 38 chicks had 2 stainless steel electrodes fitted to the
frontal bones of the skull near the midline (left rostral, right
caudal). Next day, they were trained by exposure to the stimulus not preferred in PT0 (2x1h sessions, 90min interval). Posttraining, chicks were tested as in PT0, but with 4min presentations (PT1). They then had 6h of disrupted rest (DR) in
darkness, with wheels locked. Disruption was by turning the
wheel once for 60s at random in each 30min of DR. In the first
3h of DR, chicks received: slow wave (0.75Hz, n=12, SW chicks)
or theta frequency (6Hz, n=9, TF chicks) stimulation (~2μA
peak-peak, sinusoidal), or no stimulation (17 sham implanted,
and 19 non-implanted controls). Preferences were again tested
~8h (PT2) and ~16h (PT3) after the end of DR.
The chicks did not differ significantly by group at PT0 or PT1.
Mean preference scores (% approach to TS) at PT1 (73.0±2.0%
sem) were higher (anova, P=0.001) than at PT0 (39.8±2.0%),
i.e. chicks were imprinted. There was no significant difference
between scores in PT2 and PT3, so these were pooled (PT23). Scores at PT2-3 for SW and TF chicks (73.2±3.1%) did not
differ significantly from those at PT1. In sham and control
chicks (unstimulated) the scores fell (anova, P=0.004,
64.1±3.0%). The 2 unstimulated groups of chicks did not differ significantly in any test. In a control experiment, the behavioural activity of SW, TF and unstimulated chicks (n = 4, 4, and
16 chicks respectively) in unlocked wheels during undisturbed
rest did not differ, indicating similar arousal across groups.
The results show that memory for the imprinted stimulus is
protected by SW or TF transcranial stimulation from attenuation otherwise resulting from disturbance during DR. This is
reminiscent of improved memory in human subjects following SW transcranial stimulation during sleep (Marshall et al
2004).
E. Stapleton, J.V. Gerasimenko, O.H. Petersen and
O.V. Gerasimenko
McCabe BJ (2013). WIREs Cogn Sci 4, 375-390
School of Biosciences, Cardiff University, Cardiff, S.Glamorgan, UK
Acute pancreatitis (AP) is a human disease characterised by
inflammation and necrosis of the pancreas. Intracellular activation of enzymes, the hallmark of AP, has been shown to be
the result of sustained elevations in [Ca2+]i 1,2.
Pathophysiological Ca2+ signals that precede AP, deplete the
endoplasmic reticulum (ER) Ca2+ stores. ER store depletion is
detected by the ER Ca2+ sensor, stromal interaction molecule
1 (STIM1), which activates Ca2+-release activated Ca2+ (CRAC)
channels located in the plasma membrane 3. CRAC channels,
a type of store-operated Ca2+ channels (SOCC), open in
response to Ca2+ store depletion to refill the ER in non-excitable
cells 3. However, during pathological stimulation additional
Ca2+ entering the cell will only serve to further increase already
elevated [Ca2+]i concentrations. CRAC channels are an ideal
target in order to reduce Ca2+overload.
C57BL/6 mice were killed humanely in accordance with the UK
Schedule 1 of the Animals (Scientific Procedures) Act, 1986.
[Ca2+]i measurements were made using cells loaded with the
Ca2+-sensitive dye, Fura-2.
When cells are treated with compounds that inhibit CRAC channels, such as GSK-7975A4, Ca2+ influx is markedly reduced compared with control cells and so is the necrotic cell damage associated with AP. 2-APB has traditionally been viewed as a blocker
of SOCC, but has deleterious effects in pancreatic acinar cells
by releasing Ca2+ from ER stores. Recently more potent analogues of 2-APB have been developed and when applied to
pancreatic acinar cells can reduce Ca2+ influx, in a dose dependent manner.
Calmodulin (CaM) and Ca2+-like peptides (CALPs) have been
shown to be protective against ethanol-invoked increases in
[Ca2+]i 5. CRAC channels have a known CaM binding domain
and contribute to [Ca2+]i overload; our studies using agonists,
such as CALPs, and antagonists of CaM indicate that pharmacologically targeting CaM affects Ca2+ entry.
Data collected using 2-APB analogues supports the data
obtained using GSK-7975A4, inhibiting CRAC channels reduces
Ca2+ overload in cells. Peptides, like CALP-35, also seem to have
promise in reducing Ca2+ overload.
A combination of CRAC channel blockers and CaM modulation
may be a more efficient method of reducing Ca2+ overload and
act as a potential AP therapy.
Petersen, O.H., Sutton, R., 2006. Ca2+ signalling and pancreatitis:
effects of alcohol, bile and coffee. Trends in Pharmacological Sciences,
27: 113–120.
Petersen, O.H., Gerasimenko, O.V., Gerasimenko, J.V., 2011. Pathobiology of acute pancreatitis: focus on intracellular calcium and calmodulin. F1000 Med Rep, 3: 15.
Jackson et al (2008). Curr Biol 18, 393-400
Marshall et al (2004). J Neurosci 24, 9985-9992
Supported by the BBSRC
70P
Lewis, R.S., 2011. Store-operated calcium channels: new perspectives
on mechanism and function. Cold Spring Harb Perspect Biol 3.
Gerasimenko JV, Gryshchenko O, Ferdek PE, Stapleton E, Hébert TO,
Bychkova S, Peng S, Begg M, Gerasimenko OV, Petersen OH (2013)
Ca2+ release-activated Ca2+ channel blockade as a potential tool in
anti-pancreatitis therapy PNAS110(32):13186-13191
Oral Communications
Gerasimenko JV, Lur G, Ferdek P, Sherwood MW, Ebisui E, Tepikin AV,
Mikoshiba K, Petersen OH and Gerasimenko OV (2011) Calmodulin
protects against alcohol-induced pancreatic trypsinogen activiation
elicited via Ca2+ release through inositol trisphospate receptors PNAS
108(14) 5873-5878
requirements.
Inhibition of CD38-NAADP-TPC-induced calcium signalling
blocks cholinergic excitation-mucus secretion coupling in
native human colonic crypt goblet cells
C.
A.
M. Williams1
Parris1,
M.
Loader1,
requirements.
C27
C26
Kam1,
the CD38-NAADP-TPC pathway suppressed Ca2+ signal generation and Muc2 depletion. The CD38-NAADP-TPC-calcium pathway plays a central role in cholinergic excitation-mucus secretion coupling in human colonic crypt goblet cells in situ.
N.
Juge2,
M.
Lewis3
Protective and anti-inflammatory effects of Ursodeoxycholic
acid in colonic epithelia
N.K. Lajczak, V. Saint-Criq, M.S. Mroz and S.J. Keely
and
Molecular Medicine, Royal College of Surgeons in Ireland, Dublin,
Dublin, Ireland
1Biological Sciences, University of East Anglia, Norwich, UK, 2Gut
Health and Food, Institute of Food Research, Norwich, UK and
3General Surgery, Norfolk and Norwich University Hospital,
Norwich, UK
Intestinal epithelia constitute the first line of defence with several processes contributing to this innate barrier function. For
example, fluid secretion flushes pathogens from the lumen,
defensin protects against bacterial invasion and restitution
repairs the epithelium after injury. Although bile acids are
known to be involved in the pathogenesis of Inflammatory
Bowel Disease (IBD), the regulatory mechanisms are not well
understood. The aim of this study was to investigate the effects
of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA)
on epithelial responses that contribute to barrier function.
T84 colonic adenocarcinoma cells were grown on permeable
supports and were treated with DCA (10 μM - 1 mM) and UDCA
(10 - 300 μM) for 1 - 72 h. Protein expression was investigated
by western blotting, protein secretion by ELISA and mRNA
expression by qPCR. Epithelial restitution in polarised T84 cell
monolayers was investigated using a wound healing assay.
Muscle stripped sections of human colonic mucosa were
mounted in Ussing chambers for measurements of β-defensin
secretion and ion transport by monitoring changes in short
circuit current (Isc) under voltage-clamped conditions. Data
were statistically analysed using a Student T-test or Two-way
ANOVA with Newman-Keuls Multiple Comparison Post Test.
All experiments involving human tissue had the approval of
Beaumont Hospital Ethical Committee.
When added to voltage clamped sections of human colonic
tissue, DCA, at concentrations ≥ 200 μM, stimulated Isc
responses of 11 ± 4 μA/cm2 (p < 0.05; n = 4). Furthermore,
DCA pretreatment attenuated Cl- secretory responses to the
Ca2+ dependent agonist, carbachol (CCh, 100 μM) from 219
± 71 μA/cm2 to 115 ± 26 μA/cm2 (n = 4). In T84 cells, UDCA
(50 μM) attenuated DCA (150 μM) stimulated human β
defensin-1 (HβD-1) release from 683 ± 131 pg/ml to 304 ± 94
pg/ml and human β defensin-2 (HβD-2) release from 200 ± 42
pg/ml to 21 ± 11 pg/ml (n = 8; p < 0.001). Using a specific
agonist of TGR-5 receptor (INT777), we determinated that HβD1 and HβD-2 are TGR-5 activation dependent and UDCA attenuates TGR-5 activated secretion of HβD-1 from 429 ± 95 pg/ml
to 215 ± 65 pg/ml and HβD-2 from 105 ± 23 pg/ml to 35 ± 3
pg/ml (n = 6: p < 0.01). Using specific inhibitor of NF-κB pathway (BMS-34451), we determined that NF-κB inhibition attenuated DCA (150 μM) stimulated HβD-2 secretion from 115 ±
8 pg/ml to 27 ± 8 pg/ml (n = 6; p < 0.01). Moreover UDCA (100
μM, 48 h) showed more of its protective properties by preventing DCA (150 μM) induced inhibition of epithelial restitution of mechanically wounded cell monolayers from 29 ± 15
% to 79 ± 5 % (n = 5; < 0.001).
Taken together, these data implicate colonic bile acids, DCA
and UDCA, as important regulators of intestinal epithelial bar-
The lumen of the large intestine houses approximately 1013 1014 microorganisms. In order to defend itself, the intestinal
epithelium secretes a protective coating of mucus, the major
component of which is Muc2 mucin protein. There is much
interest in the mechanism of excitation-mucus secretion coupling because compromised mucus layer formation in Muc2
knock-out mice and human disease is associated with direct
contact of bacteria with the epithelium, inflammation, bloody
diarrhoea and colon cancer. Previously, cholinergic stimulation has been shown to promote mucus secretion, but the
mechanism for excitation-mucus secretion coupling is not fully
understood. The aim of the current study was to elucidate
the mechanism of calcium (Ca2+) signal generation in native
human colonic crypt goblet cells in situ and determine the role
of Ca2+ signalling in Muc2 protein secretion. Methods: Human
colonic crypts were isolated from colorectal tissue samples
obtained at surgical resection (NRES approval) and placed in
a 3D culture system. Intracellular Ca2+ was monitored by
Fura2/Fluo-4 imaging. To complement previous qRT-PCR mRNA
expression analyses, localisation of M3AChR, CD38 (which
catalyses NAADP synthesis), TPC1&2 (i.e. Ca2+ channels stimulated by NAADP) and endolysosomes (LAMP-1 positive
puncta) within Muc2+ goblet cells was visualised by fluorescence immunolabelling and confocal microscopy. Ca2+ signal
generation and Muc2 depletion from goblet cells was assessed
following stimulation with carbachol (Cch,10μM) in the presence and absence of nicotinamide (20 mM, an inhibitor of
CD38-mediated NAADP synthesis) or NED-19 (200 μM, an
NAADP receptor inhibitor). Results: Application of Cch evoked
a Ca2+ signal at the human colonic crypt base which spread to
all cell types located along the human colonic crypt-axis, including goblet cells. Muc2+ goblet cells expressed: M3AChRs on
the basal membrane; CD38 in the cytoplasm and nucleus; cytoplasmic TPC1&2 associated with apical LAMP-1 positive intracellular puncta. Cch-induced colonic crypt Ca2+ signals were
suppressed by pre-treatment with nicotinamide (64.5±5%
reduction, N=2 subjects, n=5 crypts) and were inhibited profoundly by pre-incubation with NED-19 (85±5% reduction, N=5
subjects, n=15 crypts). Goblet cell Muc2 immunofluorescence
was dramatically reduced following a 30 minute exposure to
Cch (44±4.7 % reduction, N=4 subjects, n=4 crypts) and this
was inhibited by NED-19 (150±45%, N=4 subjects, n=4 crypts).
Conclusions: Goblets cells in the human large intestine express
the molecular machinery to synthesise NAADP. Inhibition of
71P
Oral Communications
rier function and suggest they are promising targets for the
development of new approaches to treat IBD.
receptors and may play an essential role in cancer cell adhesion to the extracellular matrix.
requirements.
requirements.
C28
C29
Role of sodium hydrogen exchanger isoform 1 in integrinsmediated adhesion and migration of cancer cells
Rapid CO2 permeation across biological membranes:
implications for CO2 venting from tissue
S. Akram1, S. Rahman2 and H. Koay3
A. Hulikova and P. Swietach
1College
Department of Physiology, Anatomy and Genetics, Oxford
University, Oxford, UK
of Medicine, Al Imam Muhammad Ibn Saud Islamic
University, Riyadh, Saudi Arabia, 2Institute of Ophthalmology,
University College London, London, UK and 3Rockhampton Base
Hospital, Queensland, QLD, Australia
Background: The Na+-H+ exchangers (NHEs) are a family of
membrane glycoproteins which transport H+ out of the cell in
exchange for Na+ with a stoichiometry of 1:1. In mammalian
cells, the NHE family consists of nine isoforms, NHE-1 to NHE9. NHE-1, the first one of the isoforms to be cloned, is ubiquitously distributed.
Several studies have shown both increased activity and protein of NHE-1 in transformed cells. Apart from its role as a principal regulator of intracellular pH (pHi) and cell volume, NHE1 has been implicated in cell proliferation, transformation and
migration.
Cell migration is a multi-step process that requires spatial asymmetry which is stimulated by Rho GTPases, phosphoinositides
and actin polymerization. Integrin family of receptors is responsible for cell surface interactions with extracellular matrix
(ECM).
Disrupting NHE-1 function leads to impaired polarity of cells
and their inability to migrate. Although NHE1 has been shown
to affect cell migration through its various functions, a role for
the exchanger in cell migration regulated by integrins has not
been extensively studied. Fact that NHE1 binds several other
proteins in the cytoplasmic regulatory domain, have led to the
hypothesis that NHE1 can act as a plasma membrane scaffold
that brings together many proteins so they can interact functionally. Thus, it is plausible to hypothesize the possible role
NHE-1 might play by direct or indirect structural interaction
with the assembly of cell adhesion molecules.
Materials and Methods:
1. Inhibition of NHE1 activity (using ethyl-isopropylamiloride(EIPA)) and its effect on pHi and cell viability (Measurement of intracellular pH, MTT assay)
2. Effect of NHE1 silencing (using siRNA transfection) and its
effect on cell viability (Western blot analysis, MTT assay)
3. Effect of NHE1 silencing and pharmacological inhibition of
its activity on cell adhesion and motility (In vitro cell adhesion assay, In vitro cell migration assay)
Results
The data obtained shows that with either EIPA treatment or
NHE-1 siRNA transfection, migratory capacity was impaired in
MDA-MB-231 human breast cancer cells. Pharmacological inhibition of NHE-1 did not significantly reduce the integrinsdependent cell adhesion in these cells. However, down-regulation of NHE-1 protein expression had significant effect on
integrins-mediated cell adhesion to fibronectin.
Conclusion: This study shows that the effect of NHE-1 on integrins-dependent cell adhesion is independent of its activity.
However, NHE-1 protein expression seems to be an important upstream event in the functional assembly of integrin
72P
The degree to which cell membranes are barriers to CO2 transport remains controversial. Proteins, such as aquaporins and
Rh-complex, have been proposed to facilitate CO2 transport
but this implies that the non-channel component of membranes must have greatly reduced CO2 permeability; otherwise, permeation across the lipid matrix would short-circuit
the channel-mediated pathway. However, other studies have
demonstrated that CO2 crosses lipid bilayers very rapidly.
To determine whether membrane CO2 permeation is rate-limiting for gas transport, the spread of CO2 across multicellular
tissue-growths (spheroids) was measured using intracellular
pH (pHi) as a spatial read-out. Experimental data were analysed
using a carefully parameterized diffusion-reaction algorithm
to extract information about CO2 dynamics. Although H+-yielding CO2 hydration is rate-limited by intracellular carbonic anhydrase (CA) activity locally, time-delays in pHi-changes at different spheroid-depths report the spread of CO2. These delays,
readily resolvable in spheroids, can ascertain whether CO2
diffuses freely or is restricted by membrane permeation. Experiments were performed in the presence of the membraneimpermeant CA inhibitor 4-aminomethyl benzenesulfonamide
(30 μM) to eliminate CA-facilitated CO2 diffusion.
Colorectal HCT116 cells have basal permeability to water
(Pf=25±1 μm/s; measured by hypo-osmotic dilution of intracellular calcein fluorescence) and NH3 (Pm,NH3=147±8 μm/s;
measured from changes in pH-sensitive cSNARF1 fluorescence
evoked by ultra-rapid (<20 ms) addition/removal of NH3/NH4+).
Furthermore, Pf and Pm,NH3 were unaffected by blockers of
aquaporins and Rh-complex (1 mM Hg2+, 2 mM p-chloromercuribenzoic acid [PCMB], 30 μM 4,4ʹ-diisothiocyano-2,2ʹ-stilbene-disulfonic acid [DIDS]), indicating the functional absence
of aquaporins and NH3 gas channels. Despite the absence of
protein-facilitated water and NH3 permeation, CO2 diffusivity
in HCT116 spheroids was fast: only 24±4% lower than in pure
water, which can be accounted for fully by volume-exclusion
due to proteins (75 g/L). Diffusivity was unaffected by PCMB
and DIDS, but reduced under hypertonic conditions (adding
300 mOsm mannitol) which increases intracellular proteincrowding. Comparably high CO2 diffusivity measurements
(~25% slower than in pure water) were obtained in spheroids
of T47D breast cells (basal water permeability; Pf=20±1 μm/s)
and NHDF-Ad fibroblasts (aquaporin 1 and 9-facilitated water
permeability; Pf=62±5 μm/s). In contrast, diffusivity of NH3, a
smaller but less lipophilic gas, was considerably slower than
in pure water (~75%), as expected from rate-limiting membrane permeation.
In conclusion, membranes, even in the functional absence of
proposed gas channels, do not restrict CO2 venting from tissue-growths.
Hulikova & Swietach FASEB J 2014 (in press)
Oral Communications
Supported by the Association for International Cancer Research
and the Royal Society
pathway causes more redistribution of the cGKII to the lipid
rafts, possibly by lipid modification.
requirements.
requirements.
C30
C31
The cGMP-dependent kinase 2 is recruited to and colocalizes
with Na+/H+ regulatory factor 2 and Na+/H+ exchanger
isoform 3 during guanylate cycle C receptor activation in
murine small intestinal brush border membrane in vivo
Hyperglycaemia-induced changes in airway surface liquid
lactate and pH
M. Luo1,2, Y. Liu1, B. Riederer1, E. Patrucco3, F. Hofmann3,
M. Donowitz4, D. Tian2, C. Yun5, H. de Jonge6, G. Lamprecht7
and U. Seidler1
Institute of Infection and Immunity, St George’s, University of
London, London, UK
1Hannover
2Tongji
Medical School, Hannover, China,
Hospital,
Huazhong University of Science and Technology, Wuhan, China,
3Forschergruppe 923, München, Germany, 4John Hopkins School
of Medicine, Baltimore, MD, USA, 5Emory University, Atlanta, GA,
USA, 6Erasmus MC, Rotterdam, Netherlands and 7University of
Rostock, Rostock, Germany
Background: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger
3 (NHE3) is regulated by the Na+/H+ Exchanger Regulatory
Factor (NHERF) family of PDZ-adapter proteins, which enables
the formation of multiprotein complexes. We previously
reported a differential association of the NHERFs to the lipid
raft and non-raft fraction of NHE3 in murine intestinal BBM.
Aim: This study was undertaken to explore the association of
NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII),
a key enzyme in hormonal and toxin-mediated inhibition of
NHE3, within lipid rafts, and to assess the effect of stimulating this signal transduction pathway on the raft assembly of
NHE3, NHERF2 and cGKII in vivo. Methods: Murine BBM was
isolated from wildtype, NHE3-deficient, cGKII-deficient, and
NHERF2-deficient mice before and after intestinal application
of a heat-stable Escherichia coli toxin (STa)/guanylin analogue,
which binds to and activates guanylate cycle C (GCC) in vivo,
followed by cervical dislocation 2 hours after drug application and removal of the small intestine. Lipid raft and non-raft
fractions were separated by Optiprep density gradient centrifugation of Triton X solubilised isolated small intestinal BBM.
The raft-associated and non-raft proteins were precipitated
and studied by Western analysis. Results: NHE3 and NHERF2
were strongly lipid raft-associated. The cGMP-dependent kinase
II, which together with NHERF2 is essential for STa/guanylinmediated NHE3 inhibition, was found in two molecular entities, a larger band of ~85 KD, which was exclusively lipid raftassociated, and a smaller band of ~72 KD that was mostly
non-raft associated. NHERF2 deletion resulted in a decreased
lipid raft association of NHE3, but not of cGKII. The application
of an oral STa/guanylin analogue to the mice and subsequent
small intestinal BBM isolation and lipid raft flotation assay
demonstrated a redistribution of cGKII, with an increase of the
lipid raft associated larger size cGKII, more cosegregation of
NHE3, NHERF2 and cGKII in the same raft fraction, and a
decrease of the smaller size, non-raft cGKII. Conclusion: The
differential association of the NHERF2, as well as cGKII, with
the raft-associated and the non-raft fraction of NHE3 in the
brush border membrane is likely one component of
STa/guanylin-mediated inhibition of NHE3. Many players of the
signalling pathway for the STa/guanylin analogues via cGKII,
leading to NHE3 inhibition, are associated with lipid rafts in
the murine small intestine, and stimulation of this signalling
J.P. Garnett, E.H. Baker and D.L. Baines
The airway epithelium is covered by a thin layer of surface liquid (ASL), which is vital for maintaining the sterility of the respiratory tract. The glucose concentration of the ASL (~0.4mM)
is much lower than that in blood and is tightly regulated by
the airway epithelium (1). ASL glucose increases with hyperglycaemia and respiratory disease (including cystic fibrosis and
chronic obstructive pulmonary disease) and this increases the
risk of respiratory bacterial infection (2,3). ASL lactate concentrations also rise with hyperglycaemia and respiratory
inflammation. The aim of this study was to investigate whether
the airway epithelium secretes lactate into ASL under hyperglycaemic conditions and if this contributes to changes in ASL
pH due to lactate-H+ co-transport.
H441, Calu-3 and primary human bronchial epithelial cells were
grown on permeable supports at air-liquid-interface to form
confluent polarised monolayers. Hyperglycaemia was mimicked by increasing the glucose concentration of the HCO3-buffered physiological salt solution in the basolateral chamber from 5 to 15 mM. ASL lactate was sampled after 6 hours
by washing the apical surface with 50 μl of salt solution and
measured using a lactate assay kit (Sigma, UK). ASL pH was
measured by adding 100 μl of salt solution to the apical surface and using a pH microelectrode. Increasing basolateral glucose resulted in a significant rise in ASL lactate across all airway epithelial cell types (P < 0.05, n=5-12), but no significant
changes in basolateral lactate concentrations were observed.
ASL pH of H441 monolayers decreased by 0.09 ± 0.02 pH units
(P < 0.05, n=4), consistent with lactate-H+ co-transport acidifying the ASL. However, an alkalinisation of 0.10 ± 0.05 pH
units (P < 0.05, n=9) was observed in the ASL of hyperglycaemic
Calu-3 monolayers. As Calu-3 cells produce HCO3--rich fluid
secretions, ASL acidification could stimulate HCO3- secretion
to neutralise the ASL. To test this hypothesis, the experiment
was repeated using a HEPES-buffered salt solution to reduce
epithelial HCO3- secretion. Under these conditions, Calu-3 ASL
pH decreased in response to elevated glucose. Inhibition of
monocarboxylate transporters MCT1 and MCT2 (lactate-H+
co-transport) with AR-C155858 (100 nM) partially overcame
the effects of hyperglycaemia on ASL lactate and pH, although
the results indicate that other transporters may be involved.
These data indicate that hyperglycaemia increases airway
epithelial lactate secretion, in part via MCT1/2 lactate-H+ cotransport, which can acidify the ASL in the absence of epithelial HCO3- secretion. This could be important in the pathogenesis of cystic fibrosis as both airway glucose and lactate
concentrations are elevated and ASL pH is more acidic due to
aberrant HCO3- secretion.
Garnett JP et al. (2012). Eur Respir J 40, 1269-1276.
Baker EH et al. (2007). J Appl Physiol 102, 1969-1975.
Philips BJ et al. (2005). Thorax 60, 761-764.
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Oral Communications
requirements.
Turner, M. J. et al., (2013). Poster presented at the 37th Congress of
the International Union of Physiological Sciences; 2013 July 21-26;
Birmingham, UK.
Garnett, J. P. et al., (2011). J Biol Chem 286, 47
C32
Chronic hypercapnia reduces cAMP-regulated fluid secretion
in airway epithelia
Work supported by an MRC Studentship awarded to MT
requirements.
M.J. Turner1, M.J. Cann2 and M.A. Gray1
1Institute for Cell and Molecular Biosciences, Newcastle University,
Newcaastle Upon Tyne, UK and 2School of Biological and
Biomedical Sciences, Durham University, Durham, UK
Hypercapnia is a symptom of chronic lung disease and is caused
by inefficient gas exchange at the alveoli. However, the effects
of elevated CO2 on airway epithelial function are not currently
well understood. The airway surface liquid (ASL) plays a major
role in lung innate immunity and it’s volume and composition is tightly regulated, predominantly by serous cells of the
submucosal glands (SMGs) in which elevations in intracellular
cAMP activate CFTR-mediated fluid and HCO3- secretion. We
have previously shown that acute hypercapnia (20 minutes)
reduces intracellular cAMP levels in Calu-3 cells, a human airway cell line derived from SMGs (1). The aim of the current
work was to investigate the effects of chronic hypercapnia (24
hrs or more) on cAMP-dependent processes in human airway
epithelia.
Calu-3 cells were grown as polarised monolayers on permeable transwell supports and the volume, pH and mucus content of fluid secreted from these cells was measured over 24
hours. CFTR-dependent HCO3- transport was assessed by measuring intracellular pH changes in response to cAMP agonists
in BCECF-AM loaded cells (2).
In normocapnia (5% CO2), 5μM forskolin stimulated a 17.1 ±
2.6% increase in fluid secretion over 24 hours (p<0.01; n = 3),
and raised the pH of the secreted fluid by 0.31 ± 0.01 (p<0.01;
n = 3). However, in response to 10% CO2, the forskolin-induced
fluid increase was 9.5 ± 0.9% (p<0.05 vs. normocapnia; n = 3),
while the pH of the secreted fluid was unchanged (0.30 ± 0.01;
p>0.05 vs normocapnia; n = 3). These results imply that CO2
exhibits differential effects on transporters regulating the volume and composition of the secreted fluid. In 5% CO2, CFTRdependent HCO3- efflux was 232.7 ± 83.8mM/min (n = 4) which
was unchanged in cells that had been exposed to 10% CO2 for
24 hours (371.3 ± 38.0mM HCO3-/min; n = 3; p>0.05 vs normocapnia) showing that chronic hypercapnia was not altering
CFTR activity to produce its effects on forskolin-stimulated
fluid secretion. Interestingly, 1mM IBMX stimulated a 16.8 ±
3.1% (p<0.01; n = 3) increase in fluid secretion over 24 hours
which was unaffected by 10% CO2 (11.8 ± 2.9%; n = 3; p>0.05
vs normocapnia) suggesting CO2 has selective effects on cAMP
generation. However, there was no effect of CO2 on mucus
secretion, as measured by the Periodic Schiffs assay, in both
non-stimulated (18.2 ± 1.0 μg/ml vs 19.2 ± 0.9 μg/ml; p>0.05;
n = 3) and forskolin-stimulated (19.2 ± 0.1 μg/ml vs 24.0 ± 4.0
μg/ml p>0.05; n = 3) conditions.
These findings demonstrate that chronic hypercapnia alters
cAMP-mediated fluid but not HCO3- or mucus transport in
human airway epithelia, through a CFTR-independent manner. This suggests that dysregulation of airway surface liquid
secretion may occur in patients suffering from hypercapnia
which could have consequences for the innate immune system of the airways.
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C33
Vasopressin regulates uptake of exosomes by kidney
collecting duct cells
W. Oosthuyzen1, A. Caporali1, J. Pound2, C.D. Gregory2,
D.J. Webb1, M.A. Bailey1 and J.W. Dear1
1Centre for Cardiovascular Science, University of Edinburgh,
Edinburgh, Edinburgh, City of, UK and 2MRC Centre for
Inflammation Research, University of Edinburgh, Edinburgh, UK
Exosomes are vesicles that are released from the kidney into
urine. They contain protein and RNA from all sections of the
nephron and represent a reservoir for biomarker discovery.
Exosomes can ‘shuttle’ protein, messenger RNA and microRNA
between cells and may represent a new physiological signalling
mechanism. To understand the mechanisms of exosome mediated signalling along the nephron we explored the uptake of
exosomes by collecting duct cells. These cells were used
because in vivo they are downstream in terms of urine flow
along the nephron and are therefore exposed to exosomes
from a range of upstream nephron segments.
To explore exosome uptake, exosomes were fluorescently
labelled then co-cultured with murine cortical collecting duct
(mCCD) cells. In a subset of studies the mCCD cells were grown
on transwell membranes to allow specific stimulation of apical or basolateral cellular compartments. To quantify exosome
transfer the supernatant exosome concentration was measured by nanoparticle tracking analysis and the intracellular
exosome concentration was measured by flow cytometry. Confocal laser scanning microscopy was used for intra-cellular visualisation and localisation of labelled exosomes.
Labelled exosomes were internalised by mCCD cells and this
uptake was significantly increased following stimulation with
the vasopressin analogue desmopressin (dDAVP) (percentage
of cells containing labelled exosomes: dDAVP 38% ± 8.4% vs
control 16.7% ± 4.8%, p<0.001). Tolvaptan, a selective vasopressin V2 receptor antagonist, decreased exosome uptake
following dDAVP stimulation (tolvaptan and dDAVP 15.6% ±
3.65% vs dDAVP 38.03% ±8.39%, p<0.001). Endothelin-1, a peptide with a known inhibitory effect on vasopressin action, and
dynamin inhibition decreased exosome uptake following
dDAVP stimulation. Using mCCD cell monolayers, the site of
dDAVP action was confirmed to be basolateral, but exosome
uptake was from both apical and basolateral compartments.
In conclusion, this study is the first to demonstrate that vasopressin regulates exosome uptake in the kidney, in a V2 receptor mediated process through clathrin-dependent endocytosis. This understanding will allow us to better define the role
of exosomes in kidney physiology and develop exosomes as
therapeutic agents for kidney disease.
requirements.
Oral Communications
C34
C35
Protein kinase D2 modulates ENaC activity and aldosteronesensitive sodium reabsorption in the kidney cortical
collecting duct
The effect of flow on placental amino acid transfer in the
isolated perfused placenta
S. Quinn, R. Dooley, M. Yusef Robles, W. Thomas and B. Harvey
Molecular Medicine, Royal College of Surgeons in Ireland, Dublin,
Ireland
A major risk factor for renal disease is hypertension which is
also indicative of impaired electrolyte homeostasis. Na+ homeostasis in the kidney can be affected by increasing synthesis
of aldosterone and activation of the mineralocorticoid receptor (MR). A dysregulation of aldosterone signalling results in
hypertension and chronic pathologies of the kidney such as
renal fibrosis and nephropathy. Aldosterone signalling is transduced via binding to MR in segments of the distal nephron
including the cortical collecting duct (CCD). Aldosterone can
modulate ENaC activity at the levels of transcription, protein
stability and subcellular trafficking (1). Here we report a novel
mechanism by which aldosterone can regulate the subcellular trafficking of ENaC subunits through activation of protein
kinase D2 (PKD2). We investigated the activation of PKD2 by
aldosterone and its effects on ENaC in the rat distal nephron
in vivo and in a murine cortical collecting duct (M1-CCD) cell
line. ENaC current was measured as the amiloride-inhibitable
transepithelial short-circuit current (IENaC). Intracellular accumulation and rapid activation of PKD2 (<10min) was induced
by aldosterone (10nM) treatment. Expression of PKD2 under
basal conditions was found to localise at the apical membrane
and in the sub-apical cytosolic space. Longer treatment with
aldosterone (30min) induced sub-cellular redistribution of
PKD2 into the trans-Golgi network. PKD2 knock-down using
shRNA in M1 cells resulted in an elevated basal ISC from 1.9 ±
0.2μA/cm2 in wild-type cells to 9.3 ± 1.4μA/cm2 in PKD2 knockdown M1 cells (n=10, p=0.002). PKD2 knock-down also
resulted in an increased amiloride-sensitive ENaC current (IENaC)
from 1.3 ± 0.3μA/cm2 in wild-type cells to 6.0 ± 1.0μA/cm2 in
PKD2 knock-down M1 cells (n=11, p=0.0001). Long term treatment (24h) of wild-type M1 cells with aldosterone increased
the basal ISC from 1.9 ± 0.2μA/cm2 to 4.6 ± 0.7 μA/cm2 (n=7,
p=0.008). The ENaC current showed an increase from 1.3 ± 0.3
μA/cm2 in wild-type M1 cells to 3.3 ± 0.5 μA/cm2 when treated
for 24h with aldosterone (n=8, p=0.001). Student’s t-tests were
performed alongside a one way ANOVA. The effect of aldosterone on both the basal ISC and IENaC is abolished with the
knock-down of PKD2. The abundance of ENaCγ in the apical
membrane showed an increase following knock-down of PKD2
in M1 cells. Studies in rats fed on a low Na+ diet showed
increased expression of ENaCγ at the apical membrane accompanied by a subcellular redistribution of PKD2 from the apical
membrane to the trans-Golgi network. The use of animals was
subject to local ethical approval in compliance with national
legislation.
Quinn S, Harvey BJ, Thomas W. Rapid aldosterone actions on epithelial sodium channel trafficking and cell proliferation. Steroids.
2014;81C:43-8.
E.M. Lofthouse1, S. Brooks1, I.P. Crocker2, J. Glazier2,
M.A. Hanson1, E.D. Johnstone2, N. Panitchob4, C.P. Please3,
C.P. Sibley2, K.L. Widdows2 and B.G. Sengers4,5
1Mother, Pregnancy and Child, University of Southampton,
Southampton, UK, 2Maternal and Fetal Health, University of
Manchester, Manchester, UK, 3Mathematical Institue, Oxford
University, Oxford, UK, 4Faculty of Engineering and the
Environment, University of Southampton, Southampton, UK and
5Life Sciences, University of Southampton, Southampton,
Hampshire, UK
Background: The rates of maternal and fetal blood flow through
the placenta are known to be rate limiting for the delivery of
oxygen to the fetus. However, the extent to which flow may
be limiting for amino acid transfer has not previously been
investigated. Amino acid transport has not been regarded to
be flow dependent. However the activity of amino acid
exchangers and facilitated transporters in the placenta will be
affected by trans-membrane gradients and so their activity
may be linked to flow.
Methods: Human placentas were collected from term, uncomplicated pregnancies delivered at the Princess Anne Hospital,
with written informed consent. The placental uptake and transfer of 14C-phenylalanine was determined in isolated perfused
human placental cotyledons from 5 placentas at different
maternal and fetal flow rates. Maternal flow was set at 10, 14
and 18 ml/min for one hour each. At each maternal flow rate,
a fetal flow rate was set at 3, 6 and 9 ml/min for 20 minutes
each. 14C-phenylalanine concentrations in the maternal and
fetal venous effluent were measured by liquid scintillation
counting. Results were analysed by a two way ANOVA.
Results: Placental uptake of 14C-phenylalanine (mol/min)
increased with increasing maternal (P=0.011) but not fetal flow
(P=0.41). The concentration of 14C-phenylalanine (mol/l) in the
fetal vein was higher at low fetal flow rates (P<0.001) but was
not associated with maternal flow (P=0.41). Total delivery
(mol/min) of 14C-phenylalanine to the fetal-side was not associated with fetal (P=0.95) or maternal flow (P=0.95). There
were no significant interactions between maternal and fetal
flow rate on 14C-phenylalanine concentration, uptake or transfer.
Conclusion: This study suggests that placental transfer of 14Cphenylalanine is independent of maternal or fetal flow and is
limited by other factors. As increased uptake at higher maternal flow rates did not correspond to increased transfer, this
suggests that 14C-phenylalanine transfer may be limited at the
basal plasma membrane.
This work was funded by project grants from the BBSRC
(BB/I011250/1 and BB/I011315/1).
requirements.
This work was supported by the European Molecular Imaging
Doctoral School (EMIDS) and the National Biophotonics
Imaging Platform Ireland (HEA PRTl14).
requirements.
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Oral Communications
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C37
The effect of maternal high fat diet and metformin during
pregnancy on placental expression of amino acid
transporter and circadian clock genes in the mouse
Changes in cerebral structure and vascular reactivity
associated with prolonged exposure to 5,050m
M. Mosquera1,2, H. Thomas1, J.K. Cleal1, F.R. Cagampang1 and
R.M. Lewis1
1Faculty of Medicine, University of Southampton, Southampton,
UK and 2Department of Physiological Sciences, Universidad del
Valle, Cali, Colombia
Introduction: Maternal obesity and diabetes are increasingly
prevalent during pregnancy and may have long-term consequences on the offspring’s health. Previous work has suggested
that in mice a maternal high fat diet can produce an insulin
resistant phenotype in the mother and reduce placental expression of specific amino acid transporter genes and circadian
clock genes. Metformin is an antidiabetic drug that improves
the glycaemic control. This study aims to determine whether
metformin treatment during pregnancy can modify the effect
of a maternal high fat diet on placental expression of amino
acid transporter and circadian clock genes in the mouse.
Methods: Female C57BL6 mice (n=22) were exposed to the
independent variables ‘diet’ (control diet, 7%kcal fat or highfat diet, 45% kcal fat) for 6 weeks before conception and during pregnancy, and ‘treatment’ (control treatment, no metformin or treatment, metformin in drinking water at
250mg/kg/day) during pregnancy. Pregnant dams were killed
by cervical dislocation on day 16 of pregnancy (pregnancy
period in mice is 21 days) and their placentas were collected,
snap frozen and stored at -80°C. These placentas came from
4-5 litters/group (2-3 placentas per dam): control diet with
control treatment (CC, n=12), control diet with metformin
treatment (Cm, n=15), high-fat diet with control treatment
(HFC, n=18), and high-fat diet with metformin treatment (HFm)
(n=21). Expression levels of the circadian clock genes Clock,
Bmal, Per2 and Cry2, and amino acid transporter genes Snat2,
Tat1 and Lat1, were measured by quantitative real-time PCR.
Data were analysed using two-way ANOVA.
Results: Maternal high fat feeding reduced the placental mRNA
expression of Snat2 (p<0.001) and Tat1 (p=0.037). Metformin
treatment reduced the expression of placental Tat1 (p< 0.001).
There was an interaction between maternal diet and metformin treatment for Lat2, with reduced expression in placentas
from dams on control diet and given metformin, and increased
placental expression in high fat fed dams on metformin
(p=0.006). Expression of the circadian clock genes Clock and
Per2 were lower in metformin treated dams, and there was an
interaction between maternal diet and metformin treatment
for Clock (p=0.031).
Conclusions: Our results show that both maternal high fat diet
and metformin treatment during pregnancy are associated
with changes in placental gene expression. However metformin treatment did not reverse changes in gene expression
as a result of feeding pregnant dams with a high fat diet, suggesting that their effects are mediated by different pathways.
Further work is required to identify these pathways.
Supported by the BBSRC and Diabetes UK
requirements.
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G.E. Foster1,3, J. Davies-Thompson2, P.B. Dominelli3,
M.K. Heran4, J. Donnelly5, G.R. duManoir1, P.N. Ainslie1,
A. Rauscher6 and W. Sheel3
1Health and Exercise Science, University of British Columbia,
Kelowna, BC, Canada, 2Ophthalmology and Visual Sciences,
University of British Columbia, Vancouver, BC, Canada, 3School of
Kinesiology, University of British Columbia, Vancouver, BC, Canada,
4Neuroradiology, University of British Columbia, Vancouver, BC,
Canada, 5Clinical Neurosciences, University of Cambridge,
Cambridge, UK and 6Radiology, University of British Columbia,
Vancouver, BC, Canada
Although high altitude exposure results in persistent impairment in neurocognitive function, it is unclear the extent to
which grey and white matter, and vasomotor function are
altered. The purpose of this study was to assess regional cerebrovascular reactivity (CVR), and the volumetric changes in
grey and white matter one week following an expedition to
5,050m. The expedition involved a week in Kathmandu
(1,400m), an 8-day trek from Lukla (2,860m) to the Pyramid
Laboratory at 5,050m where subjects remained for 3 weeks
prior to descending to Kathmandu over a 4-day period. Healthy
humans (n=6, 1 female, age= 31.8 ± 3.7 years; BMI = 27.0 ±
3.6 kg/m2; mean ± SE) underwent baseline and follow-up structural (T1-weighted imaging) and functional (blood oxygen level
dependent, BOLD) MR imaging (3T) at rest and during a CO2
CVR test. An end-tidal forcing system was used to control endtidal gases during two steps of hypocapnia (PETCO2 reduced
by 10 and 5 mmHg from baseline for 2 min by active hyperventilation) and three steps of hypercapnia (PETCO2 increased
by 5, 10, and 15 mmHg; 2 min each step). End-tidal PO2 was
clamped at 100 mmHg. CVR maps (%/mmHg) were generated
by indexing the change in BOLD signal against the change in
PETCO2. The percent change in brain volume was estimated
and global grey and white matter volumes were calculated.
Paired sample t-test and repeated measures ANOVA with
Tukey’s post hoc analysis was used to identify the effect of
high altitude on cerebral structure and CVR throughout
anatomical regions of interest. Following return to sea level,
brain volume was reduced by an estimated -0.4 ± 0.3% (p<0.01)
and global volumetric analysis revealed a selective reduction
in grey matter (-2.6 ± 1.0%; p<0.001) but not white matter (1.3 ± 0.7%; p=0.14). Although global grey matter CVR (PRE:
0.31 ± 0.03; POST: 0.35 ± 0.01 %/mmHg) and white matter
CVR (PRE: 0.18 ± 0.02; POST: 0.19 ± 0.01 %/mmHg) were
unchanged following return to sea-level, CVR was increased in
the brainstem (31 ± 12%, p<0.05), hippocampus (12 ± 3%,
p<0.01), and thalamus (10 ± 3%, p<0.01). Subsequent analysis of regions of positive and negative reactivity revealed that
this effect was the result of an improvement and/or reversal
of negative reactivity to positive reactivity. This result was
notable for grey matter, white matter, brainstem, and hippocampus. Resting minute ventilation, PETO2, PETCO2, and
SpO2 were similar at baseline and follow-up. Our results indicate a loss of grey matter tissue following exposure to high
altitude. In addition, we demonstrate regions of negative blood
flow reactivity in healthy humans that was improved following high altitude exposure. We speculate that regional angiogenesis and/or hypoxia-induced changes in vascular CO2 sensing contribute to improved vascular function in regions of
impaired cerebrovascular reactivity.
Oral Communications
requirements.
C38
Effect of post-prandial lipidaemia on cerebrovascular
function
The present research was supported by the University of South
Wales. Technical support was provided by Dr. Lewis Fall, Mr.
Dean Whitcombe and Mr. Gareth Dunseath.
requirements.
C39
C.J. Marley, D. Hodson, J.V. Brugniaux and D.M. Bailey
Faculty of Life Sciences and Education, University of South Wales,
Pontypridd, UK
Background: The debilitative effect that post-prandial lipidaemia (PPL) has on endothelial function within the systemic
arteries has been well documented (1). However, its impact
on cerebrovascular function is yet to be investigated.
Methods: Twenty young (age = 25 + 6 years) and 20 aged (age
= 67 + 5 years) healthy males participated in the study. Cerebrovascular function and circulating triglycerides were assessed
prior to, and 4 hours after the consumption of a standardised
high-fat meal (2). Middle cerebral artery velocity (MCAv; transcranial Doppler ultrasound), mean arterial pressure (MAP;
photoplethsmography) and end-tidal CO2 (PETCO2; capnography) were continuously recorded throughout each testing session, while venous blood samples were obtained from an
indwelling catheter. Cerebrovascular resistance (CVR) and cerebrovascular conductance (CVC) were calculated as MAP/MCAv
and MCAv/MAP respectively, following 5 minutes seated rest.
Cerebrovascular reactivity to carbon dioxide (CVRCO2) was
assessed in response to 3 minutes of breathing 5% CO2 (balanced air), as described previously (3). Data were analysed
using a 2 way repeated measures ANOVA and Bonferonni corrected paired sample t-tests and independent samples t-tests.
Significance was established at P < 0.05 and data are expressed
as mean + SD.
Results: Circulating triglycerides increased from 0.86 + 0.49
to 2.19 + 1.48 mmol/L in the young and from 1.44 + 0.7 to
2.96 + 1.6 mmol/L in the aged, following the consumption of
the high-fat meal (P < 0.05). PPL impaired CVRCO2 in the aged,
but not in the young (Table 1; P < 0.05). There were no changes
in resting MCAv, MAP, CVR or CVC in either age group.
Conclusion: These findings are the first to identify that the
debilitative effects of PPL on the systemic vasculature extend
to the brain. However, this effect was only present in the aged.
While the underlying mechanisms remain undetermined, these
observations have clear implications to the clinician, given the
established relationship between impaired CVRCO2 and stroke
risk.
Table 1: Changes in cerebrovascular function following a high-fat meal.
Mean + SD; */† = difference within/between age groups (P < 0.05).
Wallace et al. (2010) Int J Clin Pract; 64, 389-403.
Systemic inflammation abolishes the noradrenaline-induced
enhancement of dynamic cerebral autoregulation
R.M. Berg1,5, R.R. Plovsing2, D.M. Bailey3, N. Holstein-Rathlou5
and K. Møller4,1
1Centre of Inflammation and Metabolism, Rigshospitalet,
Copenhagen, Denmark, 2ICU 4131, Rigshospitalet, Copenhagen,
Denmark, 3Neurovascular Research Laboratory, Faculty of Life
Sciences and Education, University of South Wales, Glamorgan,
UK, 4Neuronintesive Care Unit 2093, Rigshospitalet, Copenhagen,
Denmark and 5Renal and Vascular Research Section, University of
Copenhagen, Copennhagen, Denmark
Background. In the healthy state, dynamic cerebral autoregulatory function is enhanced during acute vasopressor-induced
hypertension, which may comprise a neuroprotective mechanism during increases in blood pressure by preventing cerebral hyperperfusion and subsequent vasogenic cerebral
oedema (1). Given that sepsis may be associated with vasogenic cerebral oedema (2), we sought to investigate whether
this adaptive enhancement of dynamic cerebral autoregulation during hypertensionis affected by lipopolysaccharide (LPS)
infusion, a human-experimental model of the systemic inflammatory response during the very early stages of sepsis.
Methods. Nine healthy male volunteers aged 23 (mean; SD,
2) were included. Dynamic cerebral autoregulation was
assessed by transfer function analysis of spontaneous oscillations between mean arterial blood pressure (MAP) and middle cerebral artery blood flow velocity (MCAv) in the low frequency range (0.07–0.20 Hz) using transcranial Doppler
ultrasound (3). This was performed during normotension and
noradrenaline-induced hypertension with an intended MAPincrease of ~30 mmHg both before and after a four-hour LPS
infusion (total dose, 2 ng kg-1). The subjects were poikilopcapnic throughout. Data were analysed by Wilcoxon’s signedrank test and p-values were adjusted by Holm’s sequential Bonferroni correction.
Results. LPS induced a systemic inflammatory response which
was associated with fever and hyperventilation.A concurrent
decrease in gain and an increase in phase, indicating improved
dynamic autoregulation, were present (Table). Before LPS,
hypertension was associated with a decrease in gain with no
effect on phase, whereas neither was affected by hypertension
following LPS infusion (Table).
Conclusion. Dynamic autoregulation is likely enhanced by both
fever and hyperventilation after LPS infusion (4). The present
findings suggest that acute noradrenaline-induced hypertension does cause any further enhancement in dynamic autoregulation under these circumstances. The latter may be an early
sign of underlying cerebrovascular dysfunction, and thus be
relevant to the neuropathology of sepsis.
Patsch et al. (1983) PNAS; 80, 1449-1453.
Bailey et al. (2013) Clin Sci (London); 124, 177-189.
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Dynamic cerebral autoregulation
Sharshar et al. Intensive Care Med 33, 798–806, 2007.
Results: Hypoxia was associated with a decrease in arterial
oxygen content (8.5 ± 0.4 to 7.1 ± 0.4 mmol/L, P < 0.05)
whereas no changes were observed in CPF (51± 9 to 57 ± 7
mL/100g/min, P > 0.05) or platelet count (190 ± 51 vs. 177 ±
76 x 109/L, P > 0.05). A net cerebral output of BDNF (169 ± 300
pg/min/g) and uptake of NO2- (-126 ± 94 nmol/min/g) was
apparent at baseline during normoxia. Hypoxia was shown to
suppress (P < 0.05 vs. normoxia) both BDNF output (25± 94
pg/min/g) and NO2- uptake (-16 ± 47 nmol/min/g) due to a
reduction in JV outflow and RA inflow respectively.
Conclusions: The present findings fail to confirm the systemic
rise in BDNF previously observed during hypoxia (Hubold et
al., 2009) and further exclude the human brain as a contributory source. To the contrary, the cerebral output of BDNF was
shown to decrease during hypoxia and the concomitant reduction in NO2- uptake suggests that its cerebral formation may
be regulated by NO consumption.
Zhang et al. Am J Physiol 274, H233–H241, 1998.
Chen et al. (2013). Biomed Reports 1, 167-176.
Berg et al. Crit Care 17, R238, 2013.
Hubold et al. (2009). J Neural Transm 116, 1221-1225.
requirements.
Kety SS and Schmidt CF (1945). Am J Physiol 143, 53-66.
LPS was infused at a total dose of 2 ng kg-1. MAPsp: MAP spectral power;
MCAsp: MCAv spectral power Data are presented as median (interquartile range). Different from normotension in the same condition (i.e. baseline or LPS), * p < 0.05. Different from the corresponding intervention (i.e.
normotension or hypertension) at baseline, † p < 0.05.
Zhang et al. J Physiol 587, 2567–2577, 2009.
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Xiong et al. (1999). Eur J Neurosci 11, 1567-1576.
requirements.
Hypoxia suppresses release of brain derived neurotrophic
factor from the human brain
D.M. Bailey1, R.M. Berg2, S. Taudorf2, C. Lundby3, B. Pedersen2
and K. Møller2
1Faculty of Life Sciences and Education, University of South Wales,
South Wales, UK, 2Department of Infectious Diseases M7641,
University Hospital Rigshospitalet, Copenhagen, Denmark and
3Institute of Physiology, University of Zurich, Zurich, Switzerland
Background and Aims: Brain derived neurotrophic factor
(BDNF) is a key protein involved in neurogenesis, influencing
the regulation, growth and survival of neurons, learning and
memory (Chen et al., 2013). Its systemic concentration has
been shown to rise in response to acute hypoxia (Hubold et
al., 2009) though to what extent the human brain contributes
remains to be established. Thus, in the present study, we measured the jugular venous to radial arterial concentration difference (JV-RADIFF) to document, for the first time, the transcerebral exchange kinetics of BDNF during hypoxia. We also
chose to measure nitrite (NO2-) exchange since cellular evidence suggests that BDNF formation may be regulated by nitric
oxide (NO) (Xiong et al., 2009).
Methods: Ten healthy males were examined in normoxia and
following 12h passive exposure to poikilocapnic normobaric
hypoxia (~13% O2). Blood samples were obtained simultaneously from the RA and internal JV. Plasma was assayed for NO2using ozone-based chemiluminescence and platelet-poor BDNF
via enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). Global cerebral blood flow (CBF) was determined in the desaturation mode using inhaled nitrous oxide
(5%) as the tracer (Kety and Schmidt, 1945). Cerebral plasma
flow (CPF) was calculated as CBF x (1-haematocrit) and transcerebral exchange calculated as CPF x JV-RADIFF. Following confirmation of distribution normality using Shapiro-Wilk W tests
JV-RADIFF data were analysed with a two factor repeated measures ANOVA and post-hoc Bonferroni-corrected paired samples t-tests. Exchange data were analysed using a paired samples t-test expressed as mean ± standard deviation (SD).
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Exercise intensity is an important determinant of changes
in muscle monocarboxylate transporter abundance in active
men
C. McGinley and D.J. Bishop
Institute of Sport, Exercise and Active Living, Victoria University,
Melbourne, VIC, Australia
High-intensity interval training (HIIT) has often been found to
enhance muscle pH regulation through increases in muscle
buffer capacity (1) and H+ transport proteins (2). During exercise the largest portion of H+ regulation comes from the monocarboxylate transporters (MCT1 and MCT4) (3). The signalling
factors responsible for upregulation of H+ transport proteins
are uncertain (4), although H+ accumulation or H+ efflux may
be important stimuli (5). Maximising high non-mitochondrial
ATP turnover (H+ accumulation) and exercise duration (H+ flux)
may prove to be an optimal stimulus. We hypothesised that
more intervals performed just above the lactate threshold (LT)
would produce greater changes in MCT content than fewer
intervals closer to Wpeak. We also investigated if six weeks of
training cessation would be sufficient for MCT content to return
to baseline.
Using a two-group parallel design, 16 men (23 ± 5 y, mean ±
SD) completed 4 weeks of work-matched HIIT at either 20%
(LO, n=8) or 94% (HI, n=8) of the difference between LT and
Wpeak. VO2peak was measured pre- (0wk) and post-training
(+4wk). Repeated-sprint ability (RSA) was assessed at 0wk,
+4wk, and after 6 weeks training cessation (+10wk). Western
blotting was used to measure MCT1 and MCT4 abundance in
muscle samples taken at 0wk, +2wk, +4wk, and +10wk. Data
were analysed using a two-way mixed design ANOVA. Cohen’s
d effect sizes (ES) are presented as (ES; 90% confidence interval) of the difference scores.
MCT1 abundance (Fig.1) at +4wk increased more for LO than
HI (ES=0.47; -1.35 to 2.28). MCT4 changes followed different
time-courses for the two groups (P=0.007). HI showed an
improvement in MCT4 at +2wk but had lost this improvement
Oral Communications
at +4wk, whereas LO improved only at +4wk (Fig.2). MCT abundance returned to baseline for both groups at +10wk. LT
improved in response to training more for LO than HI (14 ±
13% vs 2 ± 9%; ES=0.36; -0.03 to 0.75). The modest increases
in Wpeak showed no difference between groups (3 ± 4% and 3
± 6% for LO and HI respectively). There was little change in
total work (2 ± 7% and -2 ± 6% for LO and HI respectively) or
fatigue decrement during the RSA test. There were no changes
in VO2peak for either group.
The large but variable increases in MCT1 abundance for both
groups provides unclear evidence as to whether prolonged H+
flux is a key factor for upregulation. Some of the equivocation in MCT4 training adaptation may be explained by differing time-courses of adaptation depending on the intensity of
HIIT. The reduction in MCT4 at +4wk and lack of improvement
in LT compared to LO hint that 4 weeks of HI training may have
caused overreaching. Finally, 6 weeks of training cessation
resulted in MCT protein abundance returning to baseline for
both groups.
Figure 2. MCT4 protein abundance for the high-intensity (HI) and low-intensity (LO) groups pre-training, after 2 and 4 weeks of training, and after a
further 6 weeks of training cessation. Data are expressed relative to a calibration curve of internal standards and normalised to pre-training values. Individual data points are plotted along with geometric mean and 95%
confidence intervals. CV of duplicates 6%.
Edge J, et al. Eur J Appl Physiol. 2006;96(1):97-105.
Burgomaster KA, et al. Am J Physiol Regul Integr Comp Physiol.
2007;292(5):R1970-6.
Juel C, et al. J Physiol. 1999;517(3):633-42.
Thomas C, et al. Am J Physiol Regul Integr Comp Physiol.
2012;302(1):R1-R14.
Weston AR, et al. Acta Physiol Scand. 1996;157(2):211-6.
Supported in part by an Exercise & Sports Science Australia
Applied Sports Science Research Grant.
requirements.
Figure 1. MCT1 protein abundance for the high-intensity (HI) and low-intensity (LO) groups pre-training, after 2 and 4 weeks of training, and after a
further 6 weeks of training cessation. Data are expressed relative to a calibration curve of internal standards and normalised to pre-training values. Individual data points are plotted along with geometric mean and 95%
confidence intervals. CV of duplicates 9%.
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Renal vasoconstriction during isometric calf exercise is
augmented in older, healthy humans
R.C. Drew, C.A. Blaha, M.D. Herr and L.I. Sinoway
Penn State Hershey Heart and Vascular Institute, Pennsylvania
State University College of Medicine, Hershey, PA, USA
Cardiovascular control during exercise is regulated in part by
reflex feedback from muscle afferent nerve fibres (McCloskey
and Mitchell, 1972). Isometric handgrip exercise causes renal
vasoconstriction in humans (Middlekauff et al., 1997) and this
effect is augmented in older, healthy humans (Momen et al.,
2004). However, it is unknown whether healthy ageing augments renal vasoconstriction during isometric calf exercise.
Exaggerated renal vasoconstriction occurs during mild, plantarflexion exercise in peripheral arterial disease patients (Drew
et al., 2013) so elucidating the effect of ageing alone on renal
responses during leg exercise is of clinical relevance. Therefore, this study examined renal vascular responses during isometric, calf exercise in healthy young and older humans. Twelve
young, healthy (Young; 6 men; mean ± SEM 25 ± 1 y) and ten
older, healthy (Older; 4 men; 62 ± 1 y) subjects rested for 5
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minutes, then performed 1.5 minutes of one-legged, isometric, calf exercise at 70% maximal voluntary contraction (MVC).
Renal blood flow velocity (RBFV; Doppler ultrasound), mean
arterial blood pressure (MAP; Finometer) and heart rate (HR;
ECG) were measured. Renal vascular resistance (RVR), an index
of renal vasoconstriction, was calculated as MAP/RBFV. Statistical analysis involved independent t-tests and two-way
ANOVA. Baseline RBFV and HR were similar between Older and
Young (Older: 54.9 ± 3.8 vs. Young: 56.7 ± 2.3 cm.sec-1 and
Older: 60 ± 2 vs. Young: 63 ± 2 b.min-1). Baseline MAP was
significantly higher in Older compared to Young (Older: 100
± 3 vs. Young: 87 ± 2 mmHg; P<0.05), therefore RVR tended
to be higher in Older compared to Young (Older: 1.9 ± 0.1 vs.
Young: 1.6 ± 0.1 arbitrary units; P=0.06). By the end of 70%
MVC exercise, Older demonstrated a similar increase in MAP
(Older: 10 ± 2 vs. Young: 9 ± 2 mmHg) but an attenuated
increase in HR (Older: 6 ± 1 vs. Young: 14 ± 3 b.min-1; P<0.05)
compared to Young. Of note, Older exhibited a greater
decrease in RBFV (Older: -11.2 ± 1.6 vs. Young: -3.8 ± 1.4
cm.sec-1; P<0.05) compared to Young by the end of 70% MVC
exercise, which precipitated an augmented increase in RVR
(Older: 39 ± 5 vs. Young: 20 ± 3 %; P<0.05) in Older compared
to Young by the end of exercise. In conclusion, these findings
suggest that renal vasoconstriction during isometric, calf exercise is augmented in older, healthy humans. This exaggerated
renal vasoconstriction during exercise in older people appears
driven by a greater reduction in renal blood flow rather than
an accentuated increase in systemic blood pressure.
Drew RC et al. (2013). Physiol Rep 1, e00154.
McCloskey DI & Mitchell JH (1972). J Physiol 224, 173-186.
Middlekauff HR et al. (1997). Circ Res 80, 62-68.
Momen A et al. (2004). Am J Physiol Heart Circ Physiol 287, H735-H740.
Supported by National Institutes of Health grants P01
HL096570 (LIS) and UL1 TR000127 (LIS).
requirements.
stant energy tap of 125 mJ delivered at 0.2 Hz via a pendulum
hammer. The MCAPs were measured using sEMG via an extracellular 7 electrode array in a linear arrangement, interelectrode distance 30mm, attached along the long axis of the VL.
The array was centred on the bipolar measurement positions
recommended by SENIAM (Freriks et al 1999). All values are
quoted as mean ± S.E.M.
In 12 female, and 14 male subjects, the latency to the MCAP
was linearly and positively correlated with the proximal distance (p<0.05 Pearson) and on average occurred 1.8 ± 0.5 ms
earlier in females (p<0.005, Unpaired t-test). Linear regression
yielded a mean MNCV that was 9.8±4.4 m s-1 faster in the
females (64.9±2.6 m s-1 ) than in the males (54.0±3.4 m s-1 )
(p<0.05, Unpaired t-test). A simple linear model, in which all
electrophysiological events prior to post-spinal excitation of
L4 alpha-motorneurons were lumped together, and which was
required to be 0.5±0.05 ms slower in males, readily accounted
for the relationship between the gender difference in latency
and proximal electrode distance when the differences in the
motor nerve conduction path length and its associated MNCV
between the two genders were considered. A model in which
the genders had the same MNCV could not describe the gender-specific change in PR latency with conduction distance.
In conclusion the faster latency for the VL component of the
patella stretch reflex in human females can be partly, if not
entirely, accounted for by gender differences in the alphamotor neuron conduction velocity. Whether the later arises
through differences in internodal distance, extent of myelination, or ion channel density remains unknown.
Vickery, H. S., & Smith, P. A. (2012). Surface electromyography reveals
males have a slower patellar reflex than females. Journal of Electromyography and Kinesiology, 22(6), 990-996.
Freriks, B., Merletti, R., Stegeman, D., Blok, J., Rau, G., DisselhorstKlug, C., & Hägg, G. (1999). European recommendations for surface
electromyography(pp. 1-122). H. J. Hermens (Ed.). The Netherlands:
Roessingh Research and Development.
requirements.
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C44
Alpha motor neurons in the human femoral nerve have a
faster conduction velocity in females than males
Telomeric region of mouse chromosome 10 affects
hypertrophy of fast-twitch muscle
P.A. Smith, A. Chana, S. Marlow, R. Nzewi and J. LLewelynDavies
A. Kilikevicius1, J. Baltusnikas1, T. Venckunas1 and A. Lionikas1,2
1Institute
University of Nottingham, Nottingham, UK
Surface electromyography (sEMG) has demonstrated that the
electrophysiological component of the patella stretch reflex
(PR) for the vastus lateralis (VL) in human females is significantly faster than that for males; an effect that cannot be
accounted for by differences in stature between the two genders (Vickery and Smith 2013). One possible explanation for
this difference is that the nerve conduction velocities for the
afferent and efferent pathways of this mono synaptic spinal
reflex is sexually dimorphic. We have investigated the later
possibility. Caucasian subjects of both genders with no known
neurophysiological deficits or injury were recruited from the
student population (Age range 20-22). Alpha-motor neuron
conduction velocity (MNCV) was determined indirectly by the
measurement of the latencies to the appearance of the muscle compound action potentials (MCAP) that are evoked in the
VL at various distances down the muscle, during the PR, due
to alpha-motor nerve innervation. The PR was elicited by a con-
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of Sport Sciences and Innovation, Lithuanian Sports
University, Kaunas, Lithuania and 2School of Medical Sciences,
College of Life Sciences & Medicine, University of Aberdeen,
Aberdeen, UK
Insight into genetics underlying muscle growth can lead to
pharmacological targets for treatment of muscle wasting conditions. The telomeric region of chromosome 10, between
markers rs3676616 and D10Utsw1, contains genes that might
be associated with signaling pathways involved in muscle
growth, Stat2 (Wang et al, 2008) and metabolism, Cs (Ratkevicius et al, 2010). The non-synonymous polymorphisms in
Stat2, and Cs genes are present between the commonly used
laboratory mouse strains, C57BL/6J (B6) and A/J, and can provide a useful research model. The aim of the study was to investigate the role of this genomic region in the compensatory
skeletal muscle growth.
Synergist ablation method was used in the B6, and congenic
B6.A-(rs3676616-D10Utsw1)/Kjn (B6.A) strain, and in the offspring (F1) of the cross between these two strains. Genotypic
Oral Communications
difference between the B6 and B6.A strains is restricted to a
~1.5 Mb region, between rs3676616 and D10Utsw1, where
the A/J allele was introgressed in the congenic strain. Males
(M; n=7-8 per strain) and females (F; n=5-9 per strain) at 12
weeks of age were used. A compensatory growth of soleus
and plantaris muscles in the left hind limb was induced by ablation of gastrocnemius. Right hind limb remained intact and
served as control. Eight weeks later animals were sacrificed,
soleus and plantaris muscles were removed from both legs
and weighed to a precision of 0.1 mg. Body weight (BW) before
ablation and at the end of experiment was measured. Data
(presented as mean ± SD) were analyzed using 2-way ANOVA
(genotype and sex) with repeated measures (treatment) where
appropriate.
The genotype did not affect (p>0.05) BW (B6: (M) 26.7±2.0
g, (F) 20.5±0.7 g; F1: (M) 25.0±1.3 g, (F) 20.9±0.8 g; B6.A:
(M) 26.0±1.3 g, (F) 21.4±1 g), soleus (B6: (M) 8.8±0.9 mg, (F)
6.8±0.6 mg; F1: (M) 8.3±0.7 mg, (F) 6.6±0.6 mg; B6.A: (M)
8.9±0.6 mg, (F) 6.9±0.7 mg) or plantaris (B6: (M) 17.9±1.3 mg,
(F) 13.0±0.9 mg; F1: (M) 17.4±1.0 mg, (F) 13.7±1.2 mg; B6.A:
(M) 18.3±1.7 mg, (F) 13.4±0.8 mg) muscle weight. During the
course of the study, BW of mice significantly increased
(p<0.001), but did not differ between strains. It indicates that
surgical intervention did not affect animal health status
adversely. The overloaded muscles were significantly heavier
(p<0.001). Soleus muscle increased by 1.5 fold independently
of genotype (p=0.64). The increase in plantaris weight was
affected by genotype (Genotype x Treatment, p<0.0001). The
B6 animals gained more weight (p<0.001), 2.2-fold, compared
to the F1 or B6.A, 1.8-fold each. There was no significant sex
effect or genotype by sex interaction in soleus or plantaris
response to ablation (p>0.05).
Our results suggest that the telomeric region of mouse chromosome 10 contains important regulator(s) of hypertrophy
in the muscles dominated by the fast twitch fibres.
Wang K, Wang C, Xiao F, Wang H, Wu Z. JAK2/STAT2/STAT3 are
required for myogenic differentiation. J Biol Chem. 2008 Dec
5;283(49):34029-36.
Ratkevicius A, Carroll AM, Kilikevicius A, Venckunas T, McDermott KT,
Gray SR, Wackerhage H, Lionikas A. H55N polymorphism as a likely
cause of variation in citrate synthase activity of mouse skeletal muscle. Physiol Genomics. 2010 Oct;42A(2):96-102.
requirements.
C45
The influence of glucagon-like-peptide-1 receptor single
nucleotide polymorphisms on gastric emptying rate in
caucasian men - a pilot study
tide-1 receptor (GLP-1R) genetic variation on gastric emptying rate in mice[3]. This pilot study investigated the effect of
GLP-1R single nucleotide polymorphisms (SNPs) on the rate of
gastric emptying in humans.
Forty-eight healthy non-smoking UK Caucasian males aged 1835 y (mean ± SD height 178.2 ± 6.9 cm, weight 75.8 ± 11.2 kg,
BMI 24 ± 3, body fat 19 ± 6%) took part in this investigation.
Following an overnight fast, participants completed a single
experimental trial involving the ingestion of 595 ml of a 6%
glucose solution containing 100 mg 13C sodium acetate. Gastric emptying rate of the solution was measured by the 13C
breath test whereby breath samples were collected at baseline and 10 min intervals for 60 min. A venous blood sample
was obtained from each participant for genetic analysis.
Twenty-seven haplotype-tagging SNPs in the GLP-1R locus were
identified from the HapMap database and genotyped using
Sequenom MassARRAY iPLEX GOLD analysis. Gastric emptying results were analysed by genotype and phenotype using
Kruskal-Wallace and Wilcoxon statistical tests, respectively.
Values are median [quartiles].
A significant effect of genotype on time of maximum emptying rate (Tlag) was seen for neighbouring SNPs rs742764 and
rs2254336. For SNP rs742764, Tlag was faster in genotype CC
than in genotype TT and TC (35 [30-36] vs. 41 [37-46] and 41
[39-45] min; P<0.01). For SNP rs2254336, Tlag was slower in
genotype AA than in genotype TT and TA (43 [39-49] vs. 36
[34-41] and 39 [35-42] min; P<0.05). An effect of phenotype
was also seen for SNP rs2254336 where Tlag was slower in
homozygotes of the major allele A than in participants with
the minor allele T (43 [39-49] vs. 39 [34-41] min; P<0.05). Further effects of phenotype were seen for SNP rs2268657 where
Tlag was faster in homozygotes of the major allele A than in
participants with the minor allele G (37 [34-39] vs. 41 [37-45]
min; P<0.05) and for SNP rs9283907 where half emptying time
(T½) was slower in homozygotes of the major allele G than in
participants with the minor allele A (67 [59-82] vs. 55 [53-61]
min; P<0.05).
The results of this study suggest that gastric emptying rate
may be influenced by SNPs within the GLP-1R gene. Replication of this pilot study in larger cohorts is required to confirm
the contribution of GLP-1R gene variation to gastric emptying rate.
Yau A et al (2013) Int J Sport Nutr Exerc Metab 23, S13
Wettergren A et al (1993) Dig Dis Sci 38, 665-673
Kumar KG et al (2008) Am J Physiol Regul Integr Comp Physiol 294, R362371.
requirements.
C46
A. Yau1, J. McLaughlin2, R.J. Maughan3, W. Gilmore1,
J.J. Ashworth1 and G. Evans1
1School of Healthcare Science, Manchester Metropolitan University,
Mancester, UK, 2Institute of Inflammation and Repair, University
of Manchester, Manchester, UK and 3School of Sport, Exercise and
Health Science, Loughborough University, Loughborough, UK
Gastric emptying limits the rate of delivery of ingested nutrients into the small intestine. The emptying rate of a glucose
solution has been shown to be highly variable between individuals[1]. The gastrointestinal hormone glucagon-like peptide-1, which is secreted following carbohydrate ingestion, has
been shown to exert inhibitory effects on gastric emptying[2].
Previous work has shown an influence of glucagon-like pep-
A change in physical activity level affects leukocyte DNA
methylation of genes implicated in cardiovascular disease
in the elderly
B. Shaw1,2, W.C. Leung2, H.S. Tapp2, A.L. Fitzpatrick3,
J.M. Saxton1 and N.J. Belshaw2
1School
of Rehabilitation Sciences, University of East Anglia,
Norwich, UK, 2Institute of Food Research, Norwich Research Park,
Norwich, UK and 3School of Public Health, University of
Washington, Seattle, WA, USA
Introduction: Increased physical activity (PA) reduces cardiovascular disease (CVD) risk but the molecular mechanisms are
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not well understood. Previous studies indicate that physical
activity (PA) can exert epigenetic effects by altering gene-specific DNA methylation. The aim of the present study was to
investigate the relationship between PA and DNA methylation
of genes known to be implicated in CVD.
Methods: Blood samples were collected at baseline and after
8 years from 253 white females (70.7 ± 0.3 yrs) and 137 white
males (71.7 ± 0.4 yrs) in the Cardiovascular Health Study. Leukocyte DNA was bisulphite-modified, and methylation determined by quantitative methylation-specific PCR (QMSP).
Anthropometric and self-report physical activity data were collected at both time points and the latter used to calculate physical activity energy expenditure (PAEE; kcal/wk). Participants
were stratified by extent of change in PAEE over the 8 years;
a decrease by >500 kcal/wk (DEC), no overall change (NOC)
and an increase by >500 kcal/wk. N-way ANCOVA with posthoc multiple t-tests with the Bonferroni correction was used
to locate differences. All results are reported as mean ± S.E.M
and deemed significant if P < 0.05.
Results: A decrease in PAEE of 500 kcal/wk or more resulted
in no change in methylation, whereas those who maintained
or increased PAEE by 500 kcal/wk or more had significantly
increased methylation of TNF (Figure 1a). Similarly, those who
increased PAEE by 500 kcal/wk had significantly reduced methylation of IL10 compared with those who decreased PAEE by
500 kcal/wk (Figure 1b). These changes were independent of
any effects of ageing or changes in waist circumference.
Discussion: Changes in PAEE of more than 500 kcal/wk were
significantly associated with the differential methylation of
TNF and IL10 in leukocytes of aged subjects. These genes are
strongly implicated in CVD risk. Importantly, PAEE was associated with a change in methylation that would presumably
benefit cardiovascular health. These results suggest that PAinduced effects on DNA methylation could underpin the
reported associations between PA and CVD risk..
Swift DL, Lavie CJ, Johannsen NM, Arena R, Earnest CP, O’Keefe JH,
Milani RV, Blair SN, Church TS. Physical activity, cardiorespiratory fitness, and exercise training in primary and secondary coronary prevention. Circ J. 2013;77:281-292.
Nakajima K, Takeoka M, Mori M, Hashimoto S, Sakurai A, Nose H,
Higuchi K, Itano N, Shiohara M, Oh T, Taniguchi S. Exercise effects on
methylation of ASC gene. International Journal of Sports Medicine.
2010;31:671-675
Cassatella MA, Meda L, Bonora S, Ceska M, Constantin G. Interleukin10 (Il-10) inhibits the release of proinflammatory cytokines from
human polymorphonuclear leukocytes - evidence for an autocrine role
of tumor-necrosis-factor and IL-1-beta in mediating the production of
IL-8 triggered by lipopolysaccharide. J Exp Med. 1993;178:2207-2211.
O’Donovan G, Blazevich AJ, Boreham C, Cooper AR, Crank H, Ekelund
U, Fox KR, Gately P, Giles-Corti B, Gill JM, Hamer M, McDermott I, Murphy M, Mutrie N, Reilly JJ, Saxton JM, Stamatakis E. The ABC of physical activity for health: A consensus statement from the British Association of Sport and Exercise Sciences. J Sports Sci. 2010;28:573-591
Bruunsgaard H, Skinhoj P, Pedersen AN, Schroll M, Pedersen BK. Ageing, tumour necrosis factor-alpha (TNF-alpha) and atherosclerosis. Clin
Exp Immunol. 2000;121:255-260.
This research was supported by grants 1R21HL104317-01A1
and HL080295 along with contracts HHSN268201200036C,
N01HC85239, N01 HC55222, N01HC85079, N01HC85080,
N01HC85081, N01HC85082, N01HC85083, N01HC85086
from the National Heart, Lung, and Blood Institute (NHLBI),
with additional contribution from the National Institute of
Neurological Disorders and Stroke (NINDS). Additional support
was provided by AG023629 from the National Institute on
Aging (NIA). A full list of principal CHS investigators and
institutions can be found at http://www.chs-nhlbi.org. This
work was also supported by the BBSRC’s core strategic grant
to the Institute of Food Research’s Gut Health and Food Safety
(BB/J004529/1) programme.
requirements.
C47
Effect of resveratrol on skeletal muscle viability and
adaptations
R. McCormick, A. Vasilaki, A. McArdle and M.J. Jackson
Skeletal Muscle Pathophysiology Research Group, Institute of
Ageing and Chronic Disease, University of Liverpool, Liverpool,
merseyside, UK
Figure 1a Maintaining (NOC) or increasing (INC) PAEE by >500 kcal/wk significantly increases TNF methylation compared with decreasing PAEE by >
500 kcal/wk (p < 0.01).
Figure 1b Increasing (INC) PAEE by >500 kcal/wk significantly decreases
IL10 methylation compared with decreasing PAEE by > 500 kcal/wk (p <
0.05).
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Skeletal muscle mass and function deteriorates considerably
with increasing age, although the mechanisms responsible for
this deterioration are poorly understood. One theory is that
as we age there is an increased state of inflammation and oxidation status of cells and that these changes are major contributors to age-related muscle degeneration. Dietary polyphenols (secondary metabolites of plants) have been proposed as
potential nutritional interventions which may modulate inflammation; a number of them have been shown to have antioxidant and anti-inflammatory properties. In particular, Resveratrol, a polyphenol found in red wine, has been shown to be
both anti-inflammatory and anti-oxidising through the up-regulation of the NAD dependent deacetylase sirtuin 1 (SIRT1).
This increase in SIRT1 activity has been shown to, in turn,
increase the production of anti-oxidant enzymes.
The aim of this study was to determine physiologically relevant concentrations of resveratrol that are functional but not
toxic to primary cells isolated from rat skeletal muscle. Further
Oral Communications
work will then use these optimum concentrations to determine the effect of this intervention on muscle function using
a 3D muscle construct.
A Syto10/DeadRed cell viability assay showed that 0.1 and 1μM
of resveratrol did not affect the viability of mononuclear
(myoblasts) or polynucleated (myotubes) primary skeletal muscle cells, up to and including 48hrs of treatment (n=6). 10μM
of resveratrol did not affect the viability of myoblasts after
48hrs; however, cell counts showed decreased cell number
(n=6; p<0.05, Students t-test). Viability of myotubes decreased
after 5days of treatment with 10μM of resveratrol (n=3).
Western blotting analysis showed that when skeletal muscle
cells were treated up to 24hrs with 0.1 and 1μM of resveratrol
SIRT1 was up regulated. 10μM of resveratrol did not increase
SIRT1 expression at any time points. When treated for up to
24 hrs with 0.1,1 and 10μM of resveratrol the anti-oxidant catalase was up-regulated dose dependently. 0.1,1 and 10μM of
resveratrol were all able to up-regulate MnSOD when treated
for 3hrs, however only 1 and 10μM was able to withstand this
up regulation for up to 12hrs. 24hrs resveratrol treatment
decreased MnSOD expression at all concentrations.
This data suggest that at 1μM and 10μM resveratrol is consistently functional in skeletal muscle cells. Since 10μM of resveratrol did not increase SIRT1 expression we hypothesise that
the up-regulation of the anti-oxidant enzymes seen following
resveratrol treatment is via different mechanisms than SIRT1
activation.
The authors would like to thank BBSRC DRINC for funding this
study.
requirements.
C48
Judgements of lifted objects and grasp aperture
A.A. Butler and S.C. Gandevia
Neuroscience Research Australia, Sydney, NSW, Australia
Accurate proprioceptive information and body representations
are required to accomplish even simple tasks, such as lifting a
cup (1-3). However it is unclear how the properties of lifted
objects affect the ‘representation’ of the hand involved in the
task. Specifically, is the perceived grasp aperture of the human
hand influenced by the size and weight of lifted objects? Vision
was excluded in all experiments. In experiment 1, 14 subjects
reported the perceived weight of lifted canisters that were of
different sizes (5.2, 6.6, 10 cm width) but the same weight
(600 g). In experiment 2, these subjects reported perceived
grasp aperture while lifting canisters that were of the same
size (width 6.6 cm) but varied weights (300, 600, 900, 1200
g). In experiment 3, 16 subjects reported perceived grasp aperture as they grasped a stationary canister at varied force levels. This investigated the role of both efferent and afferent
inputs in grasp aperture perception (4). When canisters of the
same weight but different widths were lifted (experiment 1),
perceived weight decreased by ~50% from the narrowest to
the widest (p < 0.001). When canisters of the same width but
different weights were lifted (experiment 2), perceived grasp
aperture decreased as the weight of the canister increased.
However this decrease was only 5% across the range of weights
(p < 0.001). In experiment 3, perceived grasp aperture did not
change with the level of grasp force (p > 0.05). The effect of
object properties on hand representation was much smaller
than its effect on weight perception. This small effect is not
the result of varied grasp forces. Overall, despite a distorted
perception of the weight of a grasped object, the central nervous system maintains a reasonably accurate perception of the
posture and representation of the grasping hand over a fourfold range of object weights. Presumably this accuracy in perception of grasp aperture derives from peripheral proprioceptive signals.
1. Flanagan JR & Johansson RS (2002) Encyclopedia of the Human Brain,
399-414.
2. Proske U & Gandevia SC (2012) Physiol Rev, 92, 1651-1697.
3. Longo MR, Azañón E, Haggard P (2010) Neuropsychologia, 48, 655668.
4. Gandevia SC, Smith JL, Crawford M, Proske U, Taylor JL (2006) J Physiol, 571, 703-710.
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C49
Role of long chain free fatty acid receptors in incretin
hormone secretion
C.E. Moss1,2, E. Diakogiannaki2, C. Lenaghan3, D. Smith3,
F.M. Gribble2 and F. Reimann2
1School
of Life Science, University of Nottingham, Nottingham,
UK, 2Clinical Biochemistry, University of Cambridge, Cambridge,
UK and 3CVMD iMed/Bioscience, AstraZeneca, Mölndal, Sweden
Background and aims: Therapies based on the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are widely used in the
treatment of diabetes. Clinically used agents are either
injectable peptidergic GLP-1 receptor agonists or orally available dipeptidylpeptidase-4 inhibitors, which prolong the halflife of the endogenous hormones. Enhancing endogenous hormone secretion has not yet been exploited, but appears
promising, as enhanced postprandial GLP-1 secretion after
Roux-en-Y gastric bypass surgery correlates with substantially
improved glycaemia. Two potential pharmaceutical targets to
increase GLP-1 secretion are the free fatty acid (FFA) receptors
1 and 4, present in both GLP-1 secreting L-, and GIP secreting
K-cells. In this study we investigated the role of FFA1 and 4 in
lipid stimulated GIP and GLP-1 secretion in mice.
Methods: Secretion, expressed as % of total hormone content,
was assessed in 2 h incubations of murine duodenal and colonic
cell cultures treated with post prandial micelles (PPM) (containing (mM): oleic acid (0.2), 2-monooleoyl-glycerol (0.07),
L-α-lysophosphatidylcholine (0.07), cholesterol (0.017), taurocholate (0.7)) or with taurocholate (2 mM) +/- oleic acid (0.4
mM). Responses were compared between wild-type and FFA1
or FFA4 knock-out (-/-) animals. In FFA4-/- and +/+ mice plasma
hormone was assessed after an intragastric lipid gavage of 1:1
corn and olive oils. All animal experiments were carried out
with ethical approval and in accordance with UK legislation.
Results: In duodenal cultures PPM increased GIP secretion from
1.6±0.2% to 13.4±1.0% (n=32), and GLP-1 secretion from
5.3±0.4% to 47.9±1.8% (n=24). PPM stimulated secretion was
reduced in both FFA1-/- (GIP: 1.1±0.1% to 7.5±0.7% (n=11);
GLP-1: 3.9±0.3% to 20.5±0.8% (n=11)) and FFA4-/- cultures (GIP:
1.5±0.3% to 7.4±0.9% (n=24) and GLP-1: 2.9±0.4% to
31.3±2.3%; (n=13) respectively). Taurocholate increased GIP
and GLP-1 secretion similarly from wild type and knockout cultures, but the additional presence of oleic acid, which increased
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Oral Communications
GIP and GLP-1 secretion a further 1.7 and 2.2-fold from wild
type cultures, was ineffective in FFA1-/- and FFA4-/- cultures.
Somewhat surprisingly, PPM stimulated GLP-1 secretion similarly from colonic cultures established from wild-type or FFA1/- or FFA4-/- mice, perhaps reflecting the stimulatory actions
of other PPM components. A significant reduction in plasma
GIP elevation was observed in FFA4-/- animals after lipid gavage (n=21), whilst plasma GLP-1 elevations tended to be lower,
but this failed to reach significance.
Conclusions: Both FFA1 and FFA4 underlie lipid stimulated
incretin secretion and knock-out of either receptor impaired
both GLP-1 and GIP secretion with limited receptor redundancy.
not improve glucose tolerance in Cm/HF and HFm/HF offspring. μ-CT scan showed that BF% in C/HF, HF/C and HF/HF
was increased relative to C/C by 1.66-fold (p<0.001), 1.34-fold
(p<0.01) and 1.87-fold (p<0.001), respectively. Maternal metformin treatment decreased BF% by 0.83-fold (p<0.05) in
HFm/C compared to HF/C animals.
Conclusions: Maternal metformin treatment in the obese
mother protected female offspring from increased adiposity
and reduced their fasting blood glucose. Maternal metformin
treatment may therefore reduce the long-term effects of
maternal obesity during pregnancy on offspring metabolic
health.
Transgenic mice were generated by AstraZeneca.
Supported by Diabetes UK
requirements.
requirements.
C50
C51
Metformin treatment in obese pregnant mice protects adult
offspring from increased adiposity and elevated fasting
blood glucose
Decreased energy expenditure due to maternal diet induced
obesity is prevented by maternal supplementation with a
soluble fibre (polydextrose)
H. Thomas, C.D. Byrne and F.R. Cagampang
X. Maragkoudaki, M. Naylor, J.M. Pombo, L. Poston and
P.D. Taylor
Institute of Developmental Sciences, University of Southampton
Faculty of Medicine, Southampton, UK
Introduction: Prevalence of obesity and type 2 diabetes during pregnancy is increasing, and both conditions may adversely
influence later health in the developing offspring. The anti-diabetic drug metformin is commonly prescribed for diabetes during pregnancy, and is considered safe for the developing fetus.
However, it’s uncertain whether metformin during pregnancy
affects risk of disease in the offspring in adult life.
Aims: To investigate the effects of metformin treatment of
obese mouse dams during pregnancy on their adult offspring
glucose metabolism and adiposity.
Methods: Female C57/BL6J mice (n=18) were fed a control diet
(C, 7% kcal fat) or a high-fat diet (HF, 45% kcal fat) 6 weeks
prior to mating and through pregnancy. A subset of pregnant
dams on C or HF diet were given metformin hydrochloride in
drinking water (250mg/kg bodyweight per day) through pregnancy and lactation. Female offspring were weaned onto C or
HF diet, creating 8 groups (n=5-8 per group): from untreated
dams C/C, C/HF, HF/C, & HF/HF and from metformin-treated
dams Cm/C, Cm/HF, HFm/C, & HFm/HF. Glucose tolerance in
28 week old offspring was assessed by 2 hour intraperitoneal
glucose tolerance test. Fasting concentrations and area under
the curve (AUCs) are reported. At 30 weeks of age, offspring
body fat percentage (BF%) was assessed by μ-CT scan. Data
reported as means ± SEM, or fold differences. Statistical analysis was performed by two-way ANOVA.
Results: Offspring bodyweight at 30 weeks of age was significantly increased in C/HF and HF/HF compared to C/C
(24.71±0.53g vs 34.13±1.07g, p<0.001, and 44.14 ± 1.67g,
p<0.001, respectively). Maternal metformin during pregnancy
increased bodyweight in Cm/HF vs C/HF (39.9±1g vs
34.13±1.07g, p< 0.01) but not in HFm/HF relative to HF/HF
animals. Fasting blood glucose (FBG) was significantly higher
by 1.34-fold (p<0.05) and 1.53-fold (p<0.05) in C/HF and HF/HF
offspring groups respectively compared with C/C mice. FBG in
HFm/HF offspring was significantly reduced vs HF/HF (0.72fold, p<0.05). Glucose intolerance (AUC) was raised in C/HF
offspring by 1.48-fold (p<0.01) and 2.16-fold (p<0.0001) in
HF/HF compared with C/C animals. Metformin treatment did
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King’s College London, England, Surrey, UK
Introduction: Obesity now affects nearly 1 in 3 adults in the
UK. It is estimated that 20% of pregnant women are obese.
Increasing evidence associate obesity in pregnancy with susceptibility to obesity and metabolic syndrome in the child.
Here we employ an established mouse model of maternal obesity [1] to investigate energy balance and glucose metabolism
in the offspring. Polydextrose (PDX) has been shown to
improve glucose metabolism [2] and, therefore may be beneficial in obese pregnancy.
Hypothesis: We hypothesised that (a) maternal obesity has
adverse effects on offspring energy balance and glucose
metabolism and that (b) these adverse effects will be prevented by supplementation of the maternal diet with PDX.
Moreover, we investigated whether PDX supplementation in
obese pregnancy is protective against the adverse influences
of an obesogenic dietary exposure in adulthood.
Methods: Female mice were fed a control or an obesogenic
diet [1], 6-weeks before mating and throughout pregnancy
and lactation. A cohort of the obese dams was assigned to supplementation with 5% PDX in pregnancy and lactation. Offspring were weaned onto control diet. At 3 and 6 months of
age energy intake (EI), energy expenditure (EE) and Respiratory Exchange Ratio (RER) were measured by indirect calorimetry, (Labmaster, TSE) and glucose-tolerance-tests were performed. At 3 months some of the animals were challenged for
3-weeks with an obesogenic diet before re-estimation of EI,
EE, and RER.
Results: At 3 months of age, offspring of obese dams (OffOb)
metabolic parameters did not differ from offspring of control
dams (OffCon). At 6 months OffOb were heavier (P<0.01), had
lower RER (P<0.05) and lower EE (P<0.001) compared to OffCon. OffOb had impaired glucose metabolism compared to
OffCon (P<0.05). Maternal supplementation with PDX prevented these defects. Following 3 weeks obesogenic dietary
challenge OffObs demonstrated hyperphagia and greater
increase in bodyweight than controls (P<0.05),which was prevented by maternal PDX supplementation. PDX also normal-
Oral Communications
ized the reduction in EE observed in OffOb after the obesogenic dietary challenge (P< 0.05).
Conclusions: Maternal obesity was associated with increased
bodyweight in offspring at 6 months of age, which we attribute to decreased EE. Maternal administration of PDX prevented
the effects of maternal obesity on offspring energy balance
and glucose metabolism. Maternal supplementation with PDX
protected OffObs from developing hyperphagia and decreased
EE, which lead to increased weight gain following exposure to
obesogenic diet in adulthood. Maternal obesity adversely influences offspring energy balance, which is prevented by maternal intervention with PDX. PDX may, therefore, provide a
potential therapeutic intervention in preventing the transgenerational acceleration of obesity.
Samuelsson, A.M., et al., Diet-induced obesity in female mice leads to
offspring hyperphagia, adiposity, hypertension, and insulin resistance:
a novel murine model of developmental programming. Hypertension,
2008. 51(2): p. 383-92.
Shimomura, Y., et al., Attenuated response of the serum triglyceride
concentration to ingestion of a chocolate containing polydextrose and
lactitol in place of sugar. Biosci Biotechnol Biochem, 2005. 69(10): p.
1819-23.
requirements.
C52
Kisspeptin signalling is required to maintain progesterone
levels during mouse pregnancy
W. Colledge, V. Kyle, S. Yeo and A. Herreboudt
PDN, University of Cambridge, Cambridge, UK
Kisspeptin neuropeptides stimulate release of GnRH to initiate puberty and maintain mammalian fertility. Kisspeptins are
encoded by the Kiss1 gene and directly stimulate GnRH neurons via the G-protein coupled receptor, GPR54. Transgenic
mice with inactivating mutations of Kiss1 or Gpr54 are sterile
and have hypogonadotrophic hypogonadism caused by a failure to secrete GnRH. The aim of this study was to determine
whether the sterility of mutant female mice was solely caused
by absence of central kisspeptin signalling or whether there
are additional defects in the ovaries. To study this, we used a
hormone replacement protocol to mature the reproductive
axis of Kiss1 and Gpr54 mutant mice. The mutant mice were
bred with fertile males and evaluated for maintenance of pregnancy. Although, the mutant mice were induced to ovulate,
none of the mice maintained pregnancy past E6.5 of gestation. It was found that the mutant mice had significantly lower
levels of progesterone compared to wild-type pregnant mice.
Ovary transplantation studies showed that mutant ovaries
implanted into wild-type mice could support pregnancy to
term indicating that there was no intrinsic defect in the mutant
ovaries. Progesterone replacement allowed pregnancy to
progress normally past E6.5. We hypothesise that the failure
of the mutant mice to maintain progesterone levels during
pregnancy is caused by inadequate luteotrophic stimulation
of the corpus luteum due to central kisspeptin signalling
defects.
This work was funded by a BBSRC Case Award (BB/FO1936X/1).
We would like to thank Xavier d’Anglemont de Tassigny for
help with these experiments.
requirements.
C53
Anti-inflammotory effects of Nesfatin-1 in rats with acetic
acid induced colitis and underlying mechanism
C. Cantali Ozturk1, S. Oktay2, M. Yuksel3, D. Akakin4, A. Yarat2
and O. Kasimay Cakir1
1Department of Physiology, Marmara University School of
Medicine, Istanbul, Istanbul, Turkey, 2Department of Biochemistry,
Marmara University Faculty of Dentistry, Istanbul, Turkey,
3Marmara University Vocational School of Health Related Services,
Istanbul, Turkey and 4Department of Histology and Embryology,
Marmara University School of Medicine, Istanbul, Turkey
Mucosal balance impairment, bacterial overproliferation,
cytokines, inflammatory mediators are known as responsible
for inflammatory bowel disease. Besides known anorexigenic,
neuroprotective, and anti-apoptotic effects, the major effect
of nesfatin-1 on colitis is unknown. Our aim was to investigate the possible anti-inflammatory effects of nesfatin-1 in
acetic acid induced colitis model and the underlying mechanism. Male Sprague-Dawley rats (n=48) were used. The rats
were anesthetized by intraperitoneal ketamine (100mg/kg)
and chlorpromazine (10mg/kg). For nesfatin-1 and antagonist
applications some of the rats were intracerebroventricularly
(icv) cannulated. In colitis groups, intrarectally 4% acetic acid
solution (1 ml) and 10 minutes later icv nesfatin-1 (0.05 μg/5
μl) or vehicle (5 μl) were administered. Treatments continued
for 3 days. In control group physiological saline solution was
used intrarectally. In the second part of the study, to identify
the underlying effective mechanism of Nesfatin-1, following
colitis induction; icv atosiban (oxytocin receptor antagonist,
3 μg/rat) and nesfatin-1 were administered respectively for 3
days. On the fourth day, rats were decapitated and colon tissues were sampled. Macroscopical and microscopical damage
scores of distal colon, and colonic tissue malondialdehyde
(MDA), glutathione (GSH), myeloperoxidase (MPO), superoxide dismutase, catalase (CAT), luminol and lucigenin chemiluminescence measurements were made. GraphPad Prism 5.0
was used for statistical analysis. All data were expressed as
mean ± SEM. Groups of data were compared with Mann-Whitney U non parametric test and Student t-tests. In colitis group,
MPO activity, MDA levels, luminol and lucigenin chemiluminescence measurements, macroscopic and microscopic damage scores were increased and antioxidant GSH and CAT levels were diminished compared to control group
(p<0.05-0.001). Oxidant damage due to colitis model was
decreased with Nesfatin-1 treatment, and the anti-inflammatory effects of nesfatin-1 were observed with alleviated microscopical (p<0.01) and macroscopical (p<0.001) damage scores.
Additionally, the increased MPO and MDA levels, luminol and
lucigenin chemiluminescence measurements in colitis group
were declined with Nesfatin-1 treatment (p<0.05-0.001). Nesfatin-1 might show this effect by inhibiting neutrophil infiltration and by decreasing formation of free oxygen radicals. Atosiban administration alleviated the protective effect of nesfatin-1
from macroscopic, microscopic and oxidant damage parameters and lipid peroxidation (p<0.05-0.001). The results of the
study suggest that nesfatin-1 had a protective effect from colitis induction, and the anti-inflammatory and antioxidant effects
of nesfatin-1 on colitis might occur via oxytocin receptors.
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Atsuchi K, Asakawa A, Ushikai M, Ataka K, Tsai M, Koyama K, Sato Y,
Kato I, Fujimiya M, Inui A. (2010). Centrally administered nesfatin-1
inhibits feeding behaviour and gastroduodenal motility in mice. Neuroreport, 21(15):1008-11
Sartor RB. (1999). Microbial factors in the pathogenesis of Crohn’s disease, ulcerative colitis and experimental intestinal inflamation. In: JB
Kirsner (ed), Inflammatory bowel disease. Williams and Wilkins, Baltimore, 153–78.
Stengel A, Taché Y. (2011). Minireview: Nesfatin-1 an emerging new
player in the brain-gut, endocrine, and metabolic axis. Endocrinology,
152(11): 4033-38.
Yosten GL, Samson WK. (2010). The anorexigenic and hypertensive
effects of nesfatin-1 are reversed by pretreatment with an oxytocin
receptor antagonist. Am J Physiol Regul Integr Comp Physiol,
298(6):R1642-7.
requirements.
when IL-8 and IL-6 were co-applied. However, colonic contractility in the presence of all three reagents resulted in contractile response that was larger in amplitude than IL-6 (p<0.001),
IL-8 (p<0.001) or CRF (p<0.001) alone.
These data illustrate that IL-8, IL-6 and CRF have functional
effects on colonic contractile activity and this is likely mediated by the myenteric. These findings provide evidence that
immune and stress mediators can cause changes in gastrointestinal motility which may contribute to IBS-like symptoms.
requirements.
C55
The effect of estrogen receptor agonists on pancreatic duct
ligation induced experimental acute pancreatitis model
C54
Z. Guleken1, D. Ozbeyli1, M. Acikel-Elmas2, S. Oktay3, B. Alev3,
S. Sirvanci2, A. Veli Ovunc4 and O. Kasimay Cakir1
Immune and stress factors alter colonic contractile activity
1Physiology Department, Marmara University School of Medicine,
M.M. Buckley and D. O’Malley
Physiology, University College Cork, Cork, Ireland
Irritable bowel syndrome (IBS) is a functional gastrointestinal
disorder characterized by episodic bouts of altered bowel habit,
bloating and abdominal pain, but the underlying pathophysiology is still poorly understood. There is an increase in circulating levels of both the proinflammatory cytokines IL-6 and 8
in IBS patients and as a consequence, altered cytokine profiles
have been proposed as a biomarker of IBS. Furthermore, IL-6
and IL-8 can stimulate myenteric neurons. A relationship has
been demonstrated between activation of central corticotropin-releasing factor (CRF)1 receptors and the stress-related
exacerbation of IBS symptoms. Crosstalk between IL-6, IL-8 and
CRF has also been postulated. These studies aim to determine
if immune and/or stress factors induce colonic contractile activity.
The distal colon was excised from Sprague Dawley (SD) rats
and whole mount preparations of circular muscle were prepared. The colonic tissue was then suspended from a tension
transducer in a tissue bath of CO 2 bubbled Krebs saline under
1g of tension and allowed to equilibrate. Mechanical signals
from the muscle were amplified, recorded and analysed using
Chart7.
IL-6 induced a small but robust increase in contractile activity
(0.02±0.009 mV, n=5) which was partially inhibited by the presence of the Na+ channel blocker, TTX (100nM, 0.01±0.001 mV,
n=5, p<0.01).CRF (10nM) similarly induced a contraction of
the colonic tissue (0.03±0.003 mV, n=4) and the response was
similarly reduced in the presence of TTX (0.02±0.002 mV, n=4,
p<0.01). IL-6 plus CRF together stimulated a larger contraction
(0.04±0.003 mV, n=4) than IL-6 alone (0.02±0.002 mV, n=4,
p<0.05). In these studies CRF (0.03±x0.003mV, n=4) evoked
a contraction that was larger than the IL-6 effect (p<0.05) but
was not different to the combined effect of the two reagents
(p>0.05). Application of IL-8 evoked a robust increase in contractility, resulting in large, repetitive contractions (n=5,) with
a frequency of 0.48±0.11 Hz (n=5). The frequency of contractions was increased by IL-8 (0.84±0.1 Hz n=5 P<0.01) and
was attenuated in the presence of TTX (p<0.001) but not completely inhibited .The contractile responses evoked by IL-8 were
significantly greater than those evoked by IL-6 ( p<0.001) but
no further enhancement of the IL-8-evoked response was noted
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Istanbul, Turkey, 2Histology and Embryology, Marmara University
School of Medicine, Istanbul, Turkey, 3Department of Biochemistry,
Marmara University Faculty of Dentistry, Istanbul, Turkey and
4Marmara University Vocational School of Health Related Services,
Istanbul, Turkey
It has been shown that estrogens have anti-inflammatory
effects in different experimental inflammation models, however the effect of estrogen on acute pancreatitis was not investigated. In the study it is aimed to determine the potential role
of estrogen receptor agonists on acute pancreatitis model
induced by pancreaticobiliary ligation.
In the study both sexes of Sprague-Dawley rats (n= 88,
250–300 g) were used. Animals were divided into two groups
as pancreaticobiliary duct ligated group and pancreaticobiliary duct ligation with bilateral ovariectomy group. The rats
were anesthetized by intraperitoneal ketamine (100mg/kg)
and chlorpromazine (10mg/kg) before surgeries. Laparoscopic
surgery was made to the animals in the sham group. ER-α agonist propyl-pyrazole-triol (PPT;1mg/kg/day), ER-β agonist diarylpropionitrile (DPN;1mg/kg/day) and 17-beta östradiol
(10mg/kg/day) were dissolved in olive oil and administered
intraperitonally to the groups for 3 days. At 3th day, the animals were decapitated, and the trunk blood was collected,
lung and pancreas tissues were taken. Serum TNF-α, IL–1β,
IL–6 and IL–10 levels were measured. Malondialdehyde (MDA),
glutathione (GSH), myeloperoxidase (MPO) activity, superoxide dismutase (SOD) and catalase levels were measured and
histological analyzes were made in pancreas and lung tissues.
In both tissues of male and female acute pancreatitis groups
MPO, MDA, SOD levels were significantly increased (p<0.050.01) and the antioxidant GSH levels were decreased (p<0.05)
compared to sham group. Pancreas MDA and SOD levels were
elevated in DPN and 17-beta- estradiol treated male rats with
acute pancreatitis compared to sham group (p<0,05-0,001).
After acute pancreatitis MDA and SOD levels in female rats
were decreased (p<0.01-0.001). The increased MPO levels in
pancreatitis group were decreased with PPT (p<0,05-0.001)
application. IL-1β, IL-6, and TNF-α levels of female rats and IL1β, IL-6 levels of male rats with acute pancreatitis increased
compared to sham group (p<0,05-0,001). The estrogen receptor agonists decreased the variant IL levels. These results suggest that the increased inflammatory status by experimental
pancreatitis induction was alleviated by estrogen receptor ago-
Oral Communications
nists via alpha and beta receptor subtypes in lung and pancreas tissues.
Yeo CJ, Cameron JL. Acute pancreatitis. Sabiston DC, editor. Textbook
of Surgery. 15th ed. W.B. Saunders Company; 1997. p. 1156-1165
Shields CJ, Winter DC, Redmond HP. Lung injury in acute pancreatitis: mechanisms, prevention, and therapy. Curr Opin Crit Care 2002;
8: 158-163
Strauss JF, Barbieri RL. Yen & Jaffe’s Reproductive Endocrinology.
Ankara: Günes Kitabevi: 2006,5. Baski:421-445.
requirements.
changes in whole body insulin sensitivity. Given the association between ceramide expression and impaired insulin signalling our data potentially offer insight into early adaptations
that occur in the period preceding the development of insulin
resistance.
Adams JM, Pratipanawatr T, Berria R, Wang E, DeFronzo R a, Sullards
MC & Mandarino LJ (2004). Ceramide content is increased in skeletal
muscle from obese insulin-resistant humans. Diabetes 53, 25–31.
requirements.
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C56
Six days high fat overfeeding alters skeletal muscle ceramide
expression prior to changes in whole body insulin sensitivity
in young healthy males
S. Wardle1, C. McGlory1,3, L.S. Macnaughton1, O.C. Witard1,
P.D. Whitfield2, C.N. Moran1, S. Galloway1 and K.D. Tipton1
1Health & Exercise Sciences Research Group, University of Stirling,
Stirling, UK, 2Department of Diabetes and Cardiovascular Science,
University of the Highlands and Islands, Inverness, UK and
3Department of Kinesiology, McMaster University, Hamilton, ON,
Canada
Consumption of excess energy, high fat diets are a useful model
for investigating the mechanisms that lead to obesity and Type
2 Diabetes. Ceramides have been shown to interfere with
insulin signalling and are elevated in skeletal muscle of obese,
insulin resistant individuals (Adams et al., 2004). We aimed
to investigate if the expression of skeletal muscle ceramide
species was altered in healthy males following consumption
of a diet designed to lead to insulin resistance. Twenty healthy
males (22 ± 1 y; 71.24 ± 2.16 kg (mean ± SEM) were matched
to one of two groups. Both groups participated in 6d high fat
overfeeding (150% of total kcal, 60% FAT, 25% CHO, 15% PRO)
but one group received 10% of their fats from fish oil sources
(FO) while the other consumed mostly saturated and monounsaturated fats (HF). The overfeeding period was bookended
by two identical trial days. Following an overnight fast, participants provided a basal blood sample and skeletal muscle
biopsy. The blood sample was used to measure glucose and
insulin concentrations for calculation of insulin sensitivity
indices (Matsuda Index and HOMA-IR) while the skeletal muscle biopsy was used to assess the expression of ceramide
species by LCMS-MS (results expressed as nmol/mg wet muscle). Statistical analyses were conducted using a repeated measures 2-way ANOVA with statistical significance set at p < 0.05.
As no significant differences were observed between groups,
all data presented herein refer to n = 20 (mean ± SEM) and p
values are related to changes in response to the overfeeding
period. Total ceramide expression was greater following the
overfeeding period (8.52 ± 1.24 to 12.09 ± 1.73, p = 0.02) with
significant increases in 5 of 11 species (C16:0: 0.39 ± 0.05 to
0.47 ± 0.06, p = 0.049; C22:0: 1.20 ± 0.23 to 1.82 ± 0.35, p =
0.02; C23:0: 0.56 ± 0.12 to 0.86 ± 0.17, p = 0.01; C24:1: 1.88
± 0.38 to 3.19 ± 0.55, p = 0.02; C24:0: 1.98 ± 0.33 to 2.71 ±
0.46, p = 0.03). However, irrespective of group, 6d high fat
overfeeding failed to induce any decrements in whole body
insulin sensitivity (Matsuda: 3.45 ± 0.27 to 3.24 ± 0.24, p =
0.61; HOMA-IR: 3.13 ± 0.31 to 2.67 ± 0.60, p = 0.40). High fat
overfeeding is associated with significant increases in total
ceramide expression within skeletal muscle without any
Extracellular vesicles released from hypoxic adipocytes
impair insulin signalling and glucose uptake in adipocytes
J. Mlezcko1, J. Falcon-Perez2 and S. Mora1
1University
of Liverpool, Liverpool, UK and 2CIC BioGUNE, Bilbao,
Spain
Background and aims: Exosomes and microvesicles are extracellular vesicles (EVs) shed by many cell types that mediate cell
to cell communication. EVs contain proteins and RNA species
that modulate biochemical responses in target cells.
Adipocytes have been shown to produce extracellular vesicles.
During obesity adipocytes are subject to hypoxia. The aim of
our study was to investigate whether EVs released by hypoxic
adipocytes have autocrine effects in regulating insulin action
and glucose uptake. Methods: We isolated EVs from the conditioned media of differentiated 3T3L1 adipocytes cultured
under normoxia (control cells) or from cells exposed to hypoxia
(1% O2) for 24hrs. EVs were purified from the culturing media
using centrifugation techniques and characterized using biophysical and biochemical methods, including nanoparticle
tracking analysis, cryoelectron microscopy, and immunoblotting techniques. EVs were then tested for their ability to modulate insulin action and insulin-stimulated glucose uptake in
3T3L1 adipocytes. Independent sets of differentiated 3T3L1
adipocytes were either left untreated or treated with EVs purified from control or hypoxic adipocytes for 24hr and subsequently their response to insulin stimulation was evaluated by
western blotting and glucose uptake assays. Results: We found
that 3T3L1 adipocytes release EVs of heterogeneous sizes as
determined by cryoelectron microscopy and nanoparticle
tracking analysis. Hypoxia increased the release of EVs by
adipocytes. Immunoblotting analysis showed that EV preparations were enriched in exosomal markers and markedly
devoid of other organelle markers. Adipocytes treated with
EVs obtained from hypoxic cells displayed a reduced insulinmediated activation of glucose uptake compared to those left
untreated or treated with EVs derived from control adipocytes.
No differences in the expression levels of the glucose transporter Glut4 were observed in cells treated with EVs from
hypoxic adipocytes compared to those left untreated or treated
with EVs from control cells. However, cells treated with EVs
released from hypoxic cells exhibited reduced insulin-mediated activation of phosphatidylinositol 3-kinase as seen by a
reduced phosphorylation of the downstream kinase AKT, without alterations in the total AKT levels. Conclusions: Our data
suggest that EVs released by hypoxic adipocytes contribute to
insulin resistance, as they impair insulin signalling and glucose
uptake in adipose cells.
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requirements.
In conclusion, we demonstrated for the first time that GPR17
is expressed and functional in human islets of Langerhans.
Studies are in progress to better define its role in islet cell physiology and pathology.
C58
Expression and function of the atypical purinergic receptor
GPR17 in endocrine pancreas
E.S. Di Cairano1, V. Meraviglia2, A. Ulivi2, P. Rosa2, S. Moretti1,
F. Daniele1, F. Bertuzzi3, S. LaRosa4, F. Folli5, V. Sacchi1 and
C. Perego1
1Dept of Pharmacological and Biomolecular Sciences, Università
degli Studi di Milano, Milano, Italy, 2Institute of Neuroscience, CNRBiometra, Milano, Italy, 3Niguarda Cà Granda Hospital, Milano,
Italy, 4Dept. of Pathology, Ospedale di Circolo, Varese, Italy and
5Division of Diabetes, Department of Medicine, University of Texas
Health Science Center at San Antonio, San Antonio, USA Minor
Outlying Islands
Islets of Langerhans control glucose homeostasis and metabolism by sensing blood glucose and nutrients. Factors secreted
within the islet create a parallel tuning, defined autocrineparacrine signaling, that modulates hormone release and cell
viability (Barker et al., 2013). Studying the molecular actors
of this signaling and their physiological significance may help
in understanding diabetes pathogenesis thus providing novel
therapeutic targets.
G-protein coupled receptors (GPCRs) are expressed in islets of
Langerhans, regulate hormone secretion and cell survival, and
are emerging as new targets for type-2 diabetes therapies.
In this work, we focused on the expression and role of the atypical orphan receptor GPR17, which is activated by uracil
nucleotides and can be partially antagonized by ATP, a well
known signal in islets (Ciana et al., 2006).
We first verified the GPR17 expression in endocrine cell lines
(αTC3, βTC3, RIN14B) and in human islets, then we investigated its possible role in islet physiology.
By means of RT-PCR, we detected GPR17 mRNA in human
islets, in α and δ cell lines, but not in β cell lines (n=5). We
confirmed GPR17 protein expression in α and δ cell lines and
in human islet by western blot (n=4). To investigate the specific endocrine cells where GPR17 is expressed in vivo, we carried out double immunofluorescence and confocal analysis on
human pancreas slices, using hormones as markers of the different cell populations and we confirmed the expression of
GPR17 in a sub-population of δ cells. No expression was
detected in β-cells in physiological conditions. Furthermore,
chronic incubation of human islets with 16 mM glucose, known
to cause β-cell death, causes a two-fold increase in GPR17
expression (p<0.05 T-Test; n=4), thus indicating that the receptor may be sensitive to stress conditions. To understand the
possible role of GPR17 in islet physiology we activated GPR17
with the agonist UDP-glucose, and we measured its effect on
cell viability and hormone release in cell lines and human isolated islets. 3-day incubation of αTC3 with 100 μM UDP-G
causes a significant increase in cell viability, measured by MTT
test (108±2.56% UDP-G vs ctr, p<0.005 T-Test; n= 10). A similar behavior was observed in human islets, where we found a
reduction of apoptosis in presence of 100 μM UDP-G (38±7%
UDP-G vs ctr).
Preliminary data on hormone release indicate that static acute
incubation of human islets with 100 μM UDP-G, causes a
decrease in glucagon and somatostatin release in normal glucose (5 mM) (ELISA, 2 diverse islets preparations, triplicate).
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Barker CJ, Leibiger IB, Berggren PO. The pancreatic islet as a signaling
hub. Adv Biol Regul. 2013 Jan;53(1):156-63.
Ciana P, Fumagalli M, Trincavelli ML, Verderio C, Rosa P, Lecca D, Ferrario S, Parravicini C, Capra V, Gelosa P, Guerrini U, Belcredito S, Cimino
M, Sironi L, Tremoli E, Rovati GE, Martini C, Abbracchio MP. The orphan
receptor GPR17 identified as a new dual uracil nucleotides/cysteinylleukotrienes receptor. EMBO J. 2006 Oct 4;25(19):4615-27.
requirements.
C59
The effects of maternal dexamethasone exposure on serum
analyte and oxidative stress in the kidney of male offspring
S.O. Jeje1,2, G. Ushie2 and Y. Raji1
1Department of Physiology, Cross River University Of Technology,
Okuku-Ogoja, Cross River, Nigeria and 2University of Ibadan,
Ibadan, Nigeria
The effects of maternal exposure to glucocorticoids on various organ and systems including the kidney of the offspring
have been reported in literature. The hallmark of this effect is
an increase in circulating corticosterone. Excess corticosterone
may also induce oxidative stress. This study examined the
effects of maternal dexamethasone(dex) exposure on serum
creatinine and urea, oxidative stress markers in the kidney and
kidney architecture in the offspring. Pregnant rat (180-200g)
were divided into 4 groups. Group 1 was administered
0.02ml/100gbwt/day normal saline through postnatal (PN)
day 1-21 (control). Groups 2, 3, and 4 were administered
100μg/kg bwt/day dex for PN day 1-7 (Dex1-7),1-14( Dex 114), and 1-21(Dex 1-21) respectively. All administration was
done subcutaneously. At 12weeks, male offspring were sacrificed. Evaluation of serum analyte, kidney oxidative stress and
kidney histology were done. Level of lipid peroxidation (MDA)
(Buege and Aust,1978), Catalase activity (Sinah,1971), SOD
(Mistra and Fridovich 1972) activity, Protein (Lowrey et al 1951)
were evaluated. Serum creatinine and Urea was estimated
using analytical kit (Randox lab. USA). Tissue histology (H/E
techniques). Values (Mean±SEM, n=6) are compared using one
way ANOVA. P<0.05 is significant. Serum creatinine was Higher
in
the
Dex1-7(0.234±0.018mg/dl),
Dex114(0.254±0.019mg/dl) and Dex1-21(0.293±0.0195mg/dl)
Oral Communications
when compared with the control(0.140±0.016mg/dl) (P<0.01,
0.01, 0.001 respectively). Serum Urea level was higher in the
treatment groups Dex1-7(6.995±0.44 mg/dl), Dex1-14
(4.232±0.898 mg/dl), Dex1-21(3.163±0.662) when compared
with control (0.555±0.166) (P<0.001,0.01,0.05 respectively).
However, serum urea level was higher in the Dex1-7 when compared with Dex1-14 and Dex1-21 (P<0.05,0.01 respectively) .
Kidney MDA was also increase in the Dex1-7(11.525±0.813
mole/gtissue), Dex1-14(15.95±0.92 mole/gtissue) and Dex121(17.213± 1.09 mole/g tissue) groups compared to control(5.813±0.166 mole/g tissue)(P<0.01, 0.001, 0.001 respectively). However, kidney SOD and catalase activities in the
treatment groups (Dex1-7 SOD-2.797±0.12 U/mg/ml protein,
Cat-0.95±0.030Katf; Dex1-14 SOD-1.312±0.034 U/mg/mlprotein,Cat-0.765±0.03; Dex1-21 SOD-1.292±0.03 U/mg/mlprotein,Cat-0.404±0.0212) were all lower than the control (SOD4.776±0.131U/mg/mlprotein,Cat-1.298±0.0845,)(P<0.001).
Kidney protein was lower in the Dex 1-7(4.65±0.98 mg/ml),
Dex1-14(4.21±0.24mg/ml), Dex1-21(3.29±0.94mg/ml) when
compared
with
the
control(6.98±0.04
mg/ml)
(P<0.05,0.01,0.001 respectively). Histology of the kidney
showed mild, moderate and severe tubular necrosis in the
Dex1-7, Dex1-14, Dex1-21 group respectively. Results suggest
that exposure to dex during lactation could lead to increase
oxidative stress in the kidney and increase renal necrosis.
Drake AJ, Tang JI, Nyirenda MJ 2007, Mechanisms underlying the role
of glucocorticoids in early life programming of adult disease. Clin sci
113: 219-232
Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the
autooxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247 (10), 3170–3175.
Beuge, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Meth.
Enzymol. 52, 302–310.
(at ED15-17). Endocrine actions of pituitary GH in the development and function of the chicken embryo eye are, however,
unknown. This possibility has therefore been investigated in
ED15 embryos. In ovo systemic injections of Cy3-labelled GH
(150 μg, via the chorioallantonic vein) demonstrated that GH
in the bloodstream was translocated into the neural retina and
internalized into retinal ganglion cells (RGC’s). Pituitary GH
may therefore also be functionally involved in retinal development during late embryogenesis. Cy3-labelled GH was similarly internalized into QNR/D cells (which provide an experimental model for chick embryo RGCs) (2) after its addition
into incubation media. The uptake of exogenous GH was by a
receptor-mediated mechanism and maximal after 30-60 min,
and was followed by degradation of the internalized hormone.
The exogenous (endocrine) was, however, biologically active,
since, within the QNR/D cells it promotes IGF-1 expression.
Interestingly, the labeled GH accumulated in perinuclear
regions of the QNR/D cells, but was not found in the neurite
outgrowths, in which endogenous retinal GH is located. This
suggests that exogenous (endocrine) and endogenous
(autocrine/paracrine) GH are both involved in retinal function
in late embryogenesis and they co-exist in separate intracellular compartments within retinal ganglion cells.
Harvey S (2010), Endocrine 38, 335-359
Martinez-Moreno C et al. (2014) Gen Comp Endocrinol 195, 183-189
Supported by CONACyT, Mexico (postdoctoral fellowship,
208148) and NSERC, Canada
requirements.
Sinha, K.A., 1971. Colorimetric assay of catalase. Anal. Biochem. 47,
389–394.
C61
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193,
265–275.
Transient receptor potential A1 channels contribute to
relaxation of porcine coronary arteries by hydrogen
sulphide
requirements.
C.J. Kelsall1, L.J. McCormick2, P.D. Whitfield1, M.K. Doherty1,
R.E. Morris2 and I.L. Megson1
1Diabetes
C60
Coexistence of autocrine/paracrine and endocrine growth
hormone in embryonic retinal ganglion cells
C.G. Martinez-Moreno1, M. Luna2, C. Aramburo2 and S. Harvey1
1Physiology,
University of Alberta, Edmonton, AB, Canada and
Celular y Molecular, Instituto de Neurbiologia,
Universidad Nacional Autonoma de Mexico, Juriquilla, Queretaro,
Querétaro, Mexico
2Neurobiologia
Growth hormone (GH) exerts its biological effects through the
GH receptor (GHR), which is present in the chicken embryo
eye from ED (embryonic day) 2 of the 21-day-incubation period.
In the embryo, GH gene expression also occurs in the neural
retina from ED2 and retinal GH induces the survival and differentiation of retinal ganglion cells, as these local autocrine
or paracrine actions are blocked following the immunoneutralization of endogenous GH or when retinal GH production
is reduced by siRNA knockdown (1). Retinal GH is therefore of
functional importance before the ontogenic appearance of
pituitary somatotrophs (at ED12) and the onset of pituitary
GH secretion and the appearance of GH in peripheral plasma
& Cardiovascular Science, University of Highlands &
Islands, Inverness, UK and 2School of Chemistry, University of St
Andrews, St Andrews, UK
Vascular relaxation is mediated by a range of endogenous factors, including hydrogen sulphide (H2S) and acute hypoxia.
The mechanisms by which these factors elicit vasorelaxation
have not been fully elucidated. Relaxation to H2S is O2-sensitive [1], and transient receptor potential A1 knockout mice
(TRPA1-/-) show an attenuated ear perfusion response to an
H2S donor [2]. Therefore we investigated whether relaxation
to H2S in porcine coronary arteries is mediated by TRPA1, and
if H2S and hypoxia share a common mechanism of action.
Pure H2S was administered in a controlled manner by metal
organic frameworks (MOFs). MOFs derived from 2,5-dihydroxyterephthalic acid and magnesium (Mg2(dhtp)) were prepared and pressed into 20 mg discs containing 10% Teflon (by
weight). The discs were dehydrated under vacuum at high temperature, cooled and exposed to an atmosphere of H2S. Once
loaded, MOF discs were stored individually under argon until
use. Previous studies revealed the gas is rapidly released from
the discs upon exposure to an aqueous environment at 37䊊C
[3]. Pig hearts were obtained from an abattoir and segments
of coronary artery with or without an intact endothelium (±
EC) were assessed ex vivo by wire myography. Pre-constriction
to 0.1 μM U46619 was followed by incubation with hypoxia
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(gassing chambers with 95% N2/ 5% CO2) for 1h, or HC-030031
(selective TRPA1 inhibitor; 10 μM) for 30 min, or consecutive
incubation with both, then exposure for 30 min to H2S-loaded
MOFs. Percent relaxation data are presented as mean ± SEM,
n=5-6 per group; statistical analysis was by 2-way ANOVA with
post hoc analysis.
Potent relaxation occurred in normoxic arteries in response to
H2S (+EC: 90±2; -EC: 86±1.5; ns). Hypoxia caused vasorelaxation, significantly more so in the presence of an intact
endothelium (+EC: 55±7; -EC: 22±8; p <0.05), while HC-030031
was not vasoactive. Hypoxia and inhibition of TRPA1 channels
individually attenuated relaxation to H2S, but while the effect
of hypoxia on H2S was unmodified by endothelial status (+EC:
54±19; -EC: 42±7; ns), HC-030031 completely ablated relaxation to H2S only in arteries with a functional endothelium
(+EC: -1±13, -EC: 51±12; p <0.05). Hypoxia in addition to HC030031 did not alter the degree of attenuation of relaxation
to H2S compared to hypoxia alone (+EC: 27±14; -EC: 33±5; ns
by EC status or treatment group).
The data show that TRPA1 channels mediate relaxation to H2S
in porcine coronary arteries, but that the role of these receptors may be principally associated with the endothelium. The
data do not support a common mechanism of action for
hypoxia and H2S in inducing vasorelaxation, although both
appear to be mediated, in part, by TRPA1 channel activity.
Dombkowski RA et al (2006). J Exp Biol 209: 3234-40
n=3) and analysed by Student’s t-test and one-way ANOVA
with Dunnett’s multiple comparisons.
HF dams were heavier than C dams (C 27.7g ± 0.7 vs HF 37.83g
± 1.46 p<0.0001) with higher plasma glucose (C 7.6mmol/L ±
1.3 vs HF 10.9mmol/L ± 1.3, p<0.05) and abdominal adiposity (C 0.32g ± 0.06 vs HF 1.15g ± 0.23 p<0.05). While C/HF and
HF/HF offspring were heavier than C/C and HF/C offspring (C/C
30.3g ± 2.7, C/HF 38.3g ± 7.6, HF/C 32.2g ± 5.3, HF/HF 41.9g
± 6.5, p<0.0001) there was no significant difference in systolic
blood pressure between the groups. Maternal HF-diet influenced endothelial cell IKCa expression (%; C/C 22.5 ± 4.6, C/HF
20.0 ± 1.2, HF/C 25.9 ± 2.7, HF/HF 45.1 ± 11.3 p<0.05). Colocalization of IKCa to the endothelial cell nucleus was also
increased in the HF/C group (Mander’s overlap coefficient; C/C
0.58 ± 0.05, C/HF 0.50 ± 0.02, HF/C 0.72 ± 0.01, HF/HF 0.62
± 0.05, p<0.05.) Expression of connexin 37 and the colocalization of connexin 37 and IKCa were not significantly influenced by maternal or post-weaning diet.
These data are consistent with an altered contribution of EDHsignalling in the vasodilatation of small vessels in offspring
from HF-fed dams and are indicative that an adverse developmental environment may impact on the normal growth and
maturational-related changes in endothelial function on which
pathological changes may be superimposed to produce an
‘at risk’ phenotype in the adult offspring.
This research is funded by the British Heart Foundation.
Pozsgai G et al (2012). Eur J Pharmacol 689: 56-64
Allan PK et al (2012). Dalton Trans 41: 4060-6
This work was funded by the British Heart Foundation.
requirements.
C62
Maternal high fat diet alters localization and expression of
the intermediate conductance calcium-activated potassium
channel (IKCa) in mesenteric arteries of adult mice offspring
R. Stead1, M. Musa1, A. Stanley1, R. Reynolds1, C. Torrens1,
P. Fraser2 and G. Clough1
1Faculty
of Medicine, University of Southampton, Southampton,
UK and 2School of Medicine, King’s College London, London, UK
Offspring of mothers fed a high fat diet during pregnancy show
an increased incidence of diabetes, hypertension, obesity and
vascular dysfunction with reduced endothelium-dependent
nitric oxide (NO) and hyperpolarisation (EDH)-dependent
vasodilation .The current study further investigates the impact
of a maternal high fat diet on the EDH-mediated vasodilator
signalling pathways,
Female C57/BL6 mice were fed either a high fat (HF, n=21) or
control diet (C, n=17) 6 weeks prior to conception and throughout gestation and lactation. On weaning, offspring were fed
the same or opposite diet to their dams to give four offspring
groups: C/C, C/HF, HF/C and HF/HF. Offspring bodyweight,
plasma glucose and blood pressure were measured at 15 weeks
of age, prior to culling by cardiac exsanguination. Sections of
mesenteric arterioles (0.2 mm in length) were imaged using
confocal microscopy and the endothelial expression and localization of intermediate conductance calcium activated potassium channel (IKCa) and connexin 37 quantified. Results are
expressed as mean ± SEM (C/C n= 5, C/HF n=4, HF/C n=5, HF/HF
90P
requirements.
C63
Improving the physiological relevance of calcium signalling
studies conducted on human platelets through the use of
tissue-engineered human blood vessel constructs
F.I. Musa, A.G. Harper and Y. Yang
Institute for Science and Technology in Medicine, Keele University,
Stoke-on-Trent, UK
Thrombus formation in vivo is regulated by paracrine signalling
with other cells in both the blood and the vessel wall. Thus
whilst ex vivo study of isolated human platelet suspensions creates a simplified system in which to assay platelet activation
mechanisms, they are unlikely to adequately replicate the
responses observed in vivo. Therefore experiments were performed to examine whether exposure of platelet suspensions
to simple 3D tissue-engineered constructs of either part or the
whole of the blood vessel wall was able to influence the
observed calcium signals. Proaggregatory subendothelial
matrix constructs were created by coating Poly-lactic acid
nanofibres with or without 1μg/cm2 Horm collagen. 3D vessel wall constructs was fabricated by culturing human umbilical vein endothelial cells (HUVECs) on a PLA nanofiber mesh
attached atop of a collagen hydrogel in which Human coronary artery smooth muscle cells were cultured. Platelets were
isolated from blood obtained by venepuncture of healthy volunteers under informed consent and with local ethical committee approval in accordance with the Declaration of Helsinki.
Changes in cytosolic calcium concentration ([Ca2+]cyt) were
measured in washed human platelets dually labelled with both
Fura-2/AM and DiOC6. Platelet suspensions were exposed to
partial or full vessel wall constructs for between 5-15 minutes
at 37°C. The vessel construct was then removed to examine
platelet adhesion to the surface of the construct by fluores-
Oral Communications
cence microscopy, whilst the calcium signalling capacity of the
platelet suspension was examined after stimulated with
0.2U/mL thrombin. Data are presented as mean ± SEM of the
number of samples (n) indicated. Statistical significance was
tested by Student’s t-test. Exposure of platelets to a proaggregatory subendothelial matrix constructs coated with Horm
Collagen significantly potentiated the thrombin-evoked rise in
[Ca2+]cyt to 173.7 ±11.7% (n = 4; P < 0.05) of those seen in
platelets exposed to an analogous construct made using
uncoated nanofibres. This correlated with an increased adhesion of platelets to the collagen-coated nanofibres. Exposure
of platelets to the full 3D blood vessel construct elicited a significant reduction in the thrombin-evoked rise in [Ca2+]cyt (36.7
±11.7% of control; n=6; P < 0.05), when compared to samples
not exposed to the construct.The ability of our full blood vessel construct to significantly impair thrombin-evoked rises in
[Ca2+]cyt demonstrates the need for ex vivo platelet activation
assays to more accurately reflect the in vivo environment, and
demonstrates the potential of using tissue-engineered blood
vessels constructs to help improve the physiological realism
of such studies.
FM was supported by a British Heart Foundation PhD
studentship (FS/12/48/29719)
level, Mann-Whitney 2 independent samples, U=1.19E6).
Despite this mitochondria in SHR have a slightly higher average number of neighbouring mitochondria (mean ±SEM
4.67±0.02) than those in control (WKY; 4.48±0.03; p<0.01,
one-way ANOVA). Those mitochondria in SHR are more tightly
packed (mean separation±SEM 1.91±0.011 μm apart compared to 2.21±0.016 μm in WKY; p<0.01, one-way ANOVA).
These two aspects combine to increase the extent of mitochondrial clustering within the vascular smooth muscle of SHR
compared to WKY. Resistance artery smooth muscle cells were
then co-loaded with the Ca2+-indicator fluo4AM (10 μM) plus
mitochondrial TMRE and patch-clamped in the whole cell configuration. Transient plasma-membrane depolarisation (-70 to
10 mV, 1 s) activated voltage-gated Ca2+ channels and elevated [Ca2+]c. Pixel-by-pixel analysis reveals that those pixels
that are closest to mitochondria show the highest [Ca2+]c
changes. In summary, this work has developed a novel tool for
increased resolution of mitochondrial architecture in live cells.
This tool revealed a previously unrecognised change in mitochondrial structure that may contribute to changes in Ca2+ signalling and smooth muscle function characteristic of hypertension.
Chalmers S et al. (2012). Arterioscler Thromb Vasc Biol 32, 3000-3011.
Marsboom G et al. (2012). Circ Res 110(11),1484-1497.
requirements.
requirements.
C64
C65
Flicker-assisted localisation microscopy reveals altered
mitochondrial architecture in vascular smooth muscle in
hypertension
S. Chalmers1, C. Saunter2, J.M. Girkin2 and J.G. McCarron1
1Strathclyde
Institute of Pharmacy & Biomedical Systems,
University of Strathclyde, Glasgow, UK and 2Department of Physics,
Durham University, Durham, UK
Mitochondria vary from being small puncta to forming large
interconnected networks. Mitochondrial structure is easily
visualised in cultured cells; however the precise structure of
the organelle in native cells is poorly understood due to the
cells’ complex organisation and to imaging resolution limitations. We have made use of the electrical changes during transient de- and re-polarisations of membrane potential (‘flickers’) of single mitochondria within live, native vascular smooth
muscle cells (visualised by epifluorescence imaging of the
membrane-potential sensitive indicator TMRE, 62.5 nM) to
determine the organelles’ dimensions, position and relationship with Ca2+ signals. Custom image-analysis procedures written in Python software measure the spatial and temporal
covariance of changes in fluorescence in areas around each
image pixel, to functionally resolve organelle boundaries in
clusters of visually inseparable mitochondria. We show that
the apparently wide range of mitochondrial sizes in native cells
(1-10 μm) is explained by varying numbers of multiple, tightlypacked small organelles. Alterations in mitochondrial positioning and architecture are associated with the proliferative
state of vascular smooth muscle (1, 2) and so this analysis was
applied to single smooth muscle cells freshly-isolated from
resistance arteries of spontaneously hypertensive rats (SHR)
and normotensive (WKY) control. Remarkably, the size of individual mitochondria increased in SHR (geometric mean
area±SEM 0.826±1.97 μm2, n=48, compared to 0.347±3.083
μm2, n=20, in WKY controls; significantly different at the 0.01
Ca2+ signalling is differentially altered in endothelial cells
from aortic arch and thoracic aorta in the Apolipoprotein
E knockout mouse model of atherosclerosis
C. Prendergast, J. Quayle, T. Burdyga and S. Wray
Dept. Cellular & Molecular Physiology, University of Liverpool,
Liverpool, UK
ApoE-/- mice develop hypercholesterolemia and are a widely
used model of atherosclerosis. Changes in Ca2+ are vital to the
function of endothelial cells, but how atherosclerosis changes
these signals is unclear. We examined the effect of hypercholesterolemia on Carbachol (CCh)-mediated Ca2+ signals in aortic endothelial cells from WT and ApoE-/- mice at 10 weeks
(young), before plaques; 24 weeks, when plaques are established and 30 weeks, when plaques are extensive. Using confocal microscopy, we compared CCh (0.3-10μM) responses in
thoracic aorta (TA) and the more plaque-prone aortic arch (AA).
In addition, in older ApoE-/- mice, measurements were taken
from endothelial cells immediately adjacent to plaques and
at a distance, to determine whether changes in Ca2+ response
were dependent upon proximity to lesions. Three parameters of the Ca2+ response were measured; initial Ca2+ peak size,
plateau size and percentage of cells demonstrating oscillatory
Ca2+ responses. Significance was assessed by a t-test or ANOVA
(p<0.05).
Sudan IV staining confirmed aortae from old ApoE-/- had clear
atherosclerotic plaques, whereas those from young ApoE-/showed either no or barely visible tiny areas of plaque (10w
n=10; 24w n=9; 30w n=5). No WT mice had plaques in the
aorta (10w n=10; 24w n=5; 30w n=3). Ca2+ responses (peak,
plateau) in TA from 10 week WT and ApoE-/- cells were similar,
except at 10μM CCh, where the response was larger in ApoE/- (n=4-7). In AA, Ca2+ responses were significantly larger in
ApoE-/- even at lower [CCh] (n=4-6). In 24 week ApoE-/- TA,
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responses in plaque-free regions were similar to WT, whereas
those adjacent to plaque were enhanced (at 0.3 & 10μM CCh,
n=3-4). In the plaque-prone AA, Ca2+ responses were significantly increased in ApoE-/- cells both adjacent to and away from
lesions (n=3-4). In 30 week mice, the effect of 10μM CCh was
compared (n=3-6). In plaque-free TA, Ca2+ responses were
again similar to WT, but adjacent to plaques, responses were
significantly increased in ApoE-/-. Plaques were so extensive in
the AA that plaque-free regions were not found. Ca2+ responses
were also significantly increased compared to WT. A subset of
endothelial cells responds to CCh with an oscillatory Ca2+
response. In WT mice of any age, 1-2% of cells give an oscillatory Ca2+ response. In young ApoE-/- and plaque-free regions
of older ApoE-/-, this is unchanged. However, a significant
increase in oscillations was seen in TA and AA cells adjacent
to lesions in older mice (n=5-11).
ApoE-/- Ca2+ signalling is significantly altered compared to WT
prior to plaque development and is accentuated as the mice
age. Greater differences are seen in plaque-prone AA than in
the TA and proximity to plaques enhances the changes further,
including a shift towards Ca2+ oscillations.
requirements.
C66
Altered aortic reactivity by daibetes mellitus in
ovariectomized rats: role of epoxyeicosatrienoic acids (EETs)
A. Cavka, I. Grizelj, Z. Mihaljevic, A. Cosic, S. Novak and
I. Drenjancevic
Department of Physiology and Immunology, Faculty of Medicine
University of Osijek, Osijek, Croatia
Background: Epoxyeicosatrienoic acids (EETs), the cytochrome
P450 epoxygenase metabolites of arachidonic acid, are
vasodilators believed to be the endothelium-derived hyperpolarizing factor in a number of vascular beds. EETs may play
a role in the secretion and action of insulin and the metabolism of carbohydrates and lipids. In our previous study we have
demonstrated that oestrogen may have protective effect on
acetylcholine induced relaxation (AChIR) of aortic rings in diabetic rats (1). Thus, the aim of our study was to investigate if
EETs are involved in the mechanisms of AChIR and hypoxiainduced relaxation (HIR) of aortic rings in ovariectomized (OVX)
diabetic rats.
Matherials and Methods: 26 healthy, OVX Sprague-Dawley rats
were divided in control (14 weeks old, N=13) and DM group
(8 weeks of DM, N=13). Bilateral ovariectomy (ventral
approach) was performed under anesthesia (75 mg/kg ketamine + 2.5 mg/kg midazolam i.p.) at the 5th week of age (2).
After the surgery and the next day, the rats received analgesia (15 mg/kg metamizole sodium i.m.). Diabetes mellitus (DM)
was induced by streptozotocin (60mg/kg i.p.) at the 6th week
of age, and duration of DM was 8 weeks. Prior to decapitation,
rats were anesthetized (75 mg/kg ketamine + 2.5 mg/kg midazolam i.p.). Thoracic aortic rings were used to test ACh
response (10-9-10-5 M) and response to reduced pO2 (bath
gas mixture: N2 95%, CO2 5%) after precontraction with noradrenaline (NA) for 5 minutes, in the absence/presence of the
NOS inhibitor L-NAME, COX-1, 2 inhibitor indomethacin (INDO)
and inhibitor of the CYP4A2 and CYP4A3 epoxygenation reactions (MS-PPOH) in the tissue bath. To test differences among
groups Two-way ANOVA (AChIR protocol) and One-way ANOVA
(HIR protocol) were used; p<0.05 considered significant
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(SigmaPlot v11.2, Chicago, USA). The Ethical Committee of
Faculty of Medicine University of Osijek approved the study.
Results: OVX DM rats had significantly reduced both AChIR
(P<0.001) and HIR (P=0.0017) compared to OVX controls.
While both L-NAME (P<0.001) and MS-PPOH (P<0.05) significantly blocked AChIR in OVX controls, only L-NAME blocked
AchIR in DM group (P<0.001). While in OVX controls HIR was
significantly reduced with L-NAME (P<0.05, 19.64%), INDO
(P<0.001, 47.78%) and MS-PPOH (P<0.01, 29.98%), in DM rats
only INDO significantly reduced HIR (P<0.05, 38,94%) with no
significant effect of L-NAME (15.9%) and MS-PPOH (13.39%)
administration on HIR.
Conclusion: The results of this study confirmed our previos
finding that DM impairs aortic reactivity to both ACh and
hypoxia in OVX rats. Since MS-PPOH administration significantly blocked AChIR and HIR in OVX controls, and the same
effect wasn’t observed in DM rats, this finding suggests that
diabetes may alter vascular aortic response in OVX rats by: 1)
ovariectomy per se changes the mechanisms of vasorelaxation
to ACh and hypoxia; 2) diabetes alters either production of
EETs and/or 3) bioavailability and/or sensitivity to EETs.
Cavka A, Manojlovic D, Lucic D, Grizelj I, Drenjancevic I (2013) Oestrogen has protective effect on acetylcholine induced relaxation of aortic rings in diabetic rats. Periodicum Biologorum 115(2): 21.
Ceylan-Isik AF, Erdogan-Tulmac OB, Ari N, Ozansoy G, Ren J (2009).
Effect of 17beta-oestradiol replacement on vascular responsiveness
in ovariectomized diabetic rats. Clin Exp Pharmacol Physiol. 36(11):e6571.
This study was supported by grants of Ministry of Science,
Education and Sports, Croatia, #219-2160133-2034 and # 2190000000-0328.
requirements.
C67
Mechanism of flow-induced dilation of middle cerebral
arteries in diabetic Sprague-Dawley rats
I. Grizelj, A. Cavka, Z. Mihaljevic, A. Cosic, S. Novak and
I. Drenjancevic
Department of Physiology and Immunology, Faculty of Medicine
University of Osijek, Osijek, Croatia
Background: Cerebral blood flow is autoregulated over a wide
range of arterial pressures via local myogenic, metabolic and
flow-mediated regulatory mechanisms. Substantial evidence
suggests that endothelium-dependent vasodilation in response
to various agonists is impaired in animal models and in patients
with diabetes mellitus due to reduced endothelium-derived
nitric oxide (NO) bioavailability and increased oxidative stress
in conductance and resistance arteries. However, less is known
about flow-induced dilation (FID) in cerebral resistance vessels. In this study we aimed to elucidate the mechanisms of
FID of isolated middle cerebral arteries in rats with streptozotocin (STZ)-induced diabetes mellitus.
Matherials and Methods: 17 male Sprague-Dawley (SD) rats
were organized in two groups: a) control group (N=9) and b)
DM group (8 weeks of DM, N=7). Diabetes mellitus (DM) was
induced by streptozocin 60mg/kg i.p. at 6th week of age, and
duration of DM was 8 weeks, respectively. Prior to decapitation, rats were anesthetized with 75 mg/kg ketamine+2.5
mg/kg midazolam. Middle cerebral arteries were cannulated
for vascular reactivity measurements in response to stepwise
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increasing pressure (Δ10-Δ100 cm H2O), in the absence/presence of the NOS inhibitor L-NAME, COX-1,2 inhibitor
indomethacin (INDO), selective inhibitor of microsomal
CYP450 epoxidase activity MS-PPOH, and superoxide dismutase mimetic TEMPOL. To test differences among groups Twoway ANOVA was used, P<0.05 considered significant
(SigmaPlot v11.2, Systat Software, Chicago, USA). The Ethical
Committee of Faculty of Medicine University of Osijek
approved the study.
Results: FID was reduced in DM group of rats at each pressure
gradient, but significantly at Δ20 (P<0.05), and Δ40, Δ60, Δ100
(P<0.001) compared to control rats. While L-NAME, INDO and
MS-PPOH (P<0.01 for Δ20-Δ100) significantly blocked FID in
controls, only L-NAME blocked FID in DM group (P<0.05 for
Δ40). The presence of TEMPOL improves FID in DM group at
pressure gradient Δ40 (P<0.05), while in control group does
not have this effect.
Conclusion: The results of this study demonstrate that flowinduced dilation is impaired in DM and that the mechanisms
of FID are different in control and DM group, but NO mediates
FID in both groups. Since the presence of INDO or MS-PPOH
blocked FID in control group, but has no effect in diabetic rats,
and FID is restored by TEMPOL in diabetic rats, this findings
suggest that impaired dilation of MCA in diabetes may occur
due to altered activation of COX-1,2 and cytochrome P450
epoxygenase pathways in response to changes in flow in conditions of elevated oxidative stress.
Funding: This study was supported by grant of Ministry of
Science, Education and Sports, Republic of Croatia, # 2190000000-0328 and #219-2160133-2034.
requirements.
(20 μM) was loaded preferentially into endothelial cells and
visualised with a custom built, side-viewing imaging probe
based on a GRIN relay lens. The imaging probe was optically
coupled to a custom-built fluorescence microscope, permitting direct visualisation of an area encompassing ~200
endothelial cells. The artery was pressurised (60–160 mmHg)
and endothelial [Ca2+]i measured on a per-cell basis, using a
custom image processing procedure. To allow the total
response from all responding cells to be accurately examined,
a custom analysis script (Python programming language) was
used to differentiate, and then align (in time) the response of
individual cells. ACh evoked increases in [Ca2+]i that were sensitive to 2-aminoethoxydiphenyl borate (100 μM) and cyclopiazonic acid (100 μM), suggesting the [Ca2+]i rises were derived
from InsP3-sensitive Ca2+ stores. The concentration dependence of ACh-evoked increases in the total endothelial [Ca2+]i
signal was explained by additional cells responding to ACh and
an increase in [Ca2+]i for each cell. Significantly, InsP3-evoked
[Ca2+]i was reduced in >83% of arteries (n = 6, p<0.01, oneway ANOVA with Tukey’s post hoc test) as intraluminal pressure was increased (60 to 110 to 160 mmHg). These results
provide a new method to visualise and analyse endothelial
[Ca2+]i and suggest that mechanical forces are recognised by
the endothelium and translated into biological responses, at
least in part, by changes in InsP3-mediated Ca2+ signalling.
This work was supported by the Engineering and Physical
Sciences Research Council, the British Heart Foundation and
the Wellcome Trust.
requirements.
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C68
Endothelial mechanosensing via IP3-mediated Ca2+
signalling in rat carotid arteries
C. Wilson1,2, C.D. Saunter3, J.M. Girkin3 and J.G. McCarron2
1Bioengineering, University of Strathclyde, Glasgow, Lanarkshire,
UK, 2Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, UK and 3Biophysical Sciences
Institute, University of Durham, Durham, UK
The endothelium is an interconnected network of cells whose
configuration is critical to the organ’s ability to sense mechanical forces. Haemodynamic forces act on blood vessels via
changes in endothelial [Ca2+]i to evoke a diversity of biological responses, which includes the control of arterial tone, the
extent of smooth muscle proliferation and vascular remodelling. However studying endothelial [Ca2+]i in intact and pressurised blood vessels is challenging (particularly in the larger
arteries in which atherosclerosis and re-stenosis occurs)
because of the thickness of the medial and adventitial layers.
To overcome the difficulty, we developed an imaging system,
using gradient index (GRIN) lens technology, to visualise the
endothelium from inside arteries at normal physiological pressures where the arteries’ structural integrity is maintained.
Our results show that mechanotransduction may occur via
changes in InsP3-evoked Ca2+ signals.
Segments of common carotid arteries from male Wistar rats
(150-250 g) were removed and mounted in a custom-built ateriograph superfused with MOPS buffered saline (37°C). The
Ca2+-sensitive fluorescent indicator Oregon Green BAPTA-1 AM
Can altered vascular function explain the increased risk of
stillbirth and fetal growth restriction in advanced maternal
age pregnancies?
S.C. Lean, A. Heazell, L. Peacock, J. Boscolo-Ryan, T. Mills and
R.L. Jones
Maternal and Fetal Health, University of Manchester, Manchester,
UK
Women of advanced maternal age (AMA; >35 years) are an
increasingly prevalent obstetric population and a high risk
group for fetal growth restriction (FGR) and stillbirth. Adequate perfusion of the utero-placental unit and blood flow to
the fetus are essential for a healthy pregnancy; under-perfusion of the placenta is associated with FGR, which is in turn
associated with stillbirth. It is known that ageing affects the
cardiovascular system, including a loss of vascular responsiveness and vascular wall thickening. Studies have found
increased vascular thickness and decreased uterine artery
remodelling in aged pregnant mice, in conjunction with
reduced fetal weight and increased infant death; however the
effect of AMA on utero-placental vascular function has not
yet been investigated in human pregnancies. We hypothesised
that maternal ageing alters placental and myometrial arteries function, leading to increased susceptibility to poor outcomes in these pregnancies. Tissue was collected from pregnant women aged 20-30 (control), 35-39 and ≥40 years
delivering at St Mary’s Hospital, Manchester. Placentas
(n=15/group) were collected following normal vaginal delivery or caesarean section. Myometrial samples were collected
following caesarean section (n=4/group). Placental chorionic
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plate and myometrial resistance arteries (100-500μm diameter) were dissected. Wire myography was used to assess
responses to the vasoconstrictor U46619 (thromboxane
mimetic; 10-9 to 10-5.7 M) and vasodilators sodium nitroprusside (SNP; 10-11 to 10-6 M) and bradykinin (BK; 10-10 to 10-5
M) (myometrium only). Significance is defined as p<0.05 after
2-Way ANOVA with post hoc tests. There was no effect of AMA
on placental artery constriction in response to U46619. Preconstricted placental arteries from 35-39 and ≥40 year old
mothers showed increased relaxation to SNP compared to controls. Myometrial vessels from ≥35 year old mothers showed
decreased constriction to U46619. There was no consistent
effect of vasodilators on myometrial vessels. Both placental
and chorionic plate arteries from older women exhibit altered
vascular reactivity compared with younger mothers. As SNP
acts as an NO donor, the increased response to SNP in placental
arteries from older mothers may be due to increased sensitivity to NO as a result of an age related decrease in endogenous NO. AMA seems to affect placental vascular function
despite the placenta being fetal in origin and therefore a
‘young’ organ. Decreased constriction of myometrial vessels
from AMA mothers may alter blood flow to the uterus and
could be due to age related loss in baroreceptor sensitivity or
circulating maternal factors associated with ageing. Altered
vascular control over blood flow to the placenta and baby may
contribute to the increase in poor pregnancy outcomes in AMA
pregnancies.
inhibitor of κB protein IκBα, and the inducible prostaglandin
synthase enzyme COX 2, were investigated using western blotting / densitometry. Responses between groups were compared using 1-way ANOVA with post hoc Bonferonni test
(p<0.05; means ± SEM). Experiments were completed using
biopsies of three separate patients. Following cell exposure to
IL1β, IκBα expression was considerably reduced within 15 minutes. This was succeeded at 4 hours by marked COX2 upregulation. IL1β stimulated COX2 signal at 4 hours (15.01±1.32
Arbitrary Units (Log Optical Density)) was reduced in the presence of Sc514 at 30uM (7.953±2.08AU) and 50uM
(3.86±2.04AU) and Curcumin at 30uM (8.436±2.973AU) and
50uM (1.09±0.89AU) (Fig 1). Diminution of IL1β stimulated
IκBα degradation was observed on 2 of 3 occasions with both
doses of curcumin and Sc514.In summary both inhibitors were
effective in consistently reducing IL1β-stimulated COX2 expression in human myometrial cells in a dose dependent fashion.
Inhibitor dependent prevention of IκBα degradation did not
always occur indicating that mechanisms other than the canonical NFκB pathway may be affected. It will be of interest to
ascertain the actions of other small molecule putative
inhibitors of NFκB and, also, whether these effects are stimulus-dependent.
Carolan M & Frankowska D (2011). Midwifery 37(6), 793-801
Kovo M et al. (2013). Placenta 34(4), 320-324
van der Heijden OWH et al. (2004). J Soc Gynecol Investig 11(5), 304310
Luscher TF & Tanner FC (1993). Am. J. Hypertens 6, 283S–293S.
requirements.
C70
Evaluation of small molecule inhibitors of nuclear factor
kappa B (NFκB) in abrogating cytokine-stimulated signalling
in isolated human myometrial cells
L.R. Gurney, J. Taggart, S. Robson and M. Taggart
Institute of Cellular Medicine, Newcastle University, Newcastle
Upon Tyne, UK
Preterm birth is a significant and underexposed clinical problem[1]. Current treatment strategies are inadequate and there
is a dearth of therapeutic agents available to ameliorate this
situation[2]. There is a substantial body of evidence that implicates the activation of the transcription factor NFκB as part of
a pro-inflammatory cytokine cascade within the uterine environment following infectious insult [3, 4] making the potential therapeutic value of small molecule inhibitors of the NFκB
pathway to be of interest [5]. Using an in vitro cytokine cell
stimulation model with primary human uterine cells the effectiveness of 2 such inhibitors was compared. Following written informed consent (REC:10/H0906/71) lower uterine tissue
was sampled at elective Caesarean section and myometrial
cells prepared and cultured to passages ≤4. Cells were exposed
to the cytokine stimulant IL1β (10ng/ml) alone or following 1
hour pre-incubation (and subsequent co-incubation) with two
concentrations of putative NFκB inhibitors: Sc514 and Curcumin. Cells were then lysed at selected points over a 4 hour
time course. Following protein assay, the expression of the
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Fig 1. Representative western blotting displaying COX2 protein expression
at 4 time points. Myometrial cells were exposed to either IL1β alone or
following 1 hour preincubation with indicated inhibitor (n=3).
Howey CP, K.M., Lawn JE, Born Too Soon. The Global Action Report on
Preterm Birth., ed. WHO. 2012, Geneva: March of Dimes.
Olson, D.M., et al., Emerging tocolytics: challenges in designing and
testing drugs to delay preterm delivery and prolong pregnancy. Expert
Opin Emerg Drugs, 2008. 13(4): p. 695-707.
Romero, R., et al., The role of inflammation and infection in preterm
birth. Semin Reprod Med, 2007. 25(1): p. 21-39.
Lappas, M. and G.E. Rice, The role and regulation of the nuclear factor kappa B signalling pathway in human labour. Placenta, 2007. 28(56): p. 543-56.
Karin, M., Y. Yamamoto, and Q.M. Wang, The IKK NF-kappa B system:
a treasure trove for drug development. Nat Rev Drug Discov, 2004.
3(1): p. 17-26.
L Gurney was supported with a Wellbeing of Women Research
Training Fellowship (RTF376)
requirements.
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C72
Angiotensin II as a novel regulator of urothelial and bladder
function
Molecular and functional characterisation of KCNQ- and
KCNE-encoded KV7 potassium channels in human term
pregnant myometrium
L. Adjei, J. Li and C. Wu
University of Surrey, Guildford, UK
Bladder disorders such as overactive bladders affect millions
of people worldwide but their pathogenesis is poorly understood. Recently, the role of the urothelium in bladder function
and pathology has generated intense interest, in particular
its regulation by inflammatory mediators. Angiotensin II is
trophic factor and inflammatory mediator, and is involved in
bladder obstruction and hypertrophy. However, its role in
urothelial function has never been studied. The aim of this
study was to identify the presence and localisation of
angiotensin receptor AT1 within the bladder wall and to elucidate the role of angiotensin II in urothelial and bladder function.
Guinea-pigs (male Dunkin-Hartley 450-550g) were euthanized
with schedule-1 procedure. Immuno-fluorescence was performed on frozen bladder sections with an AT1 primary antibody and an Alexa-586-conugated secondary antibody.
Mucosa-intact smooth muscle strips and mucosal strips were
isolated from the urinary bladders. The preparations were
superfused in a HEPES-buffered Tyrode’s solution for functional
measurement. The isometric tension was recorded with a tension-transducer via a bridge-amplifier. The superfusate adjacent to the tissue strip was sampled and ATP release from the
tissue was measured using a luciferin-luciferase assay.
Application of angiotensin II (200nM and 1μM) significantly
increased ATP release from the bladder mucosa (pmoles/g/min,
median (25%-75% range); control: 166 (101- 357) vs. 200nM
angiotensin II: 221 (197 - 1256); n=9, p<0.05; control: 140 (44360) vs. 1μM angiotensin II: 208 (89-641); n=14; p<0.05,
Wilcoxon signed rank test). This effect was largely inhibited
by AT1 antagonist ZD7155. Immuno-fluorescence demonstrated positive staining for angiotensin II AT1 receptor in the
bladder wall, with high intensity in the mucosal epithelium
(n=5). Angiotension II also increased the contractile activity in
mucosa-intact muscle strips (μN/mg tissue, median (25%-75%
range); control: 22.5 (2.9 – 1046.6) vs. 1μM angiotensin II:
148.0 (19.6 – 1483.7); n=7, p<0.05, Wilcoxon signed rank test).
These data provide the first evidence that angiotensin II receptor AT1 is expressed in the bladder urothelium and angiotensin
II stimulates ATP release from the urothelium via AT1 receptors. Angiotensin II is also able to generate a positive inotropic
effect on mucosa-intact smooth muscle, partly through a
paracrine effect of the released ATP. These findings suggest
the significance of angiotension II and urothelial AT1 receptor
in bladder physiology and pathophysiology.
Grundy Fund and BBSRC.
requirements.
Y. Mansour1, H.D. Mistry1, F.A. Adegoke1, C.E. Boylen1,
I.A. Greenwood2, P.D. Taylor1 and R.M. Tribe1
1Women’s
Health, King’s College London, London, UK and
2Division of Biomedical Sciences, St George’s University of London,
London, UK
Background: KV7 voltage gated potassium channels formed
by the combination of KCNQ-encoded, pore-forming α-subunits (KV7.1-7.5) and KCNE-encoded, regulatory β-subunits
(KCNE1-5) play an important role in regulating smooth muscle resting membrane potential and contractile activity. The
aims of this study were to characterise the mRNA and protein
expression of KCNQ1-5 and KCNE1-5 and determine the functional impact of a comprehensive panel of KV7-modulating
compounds.
Methods: Myometrial tissue was obtained, with informed written consent, from pregnant women undergoing elective Caesarean section at term (> 37 weeks’, not in labour, n=28).
KCNQ1-5 and KCNE1-5 mRNA expression was measured by
qRT-PCR and normalised to 3 housekeeper genes. KV7 α (7.1,
7.3 and 7.4) and β (KCNE1, 3 and 4) subunits were detected
using immunohistochemistry (IHC). Spontaneous contractile
activity [mean integral tension (MIT), contraction frequency
and amplitude] was measured in isolated myometrial strips
(n=3-7) and the effect of KV7 blockers (XE991, 20 μM; chromanol 293B, 20 μM) and activators (retigabine, 20 μM; ML213,
20 μM; ICA060973, 20 μM; acrylamide S-1, 10 μM) was compared to vehicle controls (DMSO).
Results: All KCNQ and KCNE isoforms were detected in myometrial tissue with KCNQ4 (Q4>>Q1>Q3>Q2>Q5) and KCNE4
(E4>>E3<E5<E1<E2) being the most highly expressed isoforms
(p<0.01). Protein for KCNQ1, 3 and 4 as well KCNE1, 3 and 4
were detected and localised to myometrial smooth muscle.
The KV7-selective activators retigabine and S-1 significantly
relaxed spontaneous contractions vs vehicle (mean MIT ±SEM:
25.3 ± 3.89%, 19.95 ± 8.73% vs 99.18 ± 2.59%, 94.64 ± 11.03%;
p=0.006, p=0.016 respectively). ICA069673 decreased MIT vs
vehicle controls (27.74 ± 14.96% vs 102.2 ± 12.24%). The
KV7.2/7.4-preferential activator ML213 also appeared to
decrease MIT, but this did not reach significance (p=0.057).
XE991 did not affect MIT but contraction frequency was
increased (210 ± 16.32% vs 112.3 ± 7.18%, p=0.008) and amplitude was suppressed (77.15 ± 4.9% vs 104.9% ± 7.24%,
p=0.016). Chromanol 293B had no effect on contractility vs
control (p=0.191).
Conclusions: These molecular data indicate that Kv7 channel
subunits are expressed at mRNA and protein, forming functional channels. The relaxation induced by Kv7 activation highlights the importance of these channels in modulation of uterine excitability during gestation and parturition.
Funded by an MRC project grant, MRC/KCL PhD studentship
and Action Medical Research grant. We thank the NIHR and
clinical staff involved in recruitment and sample collection,
and all the women who participated in this study.
requirements.
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Pop-Up Science: A student-led public engagement volunteer
scheme
The perceived challenges and benefits of delivering a
blended physiology and pathology module in the
undergraduate medical curriculum
E. Spurring1, A. Bacon1, F.L. Garrod1 and D.I. Lewis1,2
1School of Biomedical Sciences, University of Leeds, Leeds, UK and
2ULBERG,
The ability to storyboard or communicate with non-specialist
audiences are key skills, highly regarded by graduate employers. Recent research (Lewis, unpublished) has shown that there
are limited opportunities within UK STEM undergraduate programmes for students to be provided, either with science communication training, or to participate in public engagement
activities yet there is considerable demand from students for
such provision. In addition, the “Working towards your Future”
report (CBI & NUS, 2011) recommended that Universities provide opportunities, outside of the curriculum, for students to
develop employability skills and gain relevant work experience.
In response to this demand, ES (student) and DL (staff) founded
Pop-Up Science, a student-led, public engagement volunteer
scheme. The objective, for students to work in teams to create and deliver public engagement activites; their audience
predominantly “hard to reach” sections of the community or
those that do not normally engage with science.
This scheme was advertised to to all undergraduate students
within the Faculty of Biological Sciences and 23 students, who
collectively represented all disciplines and year groups across
the Faculty, were recruited. Students attended a public engagement training workshop, they then worked collaboratively in
self-selected groups to create sessions on, for example,
“Healthy hearts”, “Bendy bones” and “Sporting performance”.
The initial public engagement event was led by the scheme
student lead. Students on subsquent sessions were mentored
in the creation and delivery of their sessions by the student
lead or by students who had participated in earlier events. Individual sessions were delivered at venues across Yorkshire
including the Eureka (National Children’s Museum), the Yorkshire Big Bang Festival, Springtime Live (family funday at the
Yorkshire Showground) and the Yorkshire Festival (prelude to
the Tour de France Grand Départ). To gain the most out their
participation, students are required to reflect on their experiences and the skills gained, both before and after delivery of
an event, and to blog these reflections.
To date, the scheme has recieved excellent feedback from the
public, event organisers and the student volunteers; the latter recognising the key skills gained.
“became more confident in myself....better at explaining in a way
that was both fun and interesting”
“being able to think on my feet and adapt to different audiences....a challenge speaking in very lay terms, communication
skills definitely benefited…..good team-working was essential”
This scheme enables students to develop key science communication skills and gain valuable public engagement experience. It provides significant benefits for the Faculty in enhancing the student learning experience, developing graduate
attributes and in the promotion of its activities.
CBI & NUS (2011).
http://www.cbi.org.uk/media/466204/60519E9E213D34268025788F
003808EF__CBI_NUS_Employability%20report_May%202011.pdf
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S.M. Roe1 and J. Houghton2
1Centre
for Biomedical Sciences Education, School of Medicine,
Dentistry and Biomedical Science., Queens University Belfast,
Belfast, Antrim, UK and 2Centre for Medical Education, School of
Medicine, Dentistry and Biomedical Science., Queens University
Belfast, Belfast, Antrim, UK
The importance of both Physiology and Pathology Education
within the medical curriculum is enshrined within The General
Medical Councils “Tomorrow’s Doctors” document (General
Medical Council, 2009). Both subjects are emphasised in the
documents “The Doctor as a Scholar and a Scientist”, section
which stresses the need for graduates to be able to “explain
normal and abnormal human structure and function”. While
physiology underpins this knowledge, pathology makes apparent the link between the basic sciences and clinical medicine
(Marshall et al., 2004). Modern curricula, however, pose a number of challenges. With the advent of integrated, case-based
teaching, the identity of these sciences has been eroded, as
has the time spent teaching them. Indeed, the decline in
pathology in curricula has contributed to a pathology recruitment crisis with a “rapid and spectacular” rise in vacancies
for pathologists (Domizio and Wilkinson, 2006).
In QUB, medical physiology is taught during the first 2 years
in 2 modules, with pathology being taught over the same
period in “Cells, Tissues and Organs” and “Principles of Disease and Treatment” classes. Closer collaboration between the
subjects could prove beneficial. It would strengthen the identity of pathology and the link it has with the basic sciences,
while providing a context for physiology learning, giving students the “desire to learn” essential in creating active lifelong
learners (Svinicki, 1998).
To investigate the need for such collaboration, questionnaires
were distributed to 115 medical students undergoing tutorials in the final semester of second year. Questions focused on
students understanding of physiology and pathology as concepts, on their relevance to clinical skills, and whether overlap between both would be useful. A 5 point Likert scale was
used to evaluate the student response to each of the questions
with 5 indicating strong agreement with a statement and 1
strong disagreement. Ratings are given as mean mark out of
5 ± S.E.M, n = 71. In addition to this, there were 3 open-ended
questions focussing on integration of the 2 subjects.
Students responded with Likert scores over 4.5 in response to
statements on their understanding of physiology and pathology as concepts. Both subjects were felt to contribute to clinical skills (4.7 ± 0.1 in response to questions concerning the
relevance of physiology and pathology education to clinical
skills). Scoring lower were questions on the amount of overlap between physiology and pathology (3.6 ± 0.1) and the need
for an integrated physiology/pathology module (3.9 ± 0.1).
The overlap, however, was considered useful (4.0 ± 0.1).
A separate professional identity for each subject is seen as beneficial by students. Collaboration and interprofessionalism
rather than integration is perhaps more effective at this level.
Domizio, P. & Wilkinson, M. (2006). Pathology teaching in the twentyfirst century The Clinical Teacher 3 (1) 53-59
General Medical Council (2009). In Tomorrows Doctors London: General Medical Council.
Oral Communications
Marshall, R., Cartwright, N. & Mattick, K. (2004). Teaching and learning pathology: a critical review of the English literature Medical Education 38 (3) 302-313
requirements.
Svinicki, M. D. (1998). A theoretical foundation for discovery learning
Advances in Physiology Education 20 (1) S4-S7
requirements.
C76
Using Darth Vader as a case study on pulmonary
pathophysiology
R.M. Berg1, A. Ronit3, L.N. Toksvang1 and R.R. Plovsing2
C75
Enhancing employability and building bridges: Public
engagement opportunities within STEM undergraduate
degree programmes
D.I. Lewis1,2, A. Gutoreva1, L. Carlisle1, L. Cuthbert1, E. Hughes1
and L. Black1
2ULBERG,
In an increasingly technology-driven world, the public understanding of science and public engagement activities by scientists are becoming increasingly more important. Traditionally, the majority of a University’s public engagement activities
are delivered by academic staff yet there is an increasing realisation of the benefits of undergraduate students participating in, or even running, public engagement activities on behalf
of their University. The aims of this study were to investigate
the extent to which science communication training, public
engagement, outreach and service-learning activities are
embedded within STEM undergraduate programmes, both
within the UK and internationally, and to share good practice.
The principal publications databases for STEM subjects were
searched for publications which described credit-bearing science communication training, outreach, public engagement
or service-learning modules within STEM undergraduate programmes, either in the UK or globally. In addition, the programme catalogues of all UK STEM undergraduate programmes were searched for similar modules.
From the database search, 127 publications were discovered.
The majority of identified opportunities were for science students (61%), with this provision largely offered by US and Canadian HEIs (94%). Students predominantly undertook servicelearning (79%) or outreach (16%) activities; there was limited
evidence of science communication training or public engagement activities. In contrast, within the UK, of the 2542 STEM
undergraduate programme catalogues evaluated, only 301
(12%) offered their students science communication training
or the opportunity to participate in public engagement or
related activities; the majority of these for students studying
mathematics. The Institutions offering these programmes represented a complete cross-section of UK HEIs. Students predominantly engaged in outreach activities in schools (42 modules, 40%) or service-learning activities (n=34), with 22 modules
providing training in communicating to lay audiences.
This study has highlighted the limited provision, both nationally and internationally, of science communication training,
public engagement and related activities within STEM undergraduate degree programmes. Given the benefits to graduates, Institutions and Society, HEIs should re-consider their provision of these opportunities within their degree programmes.
1Centre
of Inflammation and Metabolism, Rigshospitalet,
Copenhagen, Denmark, 2Intensive Care Unit 4131, Rigshospitalet,
Copenhagen, Denmark and 3Department of Infectious Diseases,
Rigshospitalet, Copenhagen, Denmark
Background. We recently reported how Darth Vader, the infamous and enigmatic villain from the Star WarsTM franchise,
may be useful teaching tools for conveying physiological concepts to students, because he is likely to suffer from both
obstructive and restrictive respiratory failure due to severe
burns and thermal lung injury resulting from exposure to socalled pyroclastic density currents (1). He consequently
requires treatment with continuous oxygen supplementation
and positive airway pressures, which is provided by means of
a whole-body armored suit that appears to function as an
advanced bilevel positive airway pressure system (1). In the
present study, we evaluated this teaching approach during a
respiratory physiology course for medical students.
Methods. Darth Vader was introduced as a case study in a class
of 24 medical students. The students were encouraged develop
a theory that could explain Darth Vader’s breathing problems
and its putative treatment, by integrating the basic physiological concepts they would learn during the course. The theory should be prepared as a 10 minute talk that would be presented to the rest of the class during an interactive session in
the final lesson of the course. Three weeks later, the students
completed a multiple choice test relating to the core curriculum in respiratory physiology.
Results. The students formed three groups that presented their
theory during the interactive session. Apart from being entertaining to both students and evaluators, resulting in numerous applauds and cheers during the presentations, all groups
appropriately integrated respiratory mechanics, ventilationperfusion relationships, gas exchange, and acid-base regulation on a level that exceeded what was expected in the forthcoming exam. Test scores were highest in students that had
presented a theory during the final lesson, second highest in
the students that had listened to the presentations but not
participated in the competition (Figure).
Conclusion. Our findings demonstrate that using a case from
popular culture a combined collaborative format is both feasible, entertaining, and educational, and furthermore stress
the applicability of the “learning from learners” approach in
physiology education.
The financial support for this study from the Society for
Research into Higher Education is gratefully acknowledged
97P
Oral Communications
1992 institutions studied agreed that collaboration could lead
to opportunities to be able to learn additional novel research
methods that may be an asset for the student later on. Overall, the results obtained from the postgraduate students’
research may have the potential to be of higher quality and
might therefore increase the chances of publication of research
data. Furthermore, the collaborative institutions, which are
usually pre-1992 institutions, are attractive to prospective
employers and hence employment prospects are maximised.
Freestone N.S et al., (2012) Proceedings of the HEA STEM Learning and
Teaching conference 2012
Test on the core curriculum in respiratory physiology. The test was conducted three weeks after the interactive session. The abscissa shows the
number of correctly answered questions, and the ordinate the corresponding percentage of students in each group.
Plovsing & Berg, Anesthesiology 2014.
All the participating Ph.D students from the University of
Oxford (Department of Pharmacology and Department of
Chemistry) and Kingston University (Faculty of Science,
Engineering and Computing).
requirements.
requirements.
C77
Research Collaboration across the 1992 divide : the views
of postgraduate research students from pre- and post-1992
institutions
C78
Physiology teaching in the UK: Is it in decline?
N.S. Freestone and N. Mahay
Pharmacy, Kingston University, Kingston-upon-Thames, Surrey, UK
C. Sam1 and N. Freestone2
1Department
of Pharmacology, University of Oxford, Oxford, UK
and 2Faculty of Science, Engineering and Computing, Kingston
University, Surrey, UK
Previously, it has been shown that undergraduate student experience is enhanced by collaborations in teaching between institutions across the 1992 divide (Freestone et al., 2012). Postgraduate experiences during PhD programmes were
hypothesised to be similarly enhanced following collaboration
across institutions. This study investigates the views of Ph.D
students regarding such collaboration between institutions.
Ph.D students from a pre-1992 institution, the University of
Oxford (n = 10) and from a post-1992 institution, Kingston University (n = 10) were randomly chosen and semi-structured
interviews, questionnaire responses and field notes were used
as data sources. The opinions with regard to attending two
collaborative research institutions were positive from both
groups, however, the reasoning behind these views differed.
Kingston University as a newer university was perceived by
its students to have a more relaxed attitude in terms of its
research community and working ethos. Collaborative institutions were favoured greatly due to the more specialised and
established laboratories available to them however travel
between institutions was potentially a burden especially in
terms of transportation of necessary materials. Kingston University students were also keen to be exposed to a more tightly
knit and “research focused” environment where the majority
of the Principal Investigators shared an office environment
with their PhD students or postdoctoral fellows. Restricted
access to the laboratory facilities at Kingston University at unsociable hours was a common complaint from the students.
On the other hand, pre-1992 institutions such as the University of Oxford allow 24 hour access to the research workplace.
Ph.D students from the University of Oxford regard collaboration between institutions as a means of networking within
their research field giving them more exposure to other experts
in their specific area of study. However, the disadvantage here
seems to be about intellectual property rights when it comes
to collaboration between institutions. Both the pre- and post-
98P
It has been proposed that the teaching of physiology is in decline
in the UK (Naftalin, 2011). This is thought to be especially true in
the case of provision of practical skills.
This study then sought to ascertain the current situation in terms
of physiology delivery to undergraduates in a number of higher
education institutions (HEI’s) in the UK. In addition, the potential
for collaboration between HEI’s in order to sustain physiology as
a separate discipline was explored.
Sixteen institutions that offered a single physiology degree and
five other institutions which had recently merged their physiology
departments with other disciplines were sent a questionnaire. This
asked for information concerning numbers of students studying
physiology, numbers of staff delivering the programmes and the
proportion of time allotted to lectures, tutorials and practicals at
each institution. Furthermore, information on any current or future
plans for collaboration was gathered. As a follow-up to the questionnaire five semi-structured interviews were conducted with
Heads of Physiology Departments or Physiology Course Directors.
Ten HEI’s responded to the questionnaire. The data revealed that
60% of respondents felt there had not been a decline in the number of students enrolled on physiology degrees at their own institution. Paradoxically 70 % of the respondents thought that physiology education in the UK generally was in decline. 60 % of the
respondents indicated that collaboration with another HEI would
be a possible way of safeguarding the future of physiology teaching in the UK. However, only 30 % of the respondents reported any
plans to enter into such a collaboration in the near future.
Interview responses revealed that one of the reasons behind a
perceived decline in physiology teaching in the UK was due to
physiology being subsumed into other degree programmes such
as biomedical science. Additionally, loss of named Physiology
departments was attributed to the focus being shifted to more
molecular or disease-based multi-disciplinary research groupings. Strategies to protect the future of physiology teaching
included things like changing the degree name (eg. medical
physiology) but in most cases did not include any plans for collaboration. Reasons for this included lack of any perceived need
to collaborate, competition between different institutions and
logistical difficulties.
Oral Communications
Conclusion/implications: Knowing the students’ preferred
modes can enrich the learning experience as the educators
should address to the diversity of learning styles among the
students and develop appropriate learning approaches.
In conclusion, this study showed large differences in the provision
of different types of physiology teaching across the HEI’s, a decline
in the number of students studying pure physiology degrees and
loss of departments exclusively devoted to physiology research
and education. Whilst collaboration and sharing of resources and
expertise was viewed as a potential safeguard against further
retrenchment few institutions had considered this option.
Fleming ND. VARK, a Guide to Learning Styles (online).
http://www.vark-learn.com/english/page.asp?p=questionnaire [21
October 2013].
Naftalin, R.J. (2011) The Scientist.
Wehrwein EA et al. (2007). Adv Physiol Educ 31, 153–157.
This project was funded by a Physiological Society Teaching
Grant
requirements.
C79
Learning styles of first-year Romanian dental students
A.
Dumitrescu1
and D.
Badita2
1Private practice, Bucharest, Romania and 2Physiology, University
of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania
Motivation/problem statement: Learning style is defined as the
manner in which and the conditions under which learners most
efficiently and effectively perceive, process, store, and recall
what they are attempting to learn. One of the many characteristics that make up a student’s learning style is the sensory
modality by which the student prefers to take in new information. The aim of this descriptive study was to determine
the learning styles of first-year dental students using the Romanian version of the visual, auditory, read-write, kinesthetic (VARK)
questionnaire (Fleming, 1995).
Methods/procedure/approach: This study was performed at
the Department of Physiology, University of Medicine and Pharmacy “Carol Davila” Bucharest in November 2013. The Romanian version of the VARK questionnaire was administered to firstyear dental students to determine their preferred mode of
learning. Of the 276 students, 262 students (94.92%) completed
the questionnaire.
Results: Among the study sampe, the unimodality preference
was 46.6% and multimodality was 53.4% with no gender differences (chi2 = 0.018; P>0.05). Of the students who preferred
unimodal presentation of information, 16.4% of students preferred visual, 23.7% auditory, 5.3% reading/writing and 8% kinesthetic modes of information presentation. A further comparison was made of the distribution of single dominant preference
male with female dental students. Of the female unimodal learners, 14.9% were visual, 25.1%% were aural, 7.4% were reading/writing and 5.7% were kinesthetic learners. In contrast,
among males, 19.5% were visual, 20.7%% were aural, 1.1% were
reading/writing and 12.6% were kinesthetic learners (chi2 =
9.04; P>0.05). Of the 53.4% of the students who preferred a
multimodal learning style, some students preferred two modes
(bimodal, 3.45%), while some students preferred three modes
four modes (quadmodal, 43.1%) modes of information presentation. No gender differences were observed (chi2 = 0.51;
P>0.05). Of the students who preferred multimodal learning
styles, 92.6% of the students preferred all four modes of learning together. Of the students who preferred two modes of information presentation, 2.5% of the students were visual and auditory 2.5% were visual and kinesthetic, 0.8% of the students were
visual and read-write, 0.8% of the students were auditory and
read-write, and 0.8% of the students were auditory and kinesthetic. The preferred multimodal learning styles did not differ
between male and female students (chi2 = 4.52; P>0.05).
Slater JA et al .(2007). Adv Physiol Educ 31, 336-42.
Dobson JL (2010). Adv Physiol Educ 34, 197-204.
requirements.
C80
Assessment of learning style preferences of medical and
dental undergraduate students
N. Kapoor1, A. Bhagat1 and K. Marwah2
1Physiology, Govt Medical College & Hospital, Chandigarh, UT,
India and 2Physiology, HSJ Instituteof Dental sciences & Hospital,
Chandigarh, UT, India
Individuals differ in the way they learn. Learning style is a natural / preferential habitual way by which a student absorbs,
processes and comprehends information in learning situation.
It has a significant effect on the process of acquiring and retaining information. Several models for determining learning styles
have been developed. One of them, the sensory model is VARK
questionnaire based on visual (V), aural (A), read-write (R) and
kinesthetic (K) mode of learning and was used in the study.
Objectives of this study were to determine learning style preferences (LSP) of students with biology background enrolled
in medical/ dental professional college, to compare LSP of medical vs dental students and to identify gender difference if any.
241 medical and dental students,74.0% female and 26%male,
were evaluated for their LSP using VARK questionnaire. 55%
students were found to be unimodal, 9% bimodal and 40.7%
multimodal in LSP. Predominant LSP of 134 unimodal students
was Visual (27), Aural (44), Read-write (6) and kinesthetic ( 55)
. LSP between medical (n=77) and dental students (n=164)
were compared. 57.1% medical students were unimodal
whereas 54.9% dental students were unimodal. 36.3 % medical students were multimodal vs 42.7% dental students. On
comparison of LSP amongst male (n=70) and female students
(n= 171), 80% of females had predominant single modality
preference for K -39.7%, A - 23.4%, R-11.4% and V- 4.2%. Male
students also had a predominant single preferred modality of
learning (86%) and showed a similar percent distribution of
V, A and K modalities, 6.3%, 10.9% and 39.1% respectively.
However, the preference for aural mode of learning was higher
in males (31.3%) as compared to females (23.4%). Acquiring
information utilizing a single predominant modality was most
common irrespective of gender with overall predominance
of kinesthetic mode. For predominant single modality, data
for male and female students were comparable for all modalities except for aural mode which was more preferred among
male students. There was no difference in the preferred learning style among medical and dental students.
requirements.
99P
Poster Communications
PCA001
Sztajzel J. Heart rate variability: a noninvasive electrocardiographic
method to method to measure the autonomic nervous system. Swiss
Med Wkly 2004:134:514-22.
Comparison of heart rate variability in different ABO blood
groups in young adults
Bigger JT, Fleiss JL, Steinman RC, Rolnitzky LM, Kleiger RE, Rottman
JN. Frequency domain measures of heart period variability and mortality after myocardial infarction. Circulation. 1992;85:164-71.
B. Kodavanji1, C. Uppangala2, V. Anantharaya1, A. Noojibail1,
R. Kini1, N. Arunkumar1, S. Pai1 and S. Avabratha3
Stein PK and Kleger RE. Insights from the study of heart rate variability. Annu Rev med. 1999;50:249-61.
1Physiology,
Kasturba medical college, Manipal university,
Mangalore, Karnataka, India, 2Physiology, Kasturba Medical
College, Mangalore, Karnataka, India and 3Paediatrics, Father
Mullers Medical College, Mangalore, Karnataka, India
ABO blood groups have been associated with various disease
phenotypes, particularly cardiovascular diseases 1, 2. Heart
rate variability (HRV) signal is an important tool for studying
the autonomic nervous system3. Decreased HRV is a dependable predictor of future cardiovascular disease 4, 5. This study
was designed to find out the association between different A,
B, O blood groups and HRV in young adults. This cross sectional study included 120 volunteered healthy students. Institutional medical ethical committee approval was obtained and
written informed consent was taken from all the subjects. Subjects were grouped into A, B, AB and O based on their blood
groups determined by slide agglutination test. HRV was analyzed by measuring the R-R intervals using HRV soft 1.1 Version. Time domain and frequency domain method was used
to analyze HRV during normal breathing at rest for five minutes. HRV was also analyzed during deep breathing for one
minute using time domain method. In time domain method,
root of the mean of the squared successive R-R interval differences (RMSSD), and standard deviation of normal RR intervals (SDNN) in milliseconds were analyzed and in frequency
domain method low frequency (LFnu) components (0.04 to
0.15Hz) and the high frequency (HFnu) components (0.15 to
0.4 Hz) in normalized units were measured. The data were
expressed as Mean ± standard deviation. Results were analyzed
statistically by ANOVA (analysis of variance), and P-value <0.05
was taken as significant. Analysis of HRV during deep breathing showed a statistically significant (P value=0.04) increase
in SDNN value in O blood group subjects (75.67 ± 12) compared to A, B and AB blood groups subjects (72.03 ± 13, 73.21±
12, 72.49 ± 14). However, HRV analysis by time domain during normal breathing in different blood groups A, B, AB and O
did not show any statistical significant difference in SDNN value
(58.07 ± 15, 65.54 ± 21, 66.24 ± 19, and 70.95 ± 15; P
value=0.44) and RMSSD value (51.30 ± 20, 51.97 ± 17, 59.36
± 20 and 59.79 ±16; P value=0.738). Further, HRV analysis by
frequency domain method during normal breathing, also did
not show any statistically significant (P value= 0.633) difference among different A, B, AB and O blood groups in LFnu
(53.65 ±17, 52.97 ±23, 59.93 ± 23 and 51.69 ±19) and HFnu
value (46.35 ±17, 47.03 ± 23, 40.07 ± 23 and 48.31 ± 19). In
conclusion this study shows, a better HRV in O blood group
individuals during deep breathing. This may offer some protection from cardiovascular diseases but blood group alone
may not compensate for other factors that are linked to the
cardiovascular diseases. Other blood group individuals may
need to adopt more preventive measure.
Abdollahi AA, Qorbani M, Salehi A, Mansourian M. ABO Blood Groups
Distribution and Cardiovascular Major Risk Factors in Healthy population. Iranian J Publ Health. 2009; 38(3):123-6.
Sheila C, Langman MJS, Macleod IN, Mosbech J, Rahtkens K. ABO blood
groups in patients with gastric carcinoma associated with pernicious
anemia. Gut. 1971;12(6): 465–7.
100P
requirements.
PCA002
Relationship between an electrocardiogram based measure
of action potential duration (APD) restitution and
autonomic function in patients with ischaemic
cardiomyopathy
S.P. Trethewey1, W.B. Nicolson1, P.D. Brown1, P.J. Stafford2,
A.J. Sandilands2, A.L. Monaghan1, F.S. Schlindwein3,
G.P. McCann1, N.J. Samani1 and G.A. Ng1
1Department
of Cardiovascular Sciences, University of Leicester,
Leicester, UK, 2Department of Cardiology, Glenfield Hospital,
Leicester, UK and 3Department of Engineering, Bioengineering
Research Group, Leicester, UK
Introduction: Ischaemic cardiomyopathy (ICM) is a condition
characterised by left ventricular systolic dysfunction (ejection fraction<35% due to underlying coronary artery disease)
that confers increased risk of sudden cardiac death (SCD). A
promising new ECG based biomarker: Regional Restitution
Instability Index (R2I2) has been shown to be an independent
predictor of SCD in patients with ICM. R2I2 quantifies regional
heterogeneity of APD restitution, an electrophysiological property of ventricular myocardium implicated in arrhythmogenesis. Regional heterogeneity of APD restitution has been shown
to be influenced by direct modulation of sympathetic and
parasympathetic input to the heart in an isolated heart model,
however to our knowledge no attempts have been made to
evaluate this relationship in-vivo. This study explores the relationship between autonomic function, reflected by the noninvasive measurement of heart rate variability (HRV) and R2I2
in patients with ICM. Methods: Blinded, prospective, observational study of 44 ICM patients undergoing risk stratification for an implantable cardioverter defibrillator. The R2I2 technique has been described previously: an electrophysiology
study is performed and ECG surrogates for APD and diastolic
interval are used to measure regional APD restitution heterogeneity. Patients underwent 24-hour ambulatory ECG monitoring to determine HRV (Schiller medilog®DARWIN). Time
domain measures of HRV: standard deviation of ‘normal to
normal’ RR intervals (SDNN) and HRV triangular index (HRVi) were computed over the entire 24-hour period. Results: During median follow up of 22 months, 11 patients experienced
ventricular arrhythmia (VA)/SCD. R2I2 was significantly higher
in patients experiencing VA/SCD than those not
(mean±S.E.M:1.14±0.11 vs 0.84±0.05, Students t-test: p=0.01).
Patients with low HRV-i (<20) experienced a higher rate of
VA/SCD than those with high HRV-i (42% vs. 19%, Fisher’s exact
test: p=0.12). Weak negative correlation existed between R2I2
and HRV-i (Spearman’s rank correlation coefficient: -0.36,
p<0.05). Patients with high R2I2 (≥1.03) had a significantly
lower HRV-i (median [IQR}: 17.8[6.7] vs 31.9[15.4], Mann-Whitney U test: p<0.001) and SDNN (median [IQR}: 88.8[30] vs
118[47.4], Mann-Whitney U test: p<0.05) than patients with
low R2I2 (<1.03). Conclusion: A weak inverse correlation
between R2I2 and heart rate variability is seen. This finding
suggests regional heterogeneity of APD restitution may be
increased in the setting of autonomic dysfunction. Understanding this relationship will inform future application of R2I2
to patients without autonomic dysfunction and may reveal
insight into the mechanisms underlying sudden cardiac death
in these patients.
requirements.
PCA003
Cardiovascular autonomic function in healthy young adults
with parental history of type 2 diabetes
R.K. Goit1, B. Paudel2 and R. Khadka2
1Department of Physiology, Nepalgunj Medical College, Banke,
Nepal and 2Department of Physiology, B P Koirala Institute of
Health Sciences, Dharan, Nepal
Type 2 diabetes accounts for at least 90% of all cases of diabetes. It is a familiar disease with a lifetime risk of 40% if one
parent has type 2 diabetes. This disease affects not only the
elderly and middle-aged people, but also increasingly young
people. There are also marked racial differences in the prevalence of type 2 diabetes; prevalence rates in Asian and American Indians populations are particularly high. Since the autonomic nervous system plays a major role in normal
physiological function and in the pathogenesis of many medical disorders, measurement of heart rate variability (HRV) provides an easily applied non-invasive method of assessing cardiac autonomic regulation. Some studies have shown that alter
cardiac autonomic regulation in subjects with parental history
of type 2 diabetes. However, there are controversial reports.
Thus, we aimed to study the cardiac autonomic regulation as
measured by HRV in young adults with parental history of type
2 diabetes. The study was conducted on 40 young adults with
parental history of type 2 diabetes and 40 young adults without parental history of type 2 diabetes. The short term HRV
was assessed in both the groups. In time domain measures,
standard deviation of all RR intervals (SDNN) [26.5 (22-33) vs
33.5 (25-37.75) ms, P=0.014], the square root of the mean of
the sum of the squares of differences between adjacent RR
intervals (RMSSD) [25.85 (16.22-33.8) vs 30.9 (24.4-41.67) ms,
P=0.013], and percentage of consecutive RR intervals that
differ by more than 50 ms (pNN50) [4.6 (1.1-13.77) vs 12.4
(2.8-26.82) %, P=0.022] were significantly less in young adults
with parental history of type 2 diabetes. In frequency domain
measures, low frequency (LF) [115.5 (83.75-140.75) vs 141
(104.25-249.75) ms2, P=0.021], high frequency (HF) [114.5
(74.5-179) vs 182.5 (104.25-247) ms2, P=0.006] and HF [33.3
(24.52-53.22) vs 56.8 (43.02-69.17) nu, P=0.002] were significantly less in young adults with parental history of type 2 diabetes. Whereas, LF [49.8 (36.97-69.55) vs 45.2 (35.4-57.02)
nu] and LF/HF [0.98 (0.65-1.62) vs 0.85 (0.5-2.02) %] were comparable between the groups. This study indicates that parental
type 2 diabetes has an impact on the cardiac autonomic function in non-diabetic young adults.
PCA004
Plasma versus whole blood miRNA levels after reperfusion
injury in ST elevation myocardial infarction patients
following primary percutaneous coronary intervention
B. Littlejohns1, E. McAlindon2, C. Bucciarelli-Ducci2,
M. Suleiman1 and A. Baumbach2
1University of Bristol, Bristol, UK and 2NIHR Bristol Cardiovascular
Biomedical Research Unit, Bristol Heart Institute, Bristol, UK
Purpose: MicroRNAs (miRNAs) are small non-coding RNAs that
regulate gene expression by interacting with multiple mRNAs.
They can be released from tissue into plasma as a consequence
of damage and appear to reflect the degree of damage seen
in tissues including the heart. The released miRNAs are often
monitored in plasma and not in whole blood where they can
be incorporated into different blood constituents. The aim of
this work was to determine whether miRNAs measured in
whole blood reflect plasma levels. The miRNAs investigated
were miRNA-133a and miRNA-208b which have been shown
to be released from damaged cardiac tissue, miRNA-194 is
linked with the development of heart failure and miRNA-214
is associated with the failing heart, cardiac hypertrophy and
is released from rat hearts into the plasma in response to acute
myocardial infarction.
Methods: 18 patients admitted to hospital with acute ST elevation myocardial infarction (STEMI) who were undergoing
primary percutaneous coronary intervention (PPCI) were
recruited. Blood samples were collected for either plasma or
whole blood miRNA extraction from patients 24 hours after
PPCI. Total RNA was extracted from the plasma using miRNeasy
serum/plasma kit (Qiagen) and RNA from whole blood was collected and extracted using PAXgene blood miRNA kit (Qiagen). miRNAs were measured using TaqMan primers and
miRNA levels were assessed using quantitative PCR. Quantification was performed using cel-miR-39-3p (000200) as the normalising miRNA. miRNAs measured were hsa-miR-133a-3p
(002246), hsa-miR-194-5p (000493), hsa-miR-208b-3p
(002290) and hsa-miR-214-3p (002306) (Life Technologies).
The peak troponin I measurement was taken at 12 hours post
PPCI.
Results: Both miRNA-133a and miRNA-208b levels in plasma,
but not whole blood, showed a significant positive correlation
with peak troponin I release. In contrast, peak troponin I release
showed a positive correlation with miRNA-194 and miRNA-214
levels in whole blood but not in plasma.
Conclusions: These data shows that reperfusion injury-related
miRNAs (133a and 208b) in plasma correlated with cardiac
injury as measured by troponin I levels. However, troponin I
levels correlated with miRNAs related to cardiac remodelling
(e.g. failure) measured in whole blood but not in plasma.
Whether the miRNA levels in plasma or in whole blood are
indicative of long term outcome is currently being investigated.
requirements.
requirements.
101P
PCA005
PCA006
Plasma miRNA changes after reperfusion injury in ST
elevation myocardial infarction patients following primary
percutaneous coronary intervention
Vascular reactivity in normotensive male and female
Nigerians
E. McAlindon1, B. Littlejohns2, A. Baumbach1, M. Suleiman2
and C. Bucciarelli-Ducci1
1NIHR Bristol Cardiovascular Biomedical Research Unit, Bristol
Heart Institute, Bristol, UK and 2University of Bristol, Bristol, UK
Purpose: MicroRNAs (miRNAs) are small non-coding RNAs that
regulate gene expression by interacting with multiple mRNAs.
miRNAs are also released from tissue into plasma as a consequence of damage and appear to reflect both the degree of
damage seen in tissues, including the heart, and also long term
remodelling. The aim of this work was to measure miRNAs in
the plasma of ST elevation myocardial infarction (STEMI)
patients who were undergoing primary percutaneous coronary intervention (PPCI).
Methods: 50 patients admitted to hospital with STEMI and
who were undergoing PPCI were recruited. Plasma extracted
from blood samples that were collected from the patients prior
to PPCI (baseline), 24 hours after PPCI and 3 months after PPCI
was stored at -80°C. Total RNA was extracted from the plasma
using miRNeasy serum/plasma kit (Qiagen). miRNAs were
measured using TaqMan primers and miRNA levels were
assessed using quantitative PCR. Quantification was performed
using cel-miR-39-3p (000200) as the normalising miRNA. miRNAs measured were hsa-miR-133a-3p (002246), hsa-miR-1945p (000493), hsa-miR-208b-3p (002290) and hsa-miR-214-3p
(002306) (Life Technologies).
Results: Both miRNA-133a and miRNA-208b which have been
shown to increase following reperfusion injury were significantly elevated at 24 hours compared to both baseline and 3
month time points. After 3 months miRNA-208b was undetectable in most patients. In contrast, miRNAs associated with
heart failure (miRNA-194 and miRNA-214) did not show significant changes throughout. However, when looking at individual patients 60 % of them had elevated levels of miRNA-194
and miRNA-214 after PPCI.
Conclusions: These data suggest that after reperfusion there
is a release of both miRNA-133a and miRNA-208b into the
bloodstream. Even before reperfusion there is an increase in
these miRNAs suggesting that there is cellular damage prior
to PPCI. Both miRNA-194 and miRNA-214 have implications in
cardiac dysfunction and protection but it appears these are
not released into the plasma as a consequence of cardiac damage. The different patient profiles of miRNA-194 and miRNA214 with respect to long term patient outcome are currently
being investigated.
requirements.
O.A. Abidoye, S.O. Elias and G.A. Umoren
Department of Physiology, Lagos State University College of
Medicine, Ikeja, Lagos, Nigeria
Hypertension is a medical condition that does not have specific clinical manifestations until organ damage sets in and its
prevalence is said to be highest in Africa1. Hence, the need for
a study to assess and compare vascular reactivity in normotensive male and females Nigerians in order to screen for
those at risk of developing hypertension. Thirty-six apparently
healthy, normotensive male and female Nigerians, aged
18years – 37years were exposed to cold pressor test (CPT) after
informed consent was obtained from the subjects and approval
had been granted by the Grants and Experimentation Ethics
Committee of the College of Medicine of the University of
Lagos. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured before and after exposure to the
CPT. Blood pressure was determined by auscultatory method.
Subjects were instructed to immerse one foot up to the ankle
into ice slurry maintained at 4°C for 1 minute2. Vascular hyperreactivity was determined as an increase in the subject’s systolic or diastolic blood pressure ≥15 mmHg after exposure to
the CPT3. Subjects whose blood pressure showed a rise < 15
mmHg be it systolic or diastolic, were designated normoreactive. Data are expressed in mean ± SEM, using GraphPad Statistical Software, Version 5 for Windows (GraphPad Software,
San Diego, California, USA). Changes in SBP and DBP in the
subjects were analyzed using Student’s t- test and p < 0.05 was
considered significant. In the parameters measured, SBP and
DBP increased significantly (p< 0.05) after exposure to CPT.
There was no significant difference (p> 0.05) in SBP and DBP
responses to the CPT between the male and female subjects.
Systolic hyperreactivity was observed in 56% of the male subjects while 50% of them demonstrated diastolic hypereactivity. On the other hand, 50% of the female subjects showed systolic hyperreactivity while 56% of them demonstrated diastolic
hyperreactivity, indicating vulnerability of these individuals to
the development of hypertension in the future.4 Since at least,
half (50%) of the population studied, were hyperreactive in
their SBP or DBP, drastic action on preventive measures should
be targeted at those at risk of developing hypertension so as
to step-down the rising incidence and prevalence of hypertension in Africa.
World Health Organization (WHO), 2013. Global brief on hypertension: Word Health Day. Available at: www.who.int/cardiovascular_diseases/publications/global_brief_hypertension/en/
Dishman RK, Nakamura Y, Jackson EM, and Ray CA. (2003). Blood pressure and muscle sympathetic nerve activity during cold presser stress:
fitness and gender. Psycholophysiology, vol; 40(3):370-80.
Moriyama K and Ifuku, (2010). Increased cardiovascular reactivity to
the cold pressor test is not associated with increase reactivity to isometric handgrip exercise. Eur J Apply Physiol; 108: 837-843.
Matthews KA, Woodall KL, and Allen MT. (1993). Cardiovascular reactivity to stress predicts future blood pressure status. Hypertension
22:479–85.
Prof. Soga Sofola
requirements.
102P
PCA007
The effect of Azadirachta indica leaf extract on the
electrocardiogram of dogs
T.O. Omobowale, O. Adejumobi and O. Abiola
Department of Veterinary Medicine, University of Ibadan, Ibadan,
Oyo State, Nigeria
Azadirachta indica commonly referred to as “Dogonyaro” in
Nigeria has been reported to have several beneficial and medicinal properties. The plant has been used traditionally for the
treatment of gastrointestinal diseases and it has also shown
great potential as remedy for blood parasite infections including trypanosomiasis and malaria. There is however a dearth
of information on the cardiac effect of this plant. This study
was conducted to evaluate the possible effects of the ethanolic extract of A. indica leaves on the electrocardiogram of dogs.
Twenty Nigerian local dogs, obtained from a local dog breeding facility were divided into 4 groups of 5 dogs each. Group
A was the control and were administered only water orally
while groups B, C, and D were orally administered 50mg/kg,
100mg/kg and 200mg/kg respectively of the ethanolic extract
of the leaves of A. indica for a period of 7 days. Lead II ECGs
were recorded without anesthesis and analyzed prior to the
administration of the extract and also at the termination of
the experiment on day on day 7. Data were analyzed using
ANOVA at 5% level of significance.
After 7 days of administration, Group C dogs had statistically
signicantly higher (p<0.05) increase in P-R interval value. Also,
there was a statistically significant (p<0.05) increase in the values of the QTc Fridericia in both group B and C dogs. Mean Rwave amplitude value was significantly (p<0.05) lower in group
D dogs.
The use of A. indica should be with great caution as prolonged
administration could have adverse effects on the heart.
requirements.
points, corresponding to the animals in resting or active periods. Action potentials were recorded at 35-37 °C using the
amphotericin-perforated patch-clamp technique. Electrical
restitution data were obtained using pacing cycle lengths (PCL)
that were shortened until the effective refractory period (ERP)
was reached. APD was allowed to stabilize at each PCL and
the mean APD at 90% repolarisation (APD90) and DI determined from 20 events. The dynamic pacing protocol was
repeated in the presence of isoprenaline (Iso, 10 nM) and Iso
plus the non-selective nitric oxide synthase (NOS) inhibitor Nnitro-L-arginine (NNA, 500 μM). Restitution curves were fitted individually with exponential functions and mean (± SEM)
values for time constants (τ), maximum APD90 and slope at
shortest DI calculated. No significant difference (p > 0.05) in
maximum APD90 was observed under basal conditions in
active (n = 20) versus resting period (n = 24) myocytes. However, there were differences in the shapes of the electrical restitution curves in there basal conditions. τ-values were significantly (p<0.01) shorter (191.8 ± 29.9 ms n=18, and 304.7 ±
29.9 ms n=20) and maximum slopes steeper (1.0 ± 0.08 n=17,
and 0.7± 0.08 n= 19) for active versus resting period myocytes
respectively. ERPs were also significantly (P<0.05) shorter in
active (147 ±0.009 ms, n =16) than resting period (177 ± 0.009
ms, n =11) myocytes. Although resting period myocytes
responded more to Iso, maximum slopes were not as steep
and ERPs not as short as in active period myocytes. Arrhythmic events (alternans and delayed after-depolarizations) were
detected in 12 active and 3 resting period myocytes. Active
period cells responded significantly more to NNA. In conclusion, diurnal variations were observed in single cell electrical
restitution, responses to β-adrenergic receptor stimulation and
NOS activity. Our results indicate active period myocytes are
more susceptible to arrhythmias than resting period ones
requirements.
PCA009
PCA008
Enhancing NOS1 adaptor protein levels decreases
intracellular Ca2+ handling and neurotransmission in
cardiac sympathetic neurons
Diurnal variation of the electrical restitution properties of
acutely isolated guinea-pig left ventricular cardiac myocytes
C. Lu, G. Hao, N. Nikiforova, D. Li, K. Liu, N. Herring and
D. Paterson
R. Khuwaileh1, R. Caves1, N. Davies1, K. Brack2, A. Ng2 and
J. Mitcheson1
Physiology, Anatomy & Genetics, University of Oxford, Oxford,
Oxfordshire, UK
1cell physiology and pharmacology, university of leicester, Leicester,
Purpose: Genome wide association studies have implicated
neuronal nitric oxide synthase adaptor protein (NOS1AP/CAPON) as a potential molecular marker of both QT abnormalities on the ECG and sudden cardiac death (SCD). SCD is
often triggered by cardiac sympathetic stimulation which is
also able to modulate the QT interval. Interestingly, NO generated by NOS-1 reduces noradrenaline (NA) release, but this
pathway is down-regulated in animals with hypertension or
following myocardial infarction. To establish if this adaptor
protein plays a role in sympathetic neurotransmission we investigated whether endogenous NOS1-AP was present in cardiac
sympathetic neurons. We also tested the hypothesis that
enhancing neuronal NOS1-AP expression via adenoviral gene
transfer decreases sympathetic induced NA release by altering
the regulation of intracellular calcium handling.
Methods and Results: Immunohistochemistry demonstrated
that NOS1-AP resides in cardiac sympathetic neurons in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY)
UK and 2Cardiovascular science, University of Leicester, Leicester,
UK
Biological circadian rhythms have an important influence on
ventricular function. The mechanistic basis for the morning
prevalence of cardiac arrhythmias and sudden cardiac death
is unclear, but may reflect a combination of increased sympathetic activity and diurnal variation of genes important for cardiac excitability. In the current study, we investigated whether
there is any diurnal variation in single cell electrical restitution
under basal conditions and with β-adrenergic receptor stimulation. Electrical restitution is the relationship between action
potential duration (APD) and diastolic interval (DI) and
describes APD adaptations at higher heart rates. In the intact
heart, a steep slope of the restitution curve is associated with
ventricular fibrillation. Left ventricular myocytes were isolated
from adult male guinea pigs (500-700 g) at two opposing time
103P
controls, but the expression of NOS1-AP is significantly (P<0.05)
reduced in the SHR (n=6) compared to the WKY (n=6). The
peak [Ca2+]i transient of isolated stellate neurons was significantly enhanced in the SHR (n=12) compared to the WKY
(n=11). A novel noradrenergic cell specific vector (Ad.PRSx8NOS1-AP/mCherry) or its control vector were transfected into
sympathetic stellate neurons in vitro. Western blotting showed
NOS1-AP expression was up-regulated following gene transfer
in the SHR (n=6 v empty virus n=6). The peak [Ca2+]i transient
(n=19) was also reduced compared with Ad.PRSx8-mCherry
alone (n=16). NOS1 inhibition (AAAN, 10μM) significantly
increased the [Ca2+]i transient after Ad.PRSx8-NOS1AP/mCherry transfection. Electrophysiological studies showed
that the peak calcium current (ICa) density in SHR neurons
overexpressing NOS1-AP (-17.8 ± 1.2 pA/pF, n=6) was significantly reduced compared with that of the empty vector control in SHR neurons (-25.2 ± 3.1 pA/pF, n=7). Moreover,
Ad.PRSx8-NOS1-AP/mCherry reduced 3H-NA release from SHR
atria (n=7) compared with empty vector controls (n=6).
Conclusions: Artificial up-regulation of cardiac sympathetic
NOS1-AP via targeted gene transfer can directly attenuate
intracellular Ca2+ and suppress ICa, resulting in decreased
NA release in the SHR. This may provide a novel method for
decreasing enhanced cardiac sympathetic neurotransmission
in disease states where excessive NA release might trigger sudden cardiac death in patients with QT abnormalities.
requirements.
of parallel and perpendicular diffusivities. Further simulations
were conducted where a premature stimulus was applied at
the SAN. The propensity to arrhythmia was evaluated by the
formation of re-entrant scroll waves.
As the Figure illustrates, under physiological anisotropy conditions, a rapid left atrial activation was followed by the right
atrial activation. Excitation waves reached the AV border where
they terminated. Upon reduction of heterogeneity, macro reentry was seen to form. The activation of both atrial chambers
was almost simultaneous.
Fiber orientation is a mechanism for regulating atrial activation and its attenuation is proarrhythmic.
Panels from the anisotropic simulation (left) and isotropic simulation (right).
The sino-atrial node (SAN), left atrium (LA) and right atrium (RA) are shown
in the top left panel of the figure. With isotropy (i.e. no fiber orientation),
a premature pulse at the SAN was seen to give rise to a re-entrant circuit
as shown in the bottom right panel.
Maesen B. et al. (2013). Circ Arrhythm Electrophysiol. 6(5):967-75.
PCA010
Stephenson RS et al. (2012). PLoS ONE 7(4): 10.1371
Activation regulation by fiber orientation in the rabbit atria
Kharche1,2,
Zhao3,
S.R.
J.
S.
B. Smail3 and H. Zhang1
Castro1,
R.
Stevenson4,
J.
Jarvis4,
1School
of Physics and Astronomy, University of Manchester,
MANCHESTER, LANCS, UK, 2CEMPS, University of Exeter, Exeter,
Devon, UK, 3Bioengineering Institute, University of Auckland,
Auckland, New Zealand and 4Research Institute for Sport and
Exercise Sciences, Liverpool John Moores University, Liverpool,
Merseyside, UK
Atrial fibrillation (AF) is associated with alteration of atrial fiber
orientation due to structural remodelling (1). An altered fiber
orientation may lead to re-entry, a hallmark of AF.We show
that a loss of the physiological fiber orientation can initiate reentry.
A 20 μm resolution rabbit atrial anatomical model was constructed based on contrast enhanced micro-CT imaging (2).
The myofiber orientation was reconstructed and visualized
using a novel fibre tracking approach (3). A computationally
efficient Fenton-Karma excitation model for the rabbit atrial
cell type was implemented. 1D strand models were used to
estimate inter-cellular diffusion based on experimental longitudinal and transverse conduction velocities (CV) of 50 cm/s
and 13 cm/s respectively (4, 5). The estimated diffusion constants were used in the 3D anisotropic model which was simulated using the mono-domain reaction diffusion equations.
The SAN location was paced at a physiological pacing rate (2.8
Hz) for 10 beats using a stimulus of 10 pA/pF amplitude for 5
ms. BeatBox, a novel cardiac simulation environment, was used
in all simulations.
To elicit the effects of fiber orientation, computer simulations
were conducted with a reducing amount of fiber orientation
heterogeneity. This was accomplished by reducing the ratio
104P
J. Zhao et al. (2012) Circulation: Arrhythmia and Electrophysiology;5:361-370.
Litchenberg WH et al. (2000) Cardiovasc Res; 45: 379-87.
de Groot JR et al. (2003) Cardiovasc Res; 60: 288-97.
SK and HZ are supported by EPSRC grants (EP/I029664/1,
EP/I029826/1, EP/I030158/1), JZ and Smail are supported by
Emerging Researcher First Grant and Programme Grant from
Health Research Council of New Zealand, SK thanks National
Institute of Computational Sciences for HPC access. Micro-CT
scanning was performed at the EPSRC-funded Henry Moseley
X-Ray imaging Facility, University of Manchester. SC is
supported by an EPSRC doctoral funding.
requirements.
PCA011
Brainstem projection targets of the pro-opiomelanocortin
neurones in the nucleus of the solitary tract
K.A. Davis1, L.M. Powell1, N. Balthasar1, D. Olson2, S. Cerritelli1
and A.E. Pickering1
1School of Physiology & Pharmacology, Medical Sciences Building,
University of Bristol, BS8 1TD, Bristol, UK and 2University of
Michigan, Ann Arbor, MI, USA
Within the CNS, pro-opiomelanocortin (POMC) neurons are
found in the hypothalamus and the nucleus of the solitary tract
(NTS). The role of NTS POMC neurons is not clear but we presented evidence to the Society, that optogenetic activation
produces potent bradycardia and respiratory depression that
is blocked by an opioid antagonist. This suggests they may
release β-endorphin (Cerritelli et al, 2013). We also showed
that POMC neurons project to vagal preganglionic neurons in
the nucleus ambiguus (NA). Here we have extended this analysis to characterise other targets of NTS POMC neurons using
vector-mediated expression of fluorescently tagged synaptophysin, a vesicular membrane protein. Mice expressing Crerecombinase in POMC neurons (Balthasar, 2004) were anaesthetised (ketamine 70mg.kg-1/medetomidine 0.45mg.kg-1 i.p.)
for recovery stereotaxic injections of AAV-Syn-DIO-synaptophysin-mCherry, made bilaterally to the NTS (n=3). Three
weeks later, mice were killed and fixed with 4% formaldehyde
for histological examination. Double immunohistochemistry
for mCherry with either cholinacetyltransferase or neurokinin1 receptors was performed to identify putative synapses of
NTS POMC neurons and their potential post-synaptic targets.
This was visualised with conventional and confocal fluorescence microscopy. Conventional microscopy images were used
to count the numbers of synaptophysin-mCherry puncta representing putative POMC synapses in each section, for at least
8 sections. Synapses were counted within a 50x50μm ROI. A
Kruskal Wallis test with Dunn’s post test was carried out,
whereby regions showing labelling were compared with a control area; the cuneate nucleus. The viral vector labelled a discrete cluster of neurons in the NTS that co-expressed GFP indicating that the labelling was in POMC neurons. We found that
these NTS POMC neurons project specifically to several brainstem regions, involved in cardiovascular control, respiration,
pain modulation and swallowing. The pattern of labelling was
consistent across all mice. There was a high number of terminations per section in the hypoglossal nucleus (25±5, p<0.001),
NA (21±4, p<0.001), rostroventral respiratory group (19±0.14,
p<0.01) and dorsal motor nucleus of vagus (15±2, p<0.01).
The terminations were closely apposed to cholinergic neurons
in the NA, dorsal motor nucleus of vagus and hypoglossal
nucleus, and with neurokinin-1 receptor expressing cells in the
NA and ventral respiratory group. Terminations were also
observed in the raphe obscurus, periaqueductal gray and
parabrachial nucleus. Our results indicate that NTS POMC neurons project to multiple downstream cardiorespiratory centres and this connectivity could account for their potent and
diverse functional influences upon autonomic homeostasis.
Cerritelli S, Balthasar N & Pickering AE. (2013). Anatomical and functional characterisation of pro-opiomelanocortin neurones in the
nucleus of the solitary tract of the mouse. In Proceedings of The Physiological Society 37th Congress of IUPS, pp. PCC078. Birmingham.
Balthasar N, Coppari R, McMinn J, Liu SM, Lee CE, Tang V, Kenny CD,
McGovern RA, Chua SC, Jr., Elmquist JK & Lowell BB. (2004). Leptin
receptor signaling in POMC neurons is required for normal body weight
homeostasis. Neuron 42, 983-991.
Katy Davis and Laura Powell are undergraduates at UoB.
Funded by MRC and Wellcome Trust.
requirements.
PCA012
Methodological considerations
reactivity testing and analysis
for
cerebrovascular
J.A. Inskip, R. Ravensbergen, S. O’Connor and V.E. Claydon
Department of Biomedical Physiology and Kinesiology, Simon
Fraser University, Burnaby, BC, Canada
The cerebral microvasculature is exquisitely sensitive to carbon dioxide (CO2), and cerebrovascular reactivity to CO2 is
commonly quantified as a measure of cerebrovascular function. However, the details of cerebrovascular reactivity protocols and analyses vary considerably between research groups.
We aimed to examine whether these different methodologies
and analyses influence the results of cerebrovascular reactivity testing.
We used dynamic end-tidal forcing to measure cerebral reactivity to CO2 in healthy humans (n=7; 4 male) under conditions
of normoxia (PETO2=100mmHg), hypoxia (PETO2=50mmHg),
and hyperoxia (PETO2=150mmHg). Progressive hypocapnia
was obtained by voluntary hyperventilation to a target
PETCO2=20mmHg, and hypercapnia by hypoventilation with
increased inspired CO2 until PETCO2=55mmHg was achieved.
Supine beat-to-beat blood pressure (Finometer) and middle
cerebral artery blood flow velocity (MCAv; transcranial Doppler
ultrasound) were continuously recorded, as were breath-bybreath PETCO2 and PETO2. Values are mean±SEM, compared by
two-way repeated measures ANOVA.
The sensitivity of the response is reported as the gradient of
the relationship between MCAv and PETCO2 based on a linear,
segmented linear, or sigmoidal fit. There was a significant effect
of the oxygen condition on the sensitivity (P=0.012). There
was a significant effect of the curve fit on the sensitivity
(P<0.001). There was no significant interaction between condition and curve fit (P=0.156) (Figure 1). Comparisons of the
Akaike information criterion (AIC) derived for each fit revealed
a significantly worse fit across all conditions for the linear
regression (linear: 327±14; segmented linear: 313±15; and sigmoid 315±14, P<0.05). The estimated information loss was
equivalent for segmented linear and sigmoid fits.
In some studies, control of PETO2 is initiated only after completion of hyperventilation to the target nadir for PETCO2. We
evaluated whether this impacted our results. There were no
significant differences in the slopes or AIC across conditions
between early or late PETO2 forcing. However, when forcing
was initiated after hyperventilation there were no significant
differences in reactivity between conditions regardless of the
fit applied. The sensitivity was consistently larger across conditions when a sigmoid fit was applied.
Based on these analyses, the choice of curve fitting may influence the outcome of cerebral reactivity testing. Considering
the ability to detect a difference between conditions, rationale for curve fit based on the known response characteristics,
and AIC comparison, a sigmoidal fit is recommended. Initiating forcing of PETO2 after hyperventilation may influence the
ability to detect changes in cerebral reactivity during different
PETO2 conditions.
105P
stimulation to 84±5bpm post-stimulation (n = 6, p<0.01). This
was accompanied by a reduction in PR interval from 117±7ms
to 109±6ms (n=6, p<0.05).
These data indicate that there is no chronic change in underlying vagal tone and dobutamine response associated with six
weeks VNS in the healthy heart. Hoverer, a VNS induced
increase in sinus heart rate and a shortening of the PR interval. Further work will be required to corroborate these findings in the diseased heart.
[1]
Schwartz PJ & De Ferrari GM. (2009) Vagal stimulation for
heart failure: background and first in-man study. Heart Rhythm. 6:11,
pp.S76-S81
[2]
De Ferrari GM et al. (2011). Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure. European Heart Journal. 2011. 32:7, pp.847-55
requirements.
Figure 1. Impact of oxygen condition and curve fit on cerebral reactivity
to manipulation of PETCO2.
This work was supported by the Heart and Stroke Foundation
of Canada.
requirements.
PCA014
Hypertensive rats show altered hemodynamic response to
centrally infused exogenous hydrogen sulfide
M. Sikora, A. Drapala, J. Skrzypecki and M. Ufnal
Department of Experimental and Clinical Physiology, The Medical
University of Warsaw, Warsaw, Poland
PCA013
The effects of chronic vagal nerve stimulation on autonomic
function and the electrocardiogram
E. Radcliffe
University of Manchester, Manchester, UK
Vagal nerve stimulation (VNS) poses a promising approach to
the treatment of heart failure. Now in human clinical trial [1],
VNS has been shown to improve left ventricular ejection fraction, reduce New York Heart Association Classification and
improve quality of life in patients with chronic heart failure
[2]. Such findings could be attributed to a bradycardia induced
improvement in diastolic filling of the ventricles. However,
given that little is known about the underlying mechanisms
of VNS we have sought to characterise its effects in the ovine
heart. Specifically we aimed to assess the chronic effects of
VNS on (i) autonomic function and (ii) the electrocardiogram.
Welsh mountain sheep (female, aged 18 ± 6 months, 26-40kg)
were anaesthetised with isofluorane (1-4% isofluorane v/v in
oxygen). VNS devices (Cyberonics, Houston, Texas) were placed
around the right cervical vagus nerve. Following a two week
recovery period, animals were subject to six weeks VNS. VNS
burst amplitudes were set to induce a 20-30% reduction in
heart rate. Animals were fitted with telemetry jackets (EMKA,
Paris, France) throughout to provide 24 hour ECG measurements. Autonomic function was assessed pre and post stimulation in conscious, gently restrained animals using intravenous infusion of dobutamine, propranolol and atropine. All
ECG analysis was performed using automatic wave detection
software (LabChart, AD Instruments, Oxford) and checked
manually. Statistical significance was assessed using paired Ttests and values of p < 0.05 were considered significant. Values are expressed as mean ± SEM.
Six weeks VNS produced no chronic changes in vagal tone (n
= 8, p=0.51) or dobutamine stress test response (n = 6, p=0.79)
in these animals. VNS induced a chronic tachycardic effect on
natural sinus rhythm, altering heart rate from 64± 5bpm pre-
106P
Introduction: Accumulating evidence suggests that hydrogen
sulfide (H2S) is an important biological mediator involved in
various physiological processes including the regulation of the
circulatory system (1,2,3). Furthermore, it has been postulated
that disturbances in H2S homeostasis may result in hypertension (1). This may be due to the involvement of H2S signalling in both peripheral and the brain mechanisms controlling arterial blood pressure (1,2,3). In this study we checked
the hemodynamic response to intracerebrovetricular (ICV)
infusion of NaHS, a H2S donor in normotensive and hypertensive rats.
Methods: We did experiments on 12-14 weeks old, male, normotensive Wistar Kyoto rats (WKY, n=6), spontaneously hypertensive rats (SHR, n=6), angiotensin II - induced hypertensive
rats (WKY-Ang II, n=6) and high fructose diet-induced hypertensive rats (WKY-HFD, n=6). The experiments were made
according to the Directive 2010/63/EU and approved by the
Ethical Committee of the Medical University of Warsaw. In
WKY-Ang II rats hypertension was induced by osmotic
minipump driven subcutaneous infusion of ANGII
(450ng/kg/min) while in WKY-HFD rats by drinking water containing 10% fructose for 8 weeks. The animals were implanted
with a stainless steel cannula into the lateral cerebral ventricle and one week later with a catheter into the abdominal
aorta. All surgicals procedures were performed under ketamine (100mg/kg i.p.) and xylazine (10mg/kg i.p.) anesthesia.
Mean arterial blood pressure (MABP) and heart rate (HR) were
continuously recorded at baseline and during ICV infusions of
either NaHS at the rate of 300 nmol/h or vehicle (Krebs-Henseleit buffer) in rats freely moving in their home cages, three
days after the last surgery.
Results: ICV infusions of vehicle did not affect MABP and HR.
ICV infusion of NaHS in WKY rats produced an increase in MABP
and HR. In contrast, SHR rats as well as WKY-Ang II and WKYHFD rats showed significant decrease in MABP and HR during
ICV infusion of H2S donor. Autonomic ganglia blockade with
hexamethonium (30mg/kg, i.v.) blunted response to NaHS in
all the experimental groups.
Conclusion: The results provide further evidence that H2S is
involved in the neurogenic control of blood pressure. Furthermore, this study shows that hypertensive rats have altered
responsiveness to H2S, which may suggest that disturbances
in H2S signaling in the brain may be associated with hypertension.
Wang R (2012). Physiol Rev 92, 791-896.
Ufnal M & Sikora M (2011). Curr Pharm Biotechnol 12, 1322-1333.
Ufnal M et al. (2008). Acta Neurobiol Exp (Wars) 68, 382-388.
This work was supported by the National Science Center, grant
No. 2011/01/N/NZ4/03682 and Medical University of Warsaw,
grant 1MA/PM11/13.
requirements.
Conclusions: These data demonstrate that HCN channels in
the dPAG are functionally involved in the regulation of cardiorespiratory activity. Blocking PAG HCN channels reduces
heart rate and breathing rate, while facilitating HCN channel
activity increases heart rate. Furthermore our data shows that
hypertensive rats are more sensitive to HCN antagonism than
their normotensive counterparts. This suggests that PAG HCN
activity is more pronounced in hypertensive phenotype. PAG
HCN channels are thus a potential substrate of central autonomic neuroplasticity that could contribute to alterations in
cardiovascular functioning in disease states when sympathetic
activity is abnormally elevated.
Spary EJ et al (2008) J Mol Neurosci 35 211-224
Biancardi VC et al (2009) FASEB J 23 958-913
Behbehani MM (1995) Progress in Neurobiology 46 575-605
Thornton JM et al (2002) J Physiol 539 615-621
Paxinos G & Watson C (2007) The rat brain in stereotaxic coordinates,
6th ed. Academic Press: San Diego
PCA015
Wellcome Trust
Hyperpolarization-activated cyclic nucleotide gated channel
antagonism in the periaqueductal gray modulates
cardiorespiratory function
requirements.
G. Karimi, E. Mann and D. Paterson
University of Oxford, Oxford, Oxfordshire, UK
Emerging evidence suggests alterations in cardiovascular function could be in part mediated by neuroplasticity of central
autonomic networks [1-2]. In particular the periaqueductal
gray (PAG) is a powerful modulator of cardiovascular parameters [3-4]. The PAG expresses hyperpolarization-activated
cyclic nucleotide gated (HCN) channels throughout its
columns. HCN channels play essential roles in the physiology
of both the cardiovascular system and neural circuits. Therefore, we examined the functional involvement of HCN channels in the PAG in modulating cardiovascular activity in normotensive and hypertensive rats.
Methods: HCN channel modulating compounds were injected
into the PAG and changes in cardiorespiratory parameters were
assessed. Finally we compared hypertensive and normotensive animals in their response to PAG HCN channel modulation. Heart rate and breathing rate of Sprague Dawley (SD),
Wistar Kyoto (WKY) and Spontaneous Hypertensive Rats (SHR)
(270-390g) was recorded in response to in vivo stereotactic
injection via pulse oximetry of the right foot under general
anesthesia (1-3% Isoflurane). For injection into the dorsal PAG
(dPAG) the following coordinates were used [5]: -7.3mm caudal to Bregma, 0.6mm lateral from the medial suture, 4.0mm
deep from the surface of the brain. All data are presented as
mean±SEM with ANOVA for multiple comparisons.
Results: Injection of the non-specific HCN channel blocker
ZD7288 (500nl, 20μg/0.5μl; n=5) into the dPAG of SD rats,
significantly decreased heart rate from 367±7 to 308±6bpm
(p<0.001). Similarly breathing rate was also significantly
decreased (from 55±2 brpm to 44±1 brpm, n=5, p<0.01). Injection of the synthetic cGMP analog 8-Bromo-cGMP (500nl,
10mM; n=6) into the dPAG of SD rats, significantly increased
heart rate from 331±5 to 350±3bpm (p<0.005). Injecting
ZD7288, 8-Bromo-cGMP or saline outside the PAG did not
change heart rate or breathing rate. Finally, SHR rats showed
a significantly stronger reduction in heart rate in response to
ZD7288 injection (20μg/0.5μl) into the PAG then WKY rats
(SHR: from 360±6 to 285±5bpm; WKY: from 300±6 to
275±5bpm, n=9, p<0.001).
PCA016
Proteomic analysis of cardiac sympathetic neurons from
pro-hypertensive rats uncovers modifications in metabolic
processes and cytoskeletal architecture linked to abnormal
neurotransmission
R.B. Burton1, C. Schmidt2, H.E. Larsen1, C. Lu1, D. Li1, G. Hao1,
J. Shanks1, N. Nikiforova1, G. Bub1, H. Kramer1, C.V. Robinson2
and D.J. Paterson1
1Physiology, Anatomy and Genetics, University of Oxford, Oxford,
UK and 2Chemistry, University of Oxford, Oxford, Oxfordshire, UK
Introduction: Sympathetic hyper-responsiveness is an early
hallmark of hypertension and may even precede the overt clinical signs of the disease itself. Metabolic and oxidative stress
linked to impaired NO bioavailability has been implicated as a
putative cellular determinant underpinning enhanced sympathetic gain (1,2). Recent studies have shown neuronal release
of noradrenaline in animal models of hypertension (3) is related
to greater intracellular calcium transients ([Ca2+]i) (4), associated with impaired nNOS-cGMP signalling (5). However, the
molecular links to abnormal [Ca2+]i regulation are poorly
understood.
Methods: In this study, we used label-free quantitative (LFQ)
proteomics to compare cultured cardiac sympathetic stellate
neurons from pro-hypertensive spontaneously hypertensive
rat (SHR) with normotensive Wistar-Kyoto rat (WKY) pups. We
then performed adenovirus transduction of neuronal nitric
oxide synthase (nNOS) in these SHR (termed SHRnNOS) and
WKY (termed WKYnNOS) cultured neurons. Canonical protein
interaction pathways and networks were generated using Ingenuity Pathway Analysis (IPA) to study protein regulation. In
order to evidence patterns of protein enrichment or depletion
at a systemic level we also carried out functional network analyses of the proteins returned by the LFQ study using network
association data from the STRING database, followed by clustering and functional enrichment analysis using data from the
Gene Ontology.
Results: Multiple regulatory changes of proteins involved in
metabolic processes, stress responses, scaffolding, tissue archi-
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tecture and cell migration were observed in the SHR compared
to the WKY neurons. IPA analysis indicated that pathways
closely related to energy production, cellular organisation and
metabolic function were perturbed in SHR neurons. Unexpectedly we found abnormally high levels of non-muscle
Myosin 9/Myosin IIA in pro-hypertensive neurons. We also
detected down regulation of mitochondrial complexes. The
artificial up-regulation of nitric oxide via viral nNOS transduction rescued these changes observed in the SHR neurons.
Discussion: These results indicate that pre-programming of
genes involved in regulating Ca2+ handling proteins in sympathetic neurons occur even before the phenotype of sympathetic hyper-responsiveness is apparent. Enhancing nNOS
expression rescues several signalling pathways coupled to
metabolic, cytoskeletal and calcium signalling that are all linked
to abnormal neurotransmission in this genetic model of hypertension.
Hamilton, C.A., Brosnan, M.J., McIntyre, M., Graham, D. & Dominiczak,
A.F. Superoxide excess in hypertension and aging: a common cause
of endothelial dysfunction. Hypertension 37, 529-534 (2001).
Touyz, R.M. Reactive oxygen species, vascular oxidative stress, and
redox signaling in hypertension: what is the clinical significance? Hypertension 44, 248-252 (2004).
Li, D., Wang, L., Lee, C.W., Dawson, T.A. & Paterson, D.J. Noradrenergic cell specific gene transfer with neuronal nitric oxide synthase
reduces cardiac sympathetic neurotransmission in hypertensive rats.
Hypertension 50, 69-74 (2007).
Li, D., Lee, C.W., Buckler, K., Parekh, A., Herring, N. & Paterson, DJ.
Abnormal intracellular calcium homeostasis in sympathetic neurons
from young prehypertensive rats. Hypertension 59, 642-649 (2012).
Li, D., Nikiforova, N., Lu, C. J., Wannop, K., McMenamin, M., Lee, C.W.,
Buckler, K.J. & Paterson, D.J. Targeted neuronal nitric oxide synthase
transgene delivery into stellate neurons reverses impaired intracellular calcium transients in prehypertensive rats. Hypertension 61, 2027 (2013).
RABB holds an EP Abraham Cephalosporin Junior Research
Fellowship, Linacre College Oxford. This study is funded by the
BHF.
requirements.
PCA017
MicroRNA and isomiR expression profiles in a rat model of
cardiac hypertrophy
M.K. McGahon, L.J. Ferguson, D.A. Simpson and A. Collins
Centre for Experimental Medicine, Queen’s University Belfast,
Belfast, UK
MicroRNAs (miRNAs) show great promise as therapeutic
agents and targets in disease treatment, and knowledge of
how their profile varies between normal and hypertrophied
heart tissue is crucial for a better understanding of the mechanisms governing cardiac dysfunction. A small RNA cDNA
library was generated from left ventricular tissue of 8-month
old male spontaneously hypertensive heart failure rats (SHHF;
a model of cardiac hypertrophy, n=4) and Wistar-Furth controls (WF, n=5). miRNA expression was investigated using Illumina next-generation sequencing and CLC Genomics Workbench v4.0. Differences in expression (reads per million
mapped to miRBase v20; RPMM) were observed for some miRNAs between the WF and SHHF samples. These included miR133a, which is abundant in the heart, downregulated in cardiac hypertrophy/heart failure, and involved in myocardial
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remodelling. RPMM for grouped miR-133a sequences aligned
to the canonical sequence in miRBase v20, allowing for up to
3 additions or deletions at either end and up to 2 mismatches,
were 117,071.51±11891.51 (SHHF) and 172,213.62±7401.58
(WF; mean±SEM); Baggerley’s p value after false detection rate
correction <.05. We and others have shown that much of cardiac miR-133a exists as isomiRs with a single base 5’ truncation (Δ1 5’ isomiRs) that changes the seed region and the set
of predicted target genes. Downregulation of miR-133a is
reported to occur in cardiac hypertrophy/heart failure, but this
has not been investigated for individual miR-133a isomiRs. The
total RPMM (without mismatches) was 35% lower in SHHF for
all Δ1 5ʹ isomiRs (33695±3219 SHHF; 52002±3523 WF; p<.05;
t-test); and 29% lower in SHHF for all ‘canonical-seed’ isomiRs
(34521±3289 SHHF; 48780±3484 WF; p<.05; t-test). DIANAmicroT v3.0 and TargetScan Custom together predict 746 target genes for canonical-seed isomiRs and 461 genes for Δ1 5’
isomiRs, with 294 of these genes common to both isomiR
types. Of the 452 genes predicted to be targeted by canonical-seed isomiRs but not by Δ1 5ʹ isomiRs, there are 223 that
are upregulated during myocardial remodelling, according to
our analysis of the literature and microarray datasets in Gene
Expression Omnibus. Of the 167 genes predicted to be targeted by Δ1 5ʹ isomiRs, but not by canonical-seed isomiRs,
there are 81 that are upregulated during myocardial remodelling. These results provide evidence that miR-133a Δ1 5ʹ
isomiRs are downregulated in cardiac hypertrophy, and are
likely to make a selective contribution to myocardial remodelling.
British Heart Foundation grant # PG110/039/28365
requirements.
PCA018
TMAO affects hemodynamic response to Ang II and restraint
stress in rats
M. Ufnal1, R. Jazwiec2, A. Drapala1, M. Sikora1, S. Janusz1 and
M. Dadlez2
1Department of Experimental and Clinical Physiology, The Medical
University of Warsaw, Warsaw, Poland and 2Mass Spectrometry
Laboratory, Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, Warsaw, Poland
Trimethylamine N-oxide (TMAO) is the organic compound
which concentration in the blood increases after ingestion of
dietary phosphatidylcholine. Major sources of phosphatidylcholine include red meat. Resent evidence suggest that elevated fasting plasma levels of TMAO is associated with
increased risk of major adverse cardiovascular events in
humans (1). There is no data available on the effects of TMAO
on arterial blood pressure (BP), and plasma TMAO level in rats
has not been established. We checked the plasma level of
TAMO in rats as well as the effect of TMAO on BP at baseline
and during restraint stress in normotensive rats and rats with
angiotensin II (Ang II)-induced hypertension.
We did the study on four groups (n=6) of male, Sprague Dawley rats weighing 300-320g, fed standard laboratory chow. The
experiments were made according to the Directive 2010/63/EU
and approved by the Ethical Committee of the Medical University of Warsaw. Rats were implanted with telemetric transmitters (DSI) and continuous recordings of BP and animal activity were made for 7 days before and 28 days during osmotic
minipump driven subcutaneous infusion of either: saline,
TMAO (0.65 mg/hr), angiotensin II (Ang II, 2.96 μg/hr) or Ang
II + TMAO. After two weeks of infusions rats were subjected
to one hour restraint stress induced by immobilization of rats
in plastic tubes. This was repeated for the following 3 days.
After the experiments plasma was collected for evaluation of
TMAO concentration using liquid chromatography coupled
with triple-quadrupole mass spectrometry. All surgical procedures were performed under general anaesthesia with ketamine 30mg and xylazine 3mg IP.
Neither infusion of saline or TMAO affected mean arterial blood
pressure (MABP). Infusion of Ang II significantly increased
MABP. In contrast, combined infusion of Ang II and TMAO failed
to affect MABP. Restraint stress significantly increased MABP
in all the groups, however rats treated with Ang II and Ang II
+ TMAO showed higher increase in MABP than groups treated
with saline or TMAO alone. Baseline plasma concentration of
TMAO in rats was 36.3 ng/mL ± 2.8 (means±SE), which is about
10 times lower than that reported in humans (1). Chronic infusion of TMAO increased its plasma concentration to 4.6 μg/mL
± 0.4, with no effect on well-being and motor activity of rats.
In conclusion, plasma levels of TAMO are lower in rats than in
humans, which likely results from differences in a diet. This
study suggests that TMAO affects BP, however the effects are
ambiguous and may depend on the presence of Ang II or stress
hormones. As this is the first study on the hemodynamic effects
of TMAO in mammals further research is needed to clarify its
hemodynamic effects and possible involvement in cardiovascular diseases such as hypertension.
Tang WH1, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, Wu Y, Hazen
SL. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013; 368(17):1575-84.
This study was supported by the Medical University of Warsaw.
requirements.
PCA019
C-type natriuretic peptide reduces cardiac NE release and
the heart rate response to sympathetic stimulation by
inhibiting neuronal calcium handling
N. Herring, J. Shanks, D. Li, G. Hao, A. Athwal and D. Paterson
Oxford University, Oxford, UK
C-type natriuretic peptides (CNP) was originally identified in
the porcine brain and is considered the predominant neuropeptide of the nervous systems, however expression has also
been identified in extra-neuronal tissue including the heart
and vasculature. Recent data has identified a cardioprotective role for B-type natriuretic peptide (BNP) during cardiac
hypertrophy and heart failure specifically acting via the NPRA receptor. CNP is the ligand for the NPR-B receptor, and its
levels are also elevated during heart failure (Kalra, et. al. 2003).
The expression pattern of NPR-B within cardiac autonomic
innervation and the role of CNP in cardiac neural control currently remains unclear. We hypothesised that CNP, like BNP,
would inhibit sympathetic neurotransmission. Immunohistochemistry demonstrated co-staining of NPR-B with tyrosine
hydroxylase (TH) positive neurons cultured from stellate ganglia of the SD rat. Peak calcium current density in cultured stellate ganglia neurons was measured using whole cell patch
clamp recordings. A reduction in Ca2+ current was observed
with CNP (250nM; 21.7± 2.7%, n=3). Intracellular Ca2+ tran-
sients in response to transient cell depolarisation (50mM KCl,
30 sec) measured using ratiometric fluorescent imaging (fura2AM) were also significantly (p<0.01) reduced by CNP (250nM;
n=9). In isolated atria organ bath preparations CNP (250nM;
n=8) significantly reduced tritiated norepinephrine (3H-NE)
release, in response to 5Hz field stimulation, as well as the heart
rate response to stimulation of the right stellate ganglion
(250nM; n=8). This study provides evidence for the sympathoinhibitory role of CNP, which may be cardioprotective during
chronic heart failure. CNP reduces noradrenaline release and
the heart rate response to sympathetic stimulation via a reduction in neuronal calcium signalling through the NPR-B receptor.
Kalra PR, Clague JR, Bolger AP, Anker SD, Poole-Wilson PA, Struthers
AD, Coats AJ. Myocardial production of C-type natriuretic peptide in
chronic heart failure. Circulation 2003; 107:571-573.
requirements.
PCA020
Beta-blockers do not prevent the pro-arrhythmic action of
high-level sympathetic stimulation: a role for Neuropeptide
Y?
M. Kalla, G. Bub, D.J. Paterson and N. Herring
Burdon Sanderson Cardiac Centre, University of Oxford, Oxford,
Oxfordshire, UK
Purpose: Beta-blockers (BB) are the only anti-arrhythmic drugs
that improve mortality in patients with ischaemic heart disease, but a significant risk of arrhythmia remains. During highlevel sympathetic drive, such as during myocardial infarction,
co-transmitters including neuropeptide Y (NPY) can be released
with norepinephrine. We hypothesised that ventricular fibrillation threshold (VFT) would still be reduced following highlevel sympathetic stimulation even in the presence of a BB. We
also investigated whether NPY is independently pro-arrhythmic and decreases VFT, and if this mechanism is mediated by
the Y1 or Y2 receptor.
Methods: Hearts with intact stellate ganglia (SG) and sympathetic innervation were isolated from male Sprague Dawley
rats (300-350g) and Langendorff perfused in constant flow
mode. VFT was determined by pacing at a fixed cycle length
(150ms x 20 beats) followed by a 5sec 50Hz burst at increasing current amplitude (mA) until sustained VF was induced.
VF was cardioverted with 1ml of potassium chloride solution
(50mmol/L). Control experiments demonstrated that VFT
remained constant over 3 successive inductions (n=6). SG were
stimulated at 10Hz, 1msec pulse width (above capture voltage). Drugs were perfused for 15 minutes before stimulation.
Electrical restitution (RT) was derived from optically mapped
action potentials from hearts loaded with RH237 and blebbistatin (10μM). All data are presented as mean ± standard
error.
Results: Direct right SG stimulation significantly (p<0.05)
increased heart rate and left ventricular developed pressure
(LVDP) and these changes were completely blocked by metoprolol (10μmol/L). However, 2 mins of RSG stimulation in the
presence of metoprolol still significantly reduced VFT
(2.0±0.0.45 vs. 1.08±0.24mA, n=6). A similar result was
observed following 2 mins stimulation of the left SG. Perfusion with the NPY Y1 receptor antagonist BIBO3304 (1μM,
n=4) in addition to metoprolol prevented the reduction in VFT
seen with RSG stimulation. Perfusion with exogenous NPY
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(250nmol/L, n=6) also significantly reduced VFT by 50%
(2.4±0.15 vs 1.2±0.12mA) and also increased coronary vascular resistance (CVR) and LVDP. The action of NPY on VFT,
CVR and LVDP were abolished by the Y1 receptor antagonist
BIBO3304 (1μM, n=6), but not by Y2 receptor blockade with
BIIE0246 (1μM, n=6). Preliminary analysis of RT by optical mapping is suggestive of a steepening of the curve with NPY 250nM
(0.27±0.04 vs NPY 0.42±0.12, n=4).
Conclusion: Prolonged sympathetic stimulation remains proarrhythmic in the presence of BB, implicating sympathetic cotransmitters as a novel arrhythmic trigger. Addition of a NPY
Y1 receptor antagonist to the BB could prevent the pro-arrhythmic action of sympathetic stimulation. NPY can directly
decrease VFT via a Y1 receptor mediated mechanism and
steepens the RT curve.
requirements.
PCA021
The anti-fibrillatory effect of the acetylcholine analogue,
Carbamylcholine, is dependent upon muscarinic and
nicotinic receptor stimulation and the generation of NO
M. Kalla, C. Coughlan, M. Chotalia, G. Hao, G. Bub, D.J. Paterson
and N. Herring
Burdon Sanderson Cardiac Centre, University of Oxford, Oxford,
Oxfordshire, UK
Purpose: Implantable cardiac vagus nerve stimulators are a
promising new treatment for heart failure1, which may improve
both quality of life and ejection fraction. Animal studies also
suggest an anti-fibrillatory effect of stimulating the cardiac
vagus nerve that may involve a nitric oxide (NO) dependent
pathway2, although the exact site of action in the cardiac-neural axis is still debated. We investigated whether carbamylcholine (CCh), a stable analogue of the neurotransmitter acetylcholine, can mimic the effect of vagus nerve stimulation on
ventricular fibrillation threshold (VFT), and whether this mechanism is dependent on muscarinic (MR) and/or nicotinic receptor (NR) stimulation and/or generation of NO.
Methods: Hearts were isolated from adult male Sprague Dawley rats (300-350g) and Langendorff perfused in constant flow
mode (baseline perfusion pressure 54.75±7.27mmHg, LV
developed pressure 77.5±5.6mmHg, heart rate 282.8±3.4bpm,
n=6). VFT was reproducibly determined by pacing at a fixed
cycle length (150msec) for 20 beats followed by a 5sec 50Hz
burst at increasing current amplitude (mA) until sustained VF
was induced. VF was cardioverted to sinus rhythm by perfusion with 1ml of high concentration potassium chloride solution (50mmol/L). Electrical restitution (RT) was derived from
optically mapped action potentials from hearts loaded with
RH237 and blebbistatin (10μM, n=6). NO metabolites (NOx)
were measured in snap frozen coronary effluent using an ozone
chemiluminescence assay. All data are presented as mean±SEM
with ANOVA/t-test where appropriate.
Results: CCh (200nM, n=9) significantly (p<0.05) reduced baseline heart rate from 292±8 to 224±6bpm). Independent of this
heart rate change, CCh also caused a significant increase in
VFT in paced hearts that could be reversed with washout of
the drug (control 1.5±0.25 vs. CCh 2.4±0.4 mA vs. wash out
1.14±0.18 mA). CCh modified cardiac repolarisation with flattening of the RT curve (control 0.36±0.1 vs CCh 0.15±0.03,
n=6, p<0.05). The effect of CCh on VFT was completely abol-
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ished by the MR antagonist atropine (0.1μM, n=6) or the NR
antagonist, mecamylamine (10μM, n=6). The specific nNOS
inhibitor AAAN (10μM, n=6) or the soluble guanylate cyclase
inhibitor ODQ (10μM, n=6) also prevented the rise in VFT with
CCh. CCh perfusion significantly increased NOx content following the onset of bradycardia (pre bradycardia 0.001±0.02
vs post bradycardia 0.23±0.1μmol NOx, n=8).
Conclusions: These data demonstrate that the protective
effect of CCh on VFT is dependent upon muscarinic receptor
and nicotinic receptor stimulation. Generation of NO appears
to be involved in mediating this protective effect, via a nNOS,
sGC dependent pathway.
De Ferrari, G.M. et al (2011). European Heart Journal 32(7):847-55.
Brack, K.E. et al (2011). Cardiovasc Res 91(3):437-46.
requirements.
PCA022
Artificial respiratory sinus arrhythmia increases stroke
volume in rats with left ventricular dysfunction
E.L. O’Callaghan1, R.M. Lataro3, L. Zhao2, A.R. Nogaret2 and
J.F. Paton1
1Physiology and Pharmacology, University of Bristol, Bristol, Avon,
UK, 2Physics, University of Bath, Bath, UK and 3School of Medicine
of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
Respiratory sinus arrhythmia (RSA) is the physiological phenomenon whereby heart rate (HR) is modulated by respiration. Whilst the physiological function of RSA is not well
defined, mathematical modelling predicts that RSA improves
cardiac function (Ben-Tal et al. 2012). Some cardiovascular diseases, including heart failure, are associated with a loss of RSA
(La Rovere et al. 1998). We investigated the physiological
importance of RSA in rats after myocardial infarction (MI) using
a novel central pattern generator (CPG) that couples HR to respiration (Nogaret et al. 2013). Wistar rats (male, 250-300g)
were anaesthetised with ketamine (60mg kg-1) and medetomidine (250μg kg-1, both i.m.) and either the left anterior coronary artery was ligated (n = 8) or sham operation (n = 9) was
performed. After 3 days of recovery, rats were anaesthetised
with isofluorane (1.2-1.8% in pure oxygen). The CPG respiratory input signal was generated from real-time diaphragm EMG
and the stimulus output delivered to the right cervical vagus
nerve via bipolar cuff electrodes. Arterial pressures, HR, respiratory rate, expired air CO2, body temperature and instantaneous flow from the ascending aorta were monitored simultaneously. The effect of artificial RSA versus tonic bradycardia
on cardiac function was tested. Treatment effects were considered statistically significant when P<0.05 (Two-way ANOVA
between post-MI and Sham data and paired T test within postMI and Sham groups). Data are presented as mean ± SEM.
Using the CPG, RSA of magnitude between 15 and 76 beats
per minute (bpm) was generated for 5 min (average RSA amplitude 33±3bpm) in post-MI and Sham rats. Post-MI (infarct size
43±7%) rats had a prolonged duration of systole (55±2ms vs.
Sham 48±3ms, P<0.05) and reduced peak aortic flow (189±31
vs. Sham 265±19 mL/min, P<0.05) confirming left ventricular
dysfunction (Pfeffer et al. 1979). Enhancement of RSA in postMI and Sham rats significantly increased stroke volume (11±2%
and 11±4% respectively, P<0.05) to the same degree, although
this improvement was not different to tonic vagal nerve stimulation at matched average HR (8±1% vs. 9±2% in Sham). The
amplitude of RSA was not correlated to a greater increase in
stroke volume in Sham or post-MI rats (R2 = 0.21 and 0.00
respectively). We have shown that a novel CPG is capable of
inducing RSA in anaesthetised rats. Using this device we have
demonstrated that, in an acute setting, stroke volume in postMI rats increases with RSA but that this is to the same extent
as tonic vagal nerve stimulation. We next hope to understand
the physiological effects of RSA in conscious, healthy and heart
failure rats to understand the purpose of this highly conserved
physiological phenomenon.
Table 1
Ben-Tal et al. (2012). J Phys 590, 1989-2008
La Rovere MT et al. (1998). Lancet 351, 478-484
Nogaret A et al. (2013). J NeuroSci Meth 212, 124-132
Pfeffer MA et al. (1979). Circ Res 44, 503-512
Renata Lataro is supported by Fundacao de Amparo a Pesquisa
do Estado de Sao Paulo (FAPESP).
requirements.
PCA023
Electrophysiological effects of pharmacologically induced
Long QT Syndrome 1 & 2
A. Monaghan, K.E. Brack, J. Mitcheson, E. Wake and G.A. Ng
Dept of Cardiovascular Sciences, University of Leicester, Leicester,
UK
Introduction: Long QT Syndrome (LQTS) types 1 & 2 are ion
channelopathies of the slow- (IKs) or rapid- (IKr) activating
delayed rectifier K+ current that predispose individuals to cardiac arrhythmias, typified by polymorphic ventricular tachycardia known as Torsades de Pointes that can degenerate into
ventricular fibrillation (VF). Mechanisms underlying LQTS
arrhythmic-fatality are not fully known but may relate to
reduced ventricular repolarising capacity. We aimed to use
pharmacological models of LQTS 1&2 using HMR-1556 and
E4031 respectively and examine the effects on the ventricle.
Methods: Adult male Dunkin Hartley guinea pig hearts were
used (410-490g) following a non-Schedule 1 killing method
and perfused in constant flow Langendorff mode (37°C).
Monophasic action potentials (MAP) were recorded at apex
and base, measured at 90% decay (MAPD90). Effective refractory period (ERP) was measured using a single extrastimulus
protocol with VF threshold (VFT) using rapid pacing. All measurements were taken at baseline (BL) and different doses of
HMR (0.1,0.5,1μm,n=6) and E4031 (0.02,0.05,0.1μm,n=6).
Values are mean±SEM, analysed with one-way ANOVA and Bonferroni post-hoc test
Results: Both drugs reduced heart rate and increased ERP &
VFT. The drugs increased MAPD90 at both apex and base in
nearly equal amounts.
Both drugs increased slopemax, with base/apex ratio increasing in a dose-dependent manner.
Conclusion: HMR & E4031 mimic LQTS 1&2 by prolonging APD
and increasing ERP by reducing repolarisation capacity of the
ventricles. The Base/Apex slope ratio is increased by both drugs,
suggesting that the drugs have a greater effect on restitution
at the base than apex. This may be important in arrhythmogenesis in LQTS
Fig 1. RT curves recorded at base sites compare baseline with high dose of
both drugs. Hashed lines show maximum slope
requirements.
PCA024
Comparative decellularization protocols of porcine
pulmonary artery and pericardium for stem cells-based
tissue engineering
D. Iacobazzi, M. Swim, H. Jia, M. Caputo and M. Ghorbel
University of Bristol, Bristol, UK
The use of synthetic materials and biomaterials as part of tissue-engineered vascular devices, such as heart valves and vascular grafts, has long been the focus of research. However,
despite the large availability one of the limits of these materials is the low patency and elasticity associated with the risk
of trombogenicity.
Tissue engineered biological grafts have been shown to have
a more favourable outcome and better longterm patency than
currently available prostetic grafts. A decellularization process
that efficiently removes all cellular and nuclear material, while
preserving the extracellular matrix (ECM) components, is
required in order to prevent any immuno-mediated rejection
that might arise from xenogenic or allogenic tissue implantation.
The aim of this study is to optimize a decellularization protocol specific for different porcine tissues, with a view on recellularization with autologous thymus-derived Mesenchymal
Stem Cells (MSC) to produce tissue-engineered scaffolds that
can be used as a patch for cardiovascular repair.
Porcine pericardium and pulmonary artery collected from
piglet were treated with ionic detergent (SDS), non-ionic detergent (Triton) or enzymatic biologic agent (Trypsin), followed
by incubation with nuclease solution.
H&E histological analysis of decellularized matrices showed
that, while either the Triton and the Trypsin treatment effec-
111P
tively removes the nuclear content from the pericardium specimens, the Trypsin is more efficient in preserving the extracellular tissue content. However, despite the slight tissue break
caused by the Triton treatment, the MSCs growth after reseeding is not affected, as shown by the cell viability/cytotoxicity
assay. Differently, the detergent treatment has proven to be
more effective in removing the nuclear material on pulmonary
artery. In particular, the nuclei content after non-ionic detergent treatment was almost absent compared to the one
observed by treating the tissue with the ionic detergent SDS.
Negligible effects were observed on the extracellular matrix
components using either detergents. However, the Triton was
more compatible with the cells growth, as shown by viability
assay and H&E staining.
This study identifies a tissue-specific decellularization method
for native porcine pericardium and artery that maximized cellular elimination without compromising the integrity of the
tissue, and the feasibility of MSCs growth on decellularized
scaffolds either in a static and a dynamic model of cell culture,
with this latter mimicking an in vivo scenario. Future studies
involve the evaluation of the mechanical integrity and elasticity of the decellularized and recellularized tissues.
requirements.
PCA025
Umbilical cord blood derived stem cells exhibit great
potential in producing a full range of functional vascular
cells for vascular tissue regeneration used in congenital
heart surgery
H. Jia, H. Anthony, M. Caputo, M. Ghorbel, D. Iacobazzi and
S. Suleiman
university of bristol, Bristol, UK
Objectives:
In congenital heart defect corrective surgery, the commonly
used prosthetic replacement grafts limited durability and often
require repeat operations because of their lack of growth
potential. Using the child’s own stem cells to produce tissue
engineered vascular grafts may provide better grafts for surgery. Here we set out to investigate stem cells isolated from
umbilical cord blood that can produce sufficient functional
endothelial cells (ECs) and vascular smooth muscle cells (SMCs)
for tissue engineering autologous vascular grafts.
Methods
Human endothelial progenitor cells (EPCs) and mesenchymal
stem cells (MSCs) were isolated from umbilical cord blood
(UCB) by density gradient centrifugation and culturing in selective media. UCB-EPCs were characterized by their surface markers and EC functional assay (capillary formation assay). UCBMSCs were fully characterized by their cell surface markers and
multipotency. The SMC differentiation was induced by the
growth factor TGF-β1 and analysed by immune-histological,
flow cytometry and molecular biological methods. Those cells
were applied in vascular tissue engineering by seeding on a
decellularized scaffold (CorMatrix ECM) using a hollow organ
bioreactor in vitro. Pig was selected for the in vivo test. Newborn piglet peripheral blood derived MSCs were harvested and
tested for vascular tissue engineering by similar methodology.
Results:
In vitro expanded and differentiated UCB-EPCs displayed
matured EC phenotypes & EC surface makers and were capable to form capillary-like structures when plated with the BD
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matrigel support. UCB-MSCs expressed typical MSC surface
markers. Their multipotency was approved by their adipogenic
and osteogenic differentiation capability. After incubated in
SMC differentiation medium for a period of 8-12 days, histological and molecular analyses showed a consist expression of
SMC-related markers. Those matured cells had satisfactory survival rate and proliferative ability when seeded on the CorMatrix ECM. The histological staining showed that SMCs were
able to form a multi-cells layer on the scaffold. The pig peripheral blood derived MSCs showed similar multipotency, include
the smooth muscle cell differentiation potential, and the potential in vascular tissue engineering.
Conclusions:
Our investigation suggests that, readily available perinatal
tissue, like umbilical cord blood provides good stem cell sources
to use in vascular graft tissue engineering for paediatric surgery. Those stem cells are capable to produce a live vascular
graft when seeded onto a decellularized scaffold.
The Enid Liner foundation
requirements.
PCA026
Effects of ivabradine on the ventricle in isolated guinea pig
hearts
C. Zhang, K.E. Brack, E. Wake and G. Ng
Department of Cardiovascular Sciences, University of Leicester,
Leicester, UK
Purpose: Ivabradine is a novel bradycardic drug used in clinical practice. It acts on the sinoatrial node by blocking the If
channel responsible for the pacemaker potential1. Our aim was
to investigate the electrophysiological effects of Ivabradine
in the guinea pig ventricle.
Method: Hearts were isolated from adult male Dunkin Hartley guinea pigs (N=6, 0.42-0.52kg) and perfused in constant
flow Langendorff mode with Tyrode solution at 20ml/min
(37°C, pH 7.4). Left Ventricular Pressure (LVP) was recorded
with a pressure transducer connected to a fluid filled balloon
in the LV. Contact Monophasic Action Potential (MAP) electrode recorded apical and basal LV MAP duration at 90% decay
(MAPD90) during constant pacing (CP, 200ms CL). Effective
Refractory Period (ERP) was obtained using a single extra-stimulus protocol (S1 200ms CL). Restitution curves were plotted
using MAPD90 vs. the preceding diastolic interval. Ventricular
Fibrillation Threshold (VFT) was measured as the minimum
current required to induce sustained (>10s) VF with burst pacing (30x30ms). Measurements were taken at baseline (BL) and
during 0.1uM - 0.5uM Ivabradine (with constant pacing at BL
and 0.2uM). Data is shown as mean±SEM with one-way ANOVA
and Bonferroni post-hoc test or Paired T-Test. P<0.05 was taken
as significant.
Results: Ivabradine reduced HR and LVP but prolonging ERP
and VFT significantly (see Table 1). During constant pacing,
apical and basal MAPD90 were significantly prolonged with
0.2uM Ivabradine. Ivabradine had significant effects on both
the apical and basal MAPD90 restitution curves shifting them
up and to the left with a trend towards steeper maximum
slopes at high concentrations (see Figure 1).
Conclusion: 0.2uM Ivabradine significantly affects ventricular
electrophysiology in the isolated guinea pig heart despite current literature suggesting If channels to be absent in guinea
pig ventricles. The mechanisms underlying these ventricular
electrophysiological effects merit further investigation.
Table 1: Key:- * vs. BL, †vs. 0.1uM Ivabradine
ation of normal R-R intervals (SDNN) and the total power (TP)
of the power spectrum density. Repeated measures ANOVA
with post hoc pairwise comparisons using Bonferroni correction were used to test differences between conditions and
Pearson correlations accessed the inter-relationships.
Results:In 3 of 9 participants SDNN and TP maximised at 4
b.min-1, whilst the remaining 6 maximised at 6 b.min-1. Significant main effects of FR and VT were found for SDNN [F (5,
40) = 8.195; p<0.001; F (6, 48) = 13.280; p<0.001] and TP [F
(5, 40) = 11.147; p<0.001; F (6, 48) = 7.233; p<0.001] respectively. Effect sizes were moderate for all variables and conditions (ηp2=0.475-0.624). Figures 1 A-D summarise the mean
± S.E.M. for all conditions.
Discussion: There were significant independent effects of FR
and VT, upon SDNN and TP, with maxima at 6 b.min-1 for 6/9
participants, and at the highest VT for all participants. Adopting a higher VT than that used spontaneously at optimal FR
did not significantly increase HRV. Whether VTs over 40% VC
yield significantly greater HRV remains to be explored.
Figure 1: MAPD90 restitution curves in a typical experiment at Apex (A)
and Base (B) at BL, 0.1uM, 0.3uM and 0.5uM of Ivabradine. Restitution
curves (solid lines) are shown with their maximum restitution slope (dashed
lines).
Scheruebel et al. J Mol cell cardiol. 2014 Feb 25;72C :64-73
KEB is supported on a British Heart Foundation Basic Science
Research Fellowship (FS 12/2/29300)
requirements.
PCA027
Evaluation of the independent influences of breathing
frequency and tidal volume upon heart rate variability in
healthy men
P. Vargas, J. González-Alonso and A. McConnell
Centre for Sports Medicine and Human Performance, Brunel
University, Uxbridge, Middlesex, UK
Introduction: Respiratory sinus arrhythmia (RSA), a main component of heart rate variability (HRV), maximises at a breathing frequency (FR) of 4-6 b.min-1 (1), and correlates with cardiac vagal activity (2). The independent influences of FR and
concomitant changes in tidal volume (VT) and PCO2 upon HRV
and autonomic nervous system (ANS) function are unknown.
We tested the hypothesis that HRV may be maximised by controlling both FR and VT.
Methods: Nine healthy, recreationally active men participated
(27.1±2.6 yr). In part 1 the relationship between FR was
assessed and individual FR optima identified, whilst maintaining a constant VT of 30% of vital capacity (VC). In part 2
the optimal FR identified in part 1 was implemented across a
range of VT. Parts 1 and 2 included a semi-spontaneous condition in which only VT or FR were controlled, respectively.
Conditions were randomised and made in an upright-reclined
position. Mild hypercapnia was maintained under all conditions except semi-spontaneous breathing (GA-200 gas analyser,
iWorx Systems Inc.). A biofeedback system (LabView, National
Instruments Inc.) specified respiratory flow and duty cycle,
which were measured by heated pneumotachograph (Hans
Rudolph 3813, Hans Rudolph Inc.), whilst heart rate was measured via 3-lead ECG. Primary outcomes were the standard devi-
Figure. 1. SDNN (ms) for part 1 (A) and part 2 (C), and TP (ms2) for part 1
(B) and part 2 (D) of the study. Fully controlled conditions (䊏); baseline
(䉭); spontaneous FR and VT (䊊). *significantly different from baseline;
#significantly different from 10 b.min-1, p<0.05.
Song H & Lehrer PM (2003). Applied Psychophysiol Biofeedback, 28 (1),
13-23.
Kollai M & Mizsei G (1990). J Physiol, 424 (1), 329-342.
Portuguese Foundation for Science and Technology - FCT
requirements.
PCA028
Augmented vagal control in wind instrument performances
regarding perceived difficulty
C. Blasco-Lafarga, C. García-Soriano, I. Martínez-Navarro and
A. Montoya-Vieco
Physical Education and Sport Department., University of Valencia,
Valencia, Valencia, Spain
Wind instruments require continuous changes in breathing
patterns. Big expiratory effort in high notes is related to a strenuous expiratory strain similar to Valsalva (1), where acute
baroreflex stress evokes a vagal reactivation oriented to quickly
coupling cardiac and respiratory functioning (1, 2). This
improvement of the Respiratory Sinus Arrhythmia reflexes a
cardiac autonomic enhancement, increasing Heart Rate (HR)
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Variability (HRV) (1, 3). However, playing wind instruments is
also a physical and cognitive task which may exert musicians
to their maximum, leading to sympathetic arousal and autonomic worsening. Previous studies have found differences in
HRV in wind musicians regarding anxiety (4) but, to our knowledge, this is the first study looking for differences in the cardiac autonomic control regarding the Perceived Difficulty (PD).
8 male wind instrument musicians (29.13±7.33 years;
69.36±10.31 kg) were asked to rank a list of known performances in terms of PD. Later on, in two normal rehearsals with
no audience (alternate days), musicians performed a mild performance (M), and the most difficult (D) one. After 10 min of
warm up, they performed twice for 20 min, with 5 min of rest.
HR was recorded (Polar 810) in a sitting position, including
20 min previous to the performance and post-performance
registers. The last 500 beats within each 20 min in the four
sample conditions (pre; per1; per2 & post performance) were
retained for further analysis with Kubios (software 2.1). Root
Mean Square of Successive Differences (RMSSD) and Poincare
Plot indexes (SD1 and SD2) were considered due to the nonstationarity of the cardiac signal. A Repeated Measures ANOVA,
followed by Bonferroni post-hoc, was conducted to search for
significant differences regarding PD (between subject factor),
and the interaction between PD and the Time-Course of the
performance (within factor). HRV outputs were log transformed, if needed.
Univariate contrast showed higher lnRMSSD, lnSD1 and SD2
outputs in D (p<0.05), with no differences in RRi and the ratio
SD1/SD2. Regarding the interaction PD*Time-course, univariate contrast showed differences in SD1/SD2 (p<0.05), with
lnRMSSD and lnSD1 close to significance (p= 0.55 for both).
Bonferroni confirmed that both were significantly higher in
Per2_D (p=0,026).
Augmented requirements in wind musicians evoke larger
parasympathetic responses (lnRMSSD; lnSD1) mostly after
30 min playing. Vagal control is diminished in the first lap,
where musicians may be coupling HR dynamics to continuous
changes in breathing. This initial discomfort may cause autonomic reactivation. Unlike similar activities (e.g. singing) (5),
sympathetic arousal seems to be reduced regarding PD and
the interaction PD*Time-course, even while playing (i.e. SD2;
SD1/SD2). Wind instruments are healthy in terms of autonomic
control, and psychological wellness may count for it.
Elghozi JL, Girard A, Fritsch P, Laude D, Petitprez JL. Tuba players reproduce a Valsalva maneuver while playing high notes. Clinical autonomic
research : official journal of the Clinical Autonomic Research Society.
2008;18(2):96-104. Epub 2008/04/17.
Yasuma F, Hayano J. Respiratory sinus arrhythmia: why does the heartbeat synchronize with respiratory rhythm? Chest. 2004;125(2):68390. Epub 2004/02/11.
Miller EB, Goss CF. An Exploration of Physiological Responses to the
Native American Flute. arXiv preprint arXiv:14016004. 2014.
PCA029
Impact of type 2 diabetes on the autonomic profile of
hypertensive subjects
S. Aboudrar, M.M. El Bakkali, H.H. Rkain, F.F. Milouk,
L.L. Coghlan, T.T. Dakka and H.H. Benjelloun
Physiology, UM5 Souissi Faculty of Medicine and Pharmacy, Rabat,
Morocco
Purpose: High blood pressure is a very frequent medical condition, and when associated with type 2 diabetes, the risk of
coronary heart disease is higher. Autonomic nervous system
implication is of interest. The objective of this study was to
assess cardiovascular autonomic reflexes in primary hypertension with and without association to type 2 diabetes.
Method: The following groups were selected: a control group
C (n =102) including healthy subjects and a group of primary
hypertensive patients H (n=100). This group H is divided into
two subgroups, hypertensive diabetics (HD) patients (n=17)
and non diabetics hypertensive (HND) patients (n=83). Both
C and H groups were matched for sex and age. Each patients,
after 10 minutes rest blood pressure (BP) measurement, underwent cardiovascular autonomic reflexes tests as deep breathing (DB), hand grip (HG) and mental stress (SM) tests. Comparison of quantitative changes was performed using the
student’s t-test. The Pearson correlation was used to study the
association between quantitative variables.
Results: Vagal response is of 32,23 %±17,21 in group H and
41,97%±18,56 in group C (p <0,001). The subgroup HD has a
vagal response of 23,09%±11,06 and the subgroup HND was
34,15%±17,7(p<0,001). Regarding sympathetic response,
results showed that hypertensive patients have a response of
20,81%±9,66 meanwhile in the control group this response
was of 15,75%±9,82(p<0,001), without significant difference
between the two subgroups HND and HD (p=0,243).
Conclusion: Hypertensive with type 2 diabetes have a significantly higher sympathetic response when compared to controls and vagal response is significantly lower. The latter is
accentuated by the association of diabetes to hypertension
Key words: Autonomic nervous system; Primary hypertension;
Diabetes; sympathetic response; vagal response.
Valensi P GJE, Amarenco G, Sauvanet JP, Leutengger M, Attali JR. La
neuropathie autonome chez le diabétique. Diabète et Métabolisme
1997;23:1-8.
Vinik AI, & Ziegler, D. Diabetic cardiovascular autonomic neuropathy.
Circulation 2007;115:387−397
requirements.
Harmat L, Theorell T. Heart rate variability during singing and flute
playing. Music and Medicine. 2010;2(1):10-7.
Olsson EM, von Schéele B, Theorell T. Heart Rate Variability During
Choral Singing. Music and Medicine. 2013;5(1):52-9.
requirements.
PCA030
Kinetics of orthostatic blood pressure in primary
hypertension
S. Aboudrar1, F. Milouk2, M. El Bakkali1,2, H. Rkain1, T. Dakka1
and H. Benjelloun1,2
1Physiology,
UM5 Souissi Faculty of Medicine and Pharmacy,
Rabat, Morocco and 2Cardiology, UM5 SOUISSI, Rabat, Morocco
Purpose : Primary HyperTension (HT) is the most prevalent cardiovascular disorder worldwide, and has very significant morbidity and mortality. The objective of this study was to investigate in primary hypertensive patients the kinetics of the
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orthostatic Blood Pressure (BP) during the change from a
supine position to a standing position, as well as during the
standing position, using the orthostatic test.
Patients and methods: This prospective study included a group
of 107 primary hypertensive patients (mean age 55.82 ± 11.35
years, with the extremes ranging from 39 to 80 years). Orthostatic systolic BP (Ortho SBP) was recorded for 10 minutes at
the rhythm of 3 measurements per minute and compared with
the values of supine systolic pre-orthostatic (Preortho SBP).
According to the changes in Ortho SBP, three subgroups of
primary hypertensive patients were selected as below:
Subgroup A: Ortho SBP was higher than mean Preortho SBP
by 10 mmHg or more.
Subgroup B: Ortho SBP was lower than mean Preortho SBP by
20 mmHg or more.
Subgroup C: -20 mmHg < (Ortho SBP – Preortho SBP) < + 10
mmHg.
The kinetics of each groups were recorded.
Results: Mean Preortho SBP was 150.0 ± 6.2 mmHg, with the
extremes ranging from 147 to 163 mmHg. Mean Ortho SBP
was 177.5 ±17.3 mmHg, with the extremes ranging from 135
to 220 mmHg, excluding the patients with OH. The prevalence’s of subgroup A, subgroup B, and subgroup C were,
respectively, 27.1% (29/107), 15.9% (17/107), and 57.0%
(61/107) of which, 69% (20/9), 59% (10/17), and 62.3% (38/61)
were women respectively in each sub-group. The kinetics of
the Ortho SBP for the 3 subgroups was as the following: In subgroup A, in comparison to Preortho SBP, the mean ortho SBP
increases and reaches a maximum value in the first minute of
OT. Ortho HT is maintained throughout the 10 minutes duration of test. In subgroup B, SBP decreases during the whole
OT. OH is maintained throughout the duration of the OT and
in this subgroup 61.7% were women. In subgroup C, the
change in SBP is small, maintained between -9.3 and +1.67,
Conclusions: hypertensives with very similar supine SBP behavior can exhibit widely different ortho SBP. This has important
consequences for both the diagnosis and the treatment of
hypertensives. Careful and effective treatment of hypertensives requires careful consideration and assessment of orthostatic BP.
Key words: primary arterial hypertension, autonomic nervous
system, orthostatic hypertension, orthostatic hypotension.
Benjelloun H, Aboudrar S, and Jroundi I et al, [Sympathetic response
in primary hypertension]. Ann Cardiol Angeiol (Paris), 2009. 58: p.
139-143.
requirements.
PCA031
Characterization of tissue-engineered patches using
scanning electron microscopy
M. Swim, D. Iacobazzi, M. Caputo and M.T. Ghorbel
Making up approximately 1% of live births, congenital heart
defects are the most common birth defect. In the severe forms,
surgery on the vasculature is the only option for treatment.
Often multiple surgeries to replace outgrown vascular conduits are required as the patient matures. The child’s own mesenchymal stem cells (MSCs), which can differentiate into
smooth muscle cells, provide the opportunity to produce tissue engineered vascular grafts that grow, repair and remodel,
thereby eradicating the need for additional surgeries. We have
recently isolated and characterized MSCs derived from tissue
normally regarded as waste in congenital heart surgeries, the
thymus. We’re now investigating the use of these cells to tissue engineer vascular grafts that can be used in paediatric corrective heart surgery.
This study aimed to assess cell growth/viability of cells seeded
onto the CorMatrix (naturally occurring scaffold routinely used
in cardiac surgery) and to examine at high resolution the structure and topography of tissue-engineered patches.
Using live-imaging fluorescence microscopy we showed live
cells on the MSCs seeded-CorMatrix. We then inspected the
topography of the two sides of the CorMatrix using scanning
electron microscopy (SEM). Our data showed that one side
seemed to have a smoother surface than the other. However,
when seeded with MSCs, both CorMatrix sides showed a similar capacity at homing cells. These seeded MSCs formed a
layer with visible cell-cell and cell-matrix connections. Both CorMatrix sides showed the same capacity at homing endothelial cells. Higher seeding density of MSCs on the corMatrix produced a viable cell layer that perfectly covered the CorMatrix
surface.
In conclusion, we showed that thymus MSCs and endothelial
cells are able to grow on CorMatrix for potential use as a graft
for surgical repair of congenital heart defects.
requirements.
PCA032
Murine Creld1 controls cardiac development through
activation of calcineurin/NFATc1 signaling
E. Mass1, D. Wachten2 and M. Hoch1
1Program Unit Development, Genetics & Molecular Physiology,
Molecular Developmental Biology, LIMES Institute, Bonn University,
Bonn, Germany and 2Minerva Research Group, Molecular
Physiology, caesar, Bonn, Germany
Malformation of the cardiac valves and septa accounts for a
large proportion of congenital heart defects. Mutations in the
human cysteine-rich with EGF-like domains 1 (CRELD1) gene
have been described as a risk factor for the pathogenesis of
atrioventricular septum defects (AVSD) (1). However, the physiological function of CRELD1 is not known. We generated
Creld1 knockout-mice (Creld1KO) to unravel the role of Creld1
in heart development. We reveal that Creld1 is an essential
regulator of the calcineurin/NFATc1 signaling pathway (2),
which is crucial for cardiac development. Calcineurin is a phosphatase consisting of a catalytic A and a regulatory B subunit
(3), which dephosphorylates the transcription factor NFATc1
in the cytoplasm, thereby promoting its translocation to the
nucleus (4).
Our results demonstrate that Creld1 interacts with the regulatory calcineurin B subunit, thereby, controlling the phosphatase activity of the catalytic A subunit. We analyzed the
effect of Creld1 on calcineurin function using the translocation of NFATc1 to the nucleus as a read-out (Figure 1). When
expressed in cells, NFATc1 mainly remained in the cytoplasm,
only 6 % of cells displayed NFATc1 localization in the nucleus.
Stimulation with thapsigargin (Tg) activates endogenous calcineruin and caused NFATc1 translocation to the nucleus (75
%). Similarly to Tg, the presence of Creld1 dramatically
increased the number of cells that showed nuclear NFATc1
localization (51 %). Treatment of Creld1-expressing cells with
cyclosporine A (CsA), a potent calcineurin inhibitor, strongly
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reduced the number of cells showing NFATc1 in the nucleus
(1 %). The function of Creld1 is particularly important in the
cells of the endocardial cushion, the precursor of the heart
valve: whereas in wild-type embryos at developmental day
E10.5, NFATc1 translocates to the nucleus, in Creld1KO
embryos NFATc1 remains in the cytoplasm. Thereby, the
expression of NFATc1 target-genes is diminished and cell proliferation in the endocardial cushion is abolished. As a result,
cushion formation is impaired and Creld1KO embryos die at
E11.0. To analyze whether the two missense-mutations
Creld1R107H and Creld1R329C in the human CRELD1 gene
associated with AVSD (1,5) affect the action of Creld1 on calcineurin/NFATc1 signaling, we introduced the corresponding
mutations into the murine Creld1 gene. Both point mutations
dramatically reduced the translocation of NFATc1 to the
nucleus and, thereby, NFAT-dependent gene expression (Figure 1).
In summary, our results demonstrate that Creld1 controls the
calcineurin-dependent translocation of NFATc1 to the nucleus
and that mutations in Creld1 interfere with the action of Creld1
on calcineurin function. Thus, our results provide first insights
into how mutations in CRELD1 affect cellular signaling and,
thereby, the formation of cardiac valves.
Figure 1 Creld1 controls calcineurin-dependent translocation of NFATc1
to the nucleus. Quantification of NFATc1 localization in NIH3T3 cells transfected with NFATc1 alone or together with Creld1 constructs. Upon addition of 1 μM thapsigargin (Tg), NFATc1 (green) translocates to the nucleus.
The presence of Creld1 is sufficient to drive NFATc1 to the nucleus. Addition of the calcineurin inhibitor cyclosporine A (CsA, 1 μM) inhibits the
effect of Creld1-dependent translocation of NFATc1. The point mutations
Creld1R107H (R107H) and Creld1R329C (R329C) reduce the effect of Creld1
on NFATc1 translocation. The relative distribution was determined according to the absolute cell number per condition. At least n = 198 for each condition. CsA: Cyclosporin A, Tg: Thapsigargin.
Robinson, S.W., Morris, C.D., Goldmuntz, E., Reller, M.D., Jones, M.A.,
Steiner, R.D., and Maslen, C.L. (2003). Missense mutations in CRELD1
are associated with cardiac atrioventricular septal defects. Am. J. Hum.
Genet.72, 1047–1052.
Mass E., Wachten D., Aschenbrenner A.C., Voelzmann A., Hoch M.
(2014). Murine Creld1 Controls Cardiac Development through Activation of Calcineurin/NFATc1 Signaling, Dev Cell (2014),
http://dx.doi.org/10.1016/j.devcel.2014.02.012
Klee, C.B., Crouch, T.H., and Krinks, M.H. (1979). Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc.
Natl. Acad. Sci. USA 76, 6270–6273.
Crabtree, G.R., and Olson, E.N. (2002). NFAT signaling: choreographing the social lives of cells. Cell Suppl. 109, S67–S79.
Ackerman, C., Locke, A.E., Feingold, E., Reshey, B., Espana, K., Thusberg, J., Mooney, S., Bean, L.J., Dooley, K.J., Cua, C.L., et al. (2012). An
excess of deleterious variants in VEGF-A pathway genes in Down-syndrome-associated atrioventricular septal defects. American journal of
human genetics 91, 646-659.
requirements.
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PCA033
Cardiomyocyte contractile function and structural
remodelling in the left ventricle of streptozotocin-induced
type 1 diabetic rat
T. Iqbal1, A. D’Souza1, F. Howarth2, E. Adeghate2, K. Bidasse3
and J. Singh1
1Pharmacy and Forensic Science, University of Central Lancashire,
Preston, UK, 2Medicine and Health Sciences, United Arab Emirates
University, Al-Ain, United Arab Emirates and 3Pharmacology and
Neuroscience, University of Nebraska Medical Centre, Omaha, Ne,
NE, USA
Heart failure is a major cause of premature morbidity and mortality in diabetes mellitus (DM), but the underlying mechanisms are elusive and treatment remains empirical. Previously,
we reported that effects of mild hyperglycaemia in type 2
DM was associated with structural remodelling and associated
changes in inflammatory markers of the diabetic heart
(D’Souza et al, 2014). The study now investigated the role of
chronic hyperglycaemia on cardiomyocyte contractile function and histopathological changes in the left ventricle (LV) of
streptozotocin (STZ)-treated type 1 diabetic male Wistar rats
compared to age-matched controls, 2 months after STZ-administration (60 mg/kg body weight) via single intraperitoneal
injection, according to Home Office regulation. Contraction
and intracellular calcium transients [Ca2+]i were measured in
electrically stimulated ventricular myocytes by a video edgedetection system respectively. Histological and immunohistochemical studies were conducted in small portions of LV
tissues stained with Haematoxylin and Eosin and labelled with
FITC-conjugated Lectin and Masson’s trichrome stain for determination of myocyte size and quantitative assessment of fibrosis and caspase-3.
STZ-treated rats had significantly (Student’s t-test; p<0.01)
higher blood glucose values and reduced heart to body mass
ratio relative to controls. Blood glucose levels were 305±7.48
mg/dl and 89.51±3.56 mg/dl and heart to body mass ratios
were 0.24±0.11 and 0.31±0.02 for diabetic (n=8) compared
to control (n=8) rats. When electrically stimulated at 1 Hz, contractility (% of resting length) was depressed in myocytes from
diabetic rats (3.86±0.23%) compared to controls (5.82±0.34
%). Myocytes also exhibited prolonged time(s) for contraction
(129±39 ms (n=19) vs 112.47±5.01 ms (n=16) and relaxation
(48.47±2.90 ms vs 40.71±2.09 ms (n=19)(p<0.05) in DM compared to controls. Alterations in [Ca2+]i manifested as significant (p<0.05) prolonged time to peak (89.53±2.54 ms, vs
77.16±3.5 ms (n=23) and prolonged rate of decay (0.75±0.02
ms vs 0.57±0.02 ms (n=21) of the Ca2+ transient in diabetic
myocytes compared to controls. LV morphology was severely
altered by DM with significant (p<0.05) increments in fibrous
tissue proliferation (5.05±0.44% vs 3.51±0.44%) and smaller
myocyte transverse diameter (9.93±0.26 μm vs 9.11±0.25 μm)
in diabetic compared to control. In STZ-treated LV, the pathology was manifested as focal scarring, myofibrillar loss, vacuolisation and large clusters of cells showing histological signs
of apoptosis. Activity of cleaved caspase-3 (positive cells/mm2)
was also significantly increased in the STZ-treated group
(8.56±0.79) compared to control (1.86±1.30). The results indicate that STZ-induced DM can result in marked histopathological changes that are associated with functional abnormalities of the heart.
D’Souza, A, Wood, N.M., Boyett, M, Adeghate, E., Howarth, C.F.,
Bidasee, K.A. and Singh, J. (2014). Chronic effects of mild hyperglycemia on left ventricle transcriptional profile and structural remodelling in the spontaneously type 2 diabetic Goto-Kakizaki rat. Heart
Failure Review 19: 65-74,
requirements.
PCA034
requirements.
PCA035
Electrophysiological effects of carbon monoxide in murine
myocardium and their putative mechanism
D.V. Abramochkin1,3 and O.P. Konovalova2
1Department
Age-related differences in the production of reactive oxygen
species between neonatal and adult cardiomyocytes are
due to difference in catalase expression
H. Baker, B. Littlejohns, A. Maxwell and M. Suleiman
Purpose: Rat neonatal hearts (14 days old) are more resistant
to ischaemia and reperfusion (I/R) compared to adult. Generation of reactive oxygen species (ROS) during I/R is a key determinant of injury. Therefore antioxidants, including catalase
could underlie this age-related difference in vulnerability to
I/R. Consistent with this is our recent finding that cardiac catalase levels are higher in neonatal rats compared to adult. The
aim of this work was to determine whether this difference in
catalase expression alters the rate of ROS production in both
quiescent and stressed cardiomyocytes in the presence or
absence of a catalase inhibitor.
Methods: Cardiomyocytes were isolated from 14 day old
(neonate) and 250 g (adult) male Wistar rats using a collagenase and protease digestion method. Isolated cardiomyocytes were loaded with a ROS detecting fluorescent dye (DCF)
and then resuspended in a HEPES buffer containing 2.5 mM
CaCl2. Cardiomyocytes were then incubated at 37°C for 30
min with and without the addition of 20 mM 3-amino-1,2,4triazole (3-AT) (a catalase inhibitor). Cardiomyocytes were
aliquoted into a 96-well plate (2000 viable cells per well). The
wells were excited with 485 nm and emission detected at 520
nm using a fluorescent plate reader. DCF fluorescence was
measured every 2 min for 20 min and the rate of DCF fluorescence was calculated. Some wells had 10 μM H2O2 added
immediately prior to the 20 min recording period.
Results: At basal levels (without H2O2) the rate of DCF fluorescence (ROS levels) in cardiomyocytes was similar for both
age groups. However, upon addition of H2O2 the rate of DCF
fluorescence increased significantly more in the adult cardiomyocytes compared to the neonate cardiomyocytes (P <
0.001). Inhibition of catalase by 3-AT in quiescent cardiomyocytes increased the rate of ROS production to a similar degree
for both groups. In contrast, addition of H2O2 in the presence of a catalase inhibitor resulted in a significantly larger
increase in ROS levels in neonatal cardiomyocytes compared
to adult (P < 0.001).
Conclusions: During stress with H2O2, ROS production was
more marked in adult cardiomyocytes compared to neonatal
which is consistent with the higher levels of catalase in neonatal heart. The role of catalase was further confirmed by the
finding that catalase inhibition resulted in a larger increase in
ROS production in neonatal cardiomyocytes compared to
adult. These data suggest that the difference in vulnerability
between adult and neonatal hearts could be in part due to different catalase levels and that its role becomes evident during
stress.
of Human and Animal Physiology, Lomonosov
Moscow State University, Moscow, Russian Federation, 2White Sea
Biological Station of N.A.Pertsov, Lomonosov Moscow State
University, Moscow, Russian Federation and 3Department of
Fundamental and Applied Physiology, Russian National Research
Medical University, Moscow, Russian Federation
For a long time carbon monoxide (CO) has been considered as
a deadly toxic gas. However, during the last two decades CO
has become recognized as an important endogenous signaling compound regulating a number of physiological functions.
It is produced in the organism as a by-product of heme degradation catalyzed by heme oxygenases (HOs). Although CO is
a potent vasodilator and can also modulate cardiac inotropy,
little is known about its effects on cardiac electrical activity.
In the present study electrophysiological effects of CO and one
of their possible mechanisms were examined in murine working myocardium and sinoatrial node.
Standard microelectrode technique was used for registration
of myocardial action potentials (APs). RT-PCR assays were performed to estimate expression levels of phosphodiesterases
(PDEs) in murine myocardium.
Exogenous CO (10-4-10-3M) decreased AP duration in atrial
and ventricular tissue and accelerated pacemaking activity in
the sinoatrial node by facilitating diastolic depolarization.
Inhibitors of heme oxygenases (zinc and tin protoporphyrin
IX, 10-5M), which are responsible for endogenous CO production, induced the opposite effects. Therefore, endogenous
CO probably acts in the same way as exogenous.
Inhibitor of soluble guanylate cyclase (sGC) ODQ (10-5M) suppressed CO effects more than twice, while sGC activator azosidnone (10-5M-3×10-4M) and membrane-permeable cGMP analog BrcGMP (3×10-4M) induced the same effects as CO. Thus,
cGMP-dependent signaling mechanism participates in the
mediation of cardiotropic CO effects. But why effects of CO
are positive in pacemaker and negative in the working
myocardium. The answer is probably in the different activity
of phosphodiesterase subtypes in these types of cardiac tissue.
Milrinone (2×10-6M), selective inhibitor of phosphodiesterase3
(PDE3), which is negatively regulated by cGMP, blocked COinduced rhythm acceleration. In contrast to PDE3, PDE 2 is positively regulated by cGMP. Selective inhibitor of PDE2 EHNA
(2×10-6M) attenuated CO-induced AP shortening, but not the
positive chronotropic effect. Therefore, PDE2 activity prevails
in working myocardium, while PDE3 is more active in pacemaker. However, this fact cannot be explained by difference
in expression of PDE isoforms. We have found that PDE3 mRNA
is more abundant than PDE2 mRNA in all studied regions of
the heart (sinoatrial node, right auricle and right ventricular
wall).
The results suggest that cardiac effects of CO are at least partly
attributed to activation of sGC and subsequent elevation of
cGMP intracellular content. This leads to inhibition of PDE3,
increased cAMP content and positive effects in the sinoatrial
117P
node, while PDE2 stimulation in working myocardium
enhances cAMP degradation and produces negative effects.
tive removal of alkali-block by a single Cys mutation suggests
that the two gates can operate independently.
This work was supported by the Russian Foundation for Basic
Research [14-04-01564 to D.V.A.].
Swietach P, Rossini A, Spitzer KW, Vaughan-Jones RD. H+ ion activation and inactivation of the ventricular gap junction: a basis for spatial regulation of intracellular pH. Circ Res. 2007 Apr 13;100(7):104554.
requirements.
PCA036
Independent high and low pH block of connexin 43 gap
junctions
Garciarena CD, Zhuang N, Malik A, Spitzer KW, Swietach P, Moreno
A, et al. Low and high pH gating of connexin43 channels. Biophysical
Society 57th Annual Meeting, February 2-6, 2013 Philadelphia, Pennsylvania Biophysical Journal, 104(2). 2013.
Supported by BHF funding (to RDVJ).
requirements.
C.D. Garciarena1, K.W. Spitzer2, P. Swietach1, A.P. Moreno2
and R.D. Vaughan-Jones1
1Department of Physiology, Anatomy and Genetics, University of
PCA037
2Nora
Oxford, Oxford, UK and
Eccles Harrison Cardiovascular
Research and Training Institute, University of Utah, Salt Lake City,
UT, USA
Gap junctional channels comprised of connexin (Cx) subunits,
commonly couple adjacent cells in mammalian tissue. We have
shown that intracellular pH (pHi) modulates ventricular junctional conductance and permeability 1. A rise or fall of pHi from
its resting level can be inhibitory. This influence was confirmed
by measuring the pHi-sensitivity Cx43 channel conductance/permeability (the dominant ventricular isoform), with
the protein heterologously expressed in cultured cell-lines 2.
We have now investigated the possible molecular mechanism
of this dual control by pHi.
HeLa or N2a cell-pairs were stably transfected with Cx43. The
open/closed status of Cx43 channels was assessed by two
methods. (i) Cell-to-cell electrical conductance (gj) measured
with dual whole-cell patch-clamp; pHi was displaced globally
by acetate or trimethylamine [TMA] superfusion. (ii) Junctional
H+ ion permeability (PHapp); H+ ions were UV-uncaged (from
nitrobenzaldehyde) in one cell while confocally imaging
(SNARF1) a pHi-decrease in the conjoined cell; pHi was adjusted
to various starting levels before H+-uncaging, by pre-pulsing
cells with weak acid (80mM acetate) or base (30mM NH4Cl).
For the full-length Cx43 protein, gj started to change within
0.5min of a pHi displacement, stabilising in 4-8min. Displacing pHi from 7.1 to 6.4 or 7.3 reduced gj by 49±4% (n=7) and
55±30% (n=5), respectively. Corresponding PHapp values
decreased by 77±10% (n=7, at pHi 6.6) and 80±7% (n=6, at pHi
7.3). Channel modulation by pHi was independent of any Ca2+i
change (100μM AM-loaded BAPTA, n=7-13), and completely
reversible: both gj and PHapp returned to initial values (in 510min) when pHi was returned to control levels.
Cx43m257, a tailless mutant of Cx43 truncated at residue 257,
lacked pHi-sensitivity. PHapp changed by-3±22% (n=8) and 9±10% (n=5) upon pHi displacement from 6.90 to 6.5 and 7.3
respectively. The gj value also remained unaltered during
40mM TMA perfusion and washout (n=3). The C-terminal
cytosolic tail of Cx43 is thus involved in both high and low pHi
block of the channel.
An individual cysteine mutation, Cys298Ala, in the cytosolic
tail, selectively removed Cx43 alkali-block, while preserving
acid-block. PHapp decreased by 77±12% at pHi 6.6 (n=10) but
remained unaltered at pHi 7.35 (+4±26%, n=17). Reducing pHi
(80mM acetate, 4min) decreased gj by 58±7%, whereas raising pHi (40mM TMA, 4min) had no effect (n=5).
We conclude that high and low pHi gating of Cx43 channels
requires intact cytoplasmic tail regions of the protein. Selec-
118P
Modulation of sodium-dependent background current
in rabbit atrioventricular node cells by endothelin-1
S. Bose, L. Hayward, H. Cheng and J.C. Hancox
School of Physiology and Pharmacology, University of Bristol,
Bristol, UK
The atrioventricular node (AVN) plays an important role in the
co-ordination of atrial and ventricular excitation and can act
as a subsidiary pacemaker, should the sinoatrial node fail.
Recent evidence suggests that Na+-dependent background
current (IB,Na) is present and has the potential to play a role in
determining diastolic depolarisation rate in AVN pacemaker
cells (Cheng et al, 2012;2013). However, receptor-modulation
of IB,Na has so far not been identified. The peptide hormone
endothelin-1 (ET-1) has recently been demonstrated to modulate pacemaker activity of AVN cells (Choisy et al, 2012). In
this study, the effect of ET-1 on IB,Na was studied using wholecell patch clamp recording from isolated rabbit AVN cells.
AVN cells were isolated from hearts excised from male New
Zealand White rabbits (2.0-3.0 kg) using previously described
methods (Hancox et al, 1993) and in accordance with UK Home
Office legislation. Whole-cell patch clamp was used to record
IB,Na from AVN cells at 35-37°C using a descending voltage
ramp protocol from +40 mV to -100 mV, under conditions with
major time and voltage-dependent conductances inhibited
(Hagiwara et al, 1992). Under these conditions, the net current with a Cs+-containing pipette solution and Na+-containing external solution is a non-selective cation current (Cheng
et al, 2012;2013). The external [Na+]-dependent component
(IB,Na) of this net current was determined by switching between
150 mM [Na+]e and 150 mM [Tris]e (Na+-free) solutions. Twoway repeated measures ANOVA was used to compare IB,Na
before and during exposure to 10nM ET-1. Values are shown
as mean ± SEM. Rapid superfusion of cells with 10 nM ET-1 was
found to cause a small reduction in IB,Na at voltages below -15
mV (p<0.05, n=7). At -100mV, IB,Na was 1.3±0.23 pA/pF during superfusion with control solution and -0.88±0.19 pA/pF
following exposure to 10 nM ET-1. 10 nM ET-1 was also found
to reduce slope conductance within the normal diastolic membrane potential range (-60 mV to -40 mV) from 0.37±0.07 nS
to 0.12±0.04 nS (p<0.05). ET-1 did not have an effect on currents recorded in the absence of extracellular Na+, confirming that the modulatory effect was confined to the [Na+]edependent current component. To our knowledge, this is the
first demonstration of direct hormonal modulation of IB,Na.
Further work is now warranted to determine the mechanism
of ET-1 action in relation to IB,Na and whether this current can
be modulated by activation of other G-protein coupled receptors in the AVN.
Cheng H, Orchard CH, Boyett MR, Hancox JC. (2012). Sodium-dependent background current in cells from the rabbit atrioventricular node.
Proc Physiol Soc, 28, PC41.
Cheng H, Boyett MR, CH Orchard, Hancox JC. (2013). Pharmacological and pH modulation of sodium-dependent background current in
rabbit atrioventricular node cells. Proc 37th IUPS, PCA045.
Choisy SC, Cheng H, Smith GL, James AF & Hancox JC. (2012). Modulation by Endothelin-1 of Spontaneous Activity and Membrane Currents of Atrioventricular Node Myocytes from the Rabbit Heart. Plos
One 7. e33448.
Hagiwara N, Irisawa H, Kasanuki H & Hosoda S. (1992). Background
current in sinoatrial node cells of the rabbit heart. Journal of Physiology-London 448, 53-72.
Hancox JC, Levi AJ, Lee CO & Heap P. (1993). A method for isolating
rabbit atrioventricular node myocytes which retain normal morphology and function. American Journal of Physiology 265, H755-H766.
This work was funded by the British Heart Foundation
(PG/11/24/28818) and a Wolfson Foundation/Royal College of
Physicians Intercalated award for LH.
requirements.
quency (measured as sparks/1000 μm2 /second) was also
greater: 14.7 ± 3.5 in ventricular myocytes and 31.8 ± 5.4 in
SA node myocytes (p<0.05). Mean spatial width and Ca2+ spark
duration were unchanged between SA node and ventricular
myocytes.
The results show that a greater Ca2+ release occurs from the
SR during Ca2+ sparks in SA node myocytes than in ventricular myocytes. A greater subcellular SR Ca2+ release in SA node
myocytes will facilitate activation of NCX1, which helps drive
the late diastolic depolarization phase of the SA node action
potential.
Lakatta EG et al (2008). Annals of the New York Academy of Sciences
1123, 41-57.
Steele & Steele. (2014). Biophysical Journal 106 566-567.
requirements.
PCA039
Remodelling of the caveolar domain in models of right and
left heart failure in the rat
R. Norman, C. Ross, L. Trelfa, M. Drinkhill, E. White, J. Deuchars
and S. Calaghan
PCA038
A comparison of Ca2+ spark properties in rat sinoatrial node
and ventricular myocytes
L. Miller1, D. Steele2, G. Hart1, A. D’Souza1, H. Dobrzynski1 and
M. Boyett1
1Institute
of Cardiovascular Sciences, University of Manchester,
Manchester, UK and 2School of Biomedical Sciences, University
of Leeds, Leeds, UK
During the diastolic depolarization phase of the sinoatrial (SA)
node action potential, spontaneous localised Ca2+ release
events (Ca2+ sparks) from the sarcoplasmic reticulum (SR)
occur. Elevation of subcellular Ca2+ as a result of these Ca2+
sparks activates the forward mode of the Na+-Ca2+ exchanger
(NCX1), which generates an inward current and accelerates
late diastolic depolarization and helps bring the membrane
potential to the threshold potential for the firing of an action
potential (1). This is referred to as the Ca2+ clock mechanism
of SA node automaticity. In ventricular myocytes, Ca2+ sparks
can occur during diastole, but they serve no known function
other than to relieve the SR of Ca2+ overload. Consequently,
Ca2+ spark characteristics in the two cell types may differ and
the aim of this study was to investigate this.
Single myocytes were isolated by enzymatic digestion of the
SA node and ventricles of hearts taken from adult male Wistar rats weighing approximately 200-225 g. All procedures
were in accordance with the Animals (Scientific Procedures)
Act 1986. Changes in [Ca2+]i were detected by loading cells
with 5 μM Fluo-8 AM. Cells were bathed in Tyrode’s solution
(containing in mM: 140 NaCl, 5.4 KCl, 1 MgCl2, 5 HEPES, 5.5
glucose, 1.8 CaCl2). Confocal Ca2+ imaging was performed
using an Andor Revolution XD confocal system with a Yokogawa CSU spinning disk. Ca2+ sparks were analysed from x-y
image stacks using recently published xySpark software from
Steele and Steele (2).
Mean Ca2+ spark amplitude (ΔF/F0) was 0.39 ± 0.03 in ventricular myocytes (n = 8) and 0.67 ± 0.08 in SA node myocytes
(p<0.05, n=17), an increase of 72%. Average Ca2+ spark fre-
Faculty of Biology, University of Leeds, Leeds, UK
Heart failure (HF) is a major health problem in the UK today
which puts a major burden on the NHS. A hallmark of HF is a
desensitisation in the β-adrenoceptor (β-AR) response, associated with aberrant spatial control of intracellular cAMP. Caveolae, flask-shaped invaginations of the plasma membrane, are
key controllers of β-AR signalling. Many of the proteins involved
in the β-AR cascade are present in caveolar domains or interact with caveolar proteins. The molecular changes which occur
to the β-AR cascade in HF are not yet fully understood. Using
two different models of HF, one induced by pulmonary hypertension causing right ventricular (RV) pressure-overload and
one induced via aortic banding causing left ventricular (LV)
pressure-overload, we can begin to address these molecular
changes in relation to the caveolae. RV HF model: Male Wistar rats (190-230g) received an i.p. injection of 60 mg/kg
monocrotaline (MCT) or saline. MCT induces RV hypertrophy
which progresses to RV HF by 21-28 days post-injection. LV
HF model: Male Wistar rats (80 ± 20g) were anaesthetized
through inhalation with isoflurane (3.5% in medical O2) and
partial thoracotomy performed were a metal clip was
(I.D=0.6mm) placed around the ascending aorta. Sham animals underwent identical surgery minus aortic banding. When
ejection fraction (EF) fell >45% (19-26 weeks post-surgery),
pressure-volume loops were measured under anaesthesia (as
above). Sham animals were time matched. Ventricular muscle was homogenized in Laemmli sample buffer for Western
blotting to assess total protein expression and homogenised
in detergent-free buffer (500mM Na2CO3) and sonicated, then
separated on a discontinuous sucrose gradient to assess protein distribution. Proteins of interest include caveolar proteins
caveolin (Cav) 1, Cav 3, cavin 1 and cavin 4 which are crucial
for caveolar formation. Figure 1 shows changes in expression
of the caveolar proteins in the RV and LV HF models. There was
a decrease in Cav 1, Cav 3 and Cavin 1 expression (P<0.01) and
a trend for an increase in cavin 4 (P=0.2) in the RV HF model
compared with controls. The proportion of Cav 3 and cavin 1
found in the buoyant caveolae containing fractions was also
reduced in this model. In the LV model there was a decrease
119P
in Cav 1 and an increase in cavin 4 (P<0.05) compared with
sham animals. For both models, the largest change was in Cav1
expression. Of note, Cav 1 expression has previously been seen
to decreased in human HF and increased expression after
mechanically unloading of the heart with a LV assisted device
1. Increased cavin 4 expression in the LV failure model reflects
recent reports of a link between cavin 4 and hypertrophic signalling 2. Understanding the changes occurring in the controlled microenvironment of the caveolae in HF may aid in
explaining the aberrant βAR signalling seen and reveal possible pharmacological targets.
(with respective IC50 values of 3.9±0.6 μM and 12.9±1.4 μM).
With high (94 mM) [K+]e, the IC50 values for (S+) and (R-)DISO
were 16.9 ±3.0 μM and 61.1±7.4 μM respectively. The F656A
mutant of the key S6 binding residue for DISO reduced the
potency of IhERG by both DISO enantiomers, with a ratio of
F656A/ wild-type IC50 of ~29 for (S+)DISO and ~ 20 for (R)DISO. Our data provide evidence for stereoselectivity of IhERG
block by DISO: (S+) DISO is more potent against IhERG than is
(R-) DISO. This may correlate with a slightly stronger interaction with the F656 aromatic residue for (S+) than for (R-) DISO.
El Harchi A et al. (2012). J Mol Cell Cardiol 52(1),185-95.
Grilo LS & Abriel H (2010). Front Pharmacol 1,137.
Lin C et al. (2009).Cardiology 113(1):59-65.
Paul AA et al. (2001). Biochem Biophys Res Commun 280(5), 124350.
Funded by the British Heart Foundation
Figure 1. A. Effect of RV failure on caveolar protein expression. B. Effect of
LV failure on caveolar protein expression. All values are normalized to
GAPDH. Data are mean + S.E.M (n=6; *P<0.05; **P<0.01; ***P<0.001, Student’s t-test).
Uray, I. P., J. H. Connelly, O. H. Frazier, H. Taegtmeyer, and P. J. A. Davies.
2003. Mechanical unloading increases caveolin expression in the failing human heart. Cardiovascular Research 59:57-66.
Ogata, T., D. Naito, N. Nakanishi, Y. K. Hayashi, T. Taniguchi, K. Miyagawa, T. Hamaoka, N. Maruyama, S. Matoba, K. Ikeda, H. Yamada, H.
Oh, and T. Ueyama. 2014. MURC/Cavin-4 facilitates recruitment of ERK
to caveolae and concentric cardiac hypertrophy induced by α1-adrenergic receptors. Proceedings of the National Academy of Sciences
111:3811-3816.
requirements.
PCA040
Stereoselective block of hERG channel by disopyramide
Y. Zhang1, A. El Harchi11, C.E. Dempsey2 and J.C. Hancox1
1School of Physiology and Pharmacology and Cardiovascular
Research Laboratories, Medical Sciences Building, University of
Bristol, Bristol, UK and 2School of Biochemistry, Medical Sciences
Building, University of Bristol, Bristol, UK
The hERG channel mediates the IKr potassium current, which
regulates ventricular action potential duration and, thereby,
the duration of the QT interval. HERG is a target for Class Ia
and III antiarrhythmic drugs as well as for non-cardiac drugs
associated with drug-induced Long QT Syndrome. Prior work
has established that the Class Ia agent disopyramide (DISO)
inhibits hERG current (IhERG) significantly at clinically relevant
concentrations by binding within the channel’s inner cavity
(Paul et al, 2001; El Harchi et al, 2012). DISO exists as a racemic
mixture of (S+) DISO and (R-) DISO. However, whilst stereoselectivity has been established for some hERG-blocking compounds (Lin et al, 2009; Grilo and Abriel, 2010), little is known
for DISO in this regard. In this study, we have investigated the
potential for stereoselective IhERG inhibition by DISO.
Whole-cell patch-clamp measurements of IhERG were made
at 37°C from hERG-expressing HEK293 cells. All observations
reported here are from at least 5 replicates. With a conventional voltage-step protocol, observing IhERG tails at -40 mV
following depolarisation to +20 mV, (S+)DISO was found to
block wild-type IhERG ~3 -fold more potently than did (R-)DISO
120P
requirements.
PCA041
Caveolin-3 dependent regulation of ICa is altered in
ventricular myocytes from failing rat hearts
S. Bryant, C.H. Orchard and A.F. James
Physiology and Pharmacology, University of Bristol, Bristol, UK
Stimulation of t-tubular ICa in cardiac ventricular myocytes by
both basal and β2-adrenoceptor-stimulated protein kinase A
(PKA) activity is dependent on caveolin-3 (Cav-3) binding
(Bryant et al., 2014). It is unknown whether Cav-3-dependent
stimulation of ICa is altered in heart failure (HF). We have therefore investigated the regulation of ICa by Cav-3 and PKAdependent mechanisms in HF.
and conducted in accordance with UK legislation. Coronary
artery ligation (CAL) was performed in male Wistar rats (200250g) under anaesthesia (ketamine 75 mg/kg, medetomidine
0.5 mg/kg, i.p.) with appropriate analgesia (buprenorphine
0.05 mg/kg s.c.). 17 weeks post-surgery, hearts were excised
from ligated (CAL) or sham-operated (Sham) animals under
pentobarbitone (140 mg/kg i.p.) anaesthesia and left ventricular myocytes isolated. ICa was recorded using whole-cell patchclamp in intact cells and following acute detubulation (2225°C). PKA was inhibited using 20 μM H-89; β2-adrenoceptors
were stimulated using the β2-agonist zinterol (3 μM) in combination with 10 μM atenolol. Cav-3 binding was disrupted
using the peptide C3SD as described previously (Bryant et al.,
2014). Data are expressed as mean±SEM (n cells). Statistical
analyses were performed using Student’s t-test or ANOVA with
the appropriate Bonferroni post hoc test; ** p<0.01, ***
p<0.001.
Under basal conditions ICa density was not different between
Sham and CAL myocytes (Fig 1A); however, after inhibition of
PKA ICa density was smaller in CAL than in Sham myocytes (Fig
1B), suggesting that PKA stimulation helps maintain ICa density in CAL myocytes.
Measurement of ICa density in intact and detubulated myocytes
showed that the difference in ICa density between t-tubular
(TT) and surface sarcolemmal (SS) membrane observed in
Sham cells was reduced in CAL myocytes (Fig 1A). H-89 abolished this difference in ICa density in CAL but not Sham
myocytes (Fig 1B). C3SD decreased basal ICa in Sham (con -
6.5±0.4 (16) vs C3SD -4.9±0.3 (16) pA/pF, p<0.001) but not in
CAL myocytes (con -5.6±0.4 (14) vs C3SD -5.8±0.5 (14) pA/pF,
ns) suggesting loss of Cav-3-dependent stimulation of ICa in
CAL.
Zinterol increased ICa in intact but not detubulated Sham
myocytes (by 46±8% (7), p<0.01 and 2±5% (7), ns, respectively),
but increased ICa in both intact and detubulated CAL myocytes
(by 39±12% (5), p<0.01 and 37±2% (4), p<0.01).
These data suggest loss of Cav-3 dependent localization of PKA
activity at the t-tubules in HF, accompanying redistribution
of β2-adrenoceptors to the SS (Nikolaev et al., 2010).
currently unknown if TBQ also has effects on other components of ventricular intracellular Ca handling. The aim of these
experiments was therefore to characterise the effects of TBQ
on Ca handling in the rat ventricular myocyte and assess its
suitability as specific inhibitor of SERCA.
Male Wistar rats were killed in accordance with the The Home
Office Animal (Scientific Procedures) Act 1986 for enzymatic
isolation of ventricular myocytes which were subsequently
loaded with the Ca indicator Fluo-3AM to measure global intracellular Ca. Myocytes were voltage clamped via perforated
patch and paced at 0.5 Hz to measure sarcolemmal Ca fluxes.
TBQ produced a concentration dependent decrease of the rate
of decay of systolic Ca, consistent with a decrease in SERCA
activity. This was fully reversible at all concentrations tested.
For most experiments we used 10 μM TBQ as this produced a
large effect without completely abolishing the systolic Ca transient. 10 μM TBQ produced a 48 ± 5 and 54 ± 6 % decrease in
the amplitude and the rate of decay of the systolic Ca transient respectively. However, at 10 μM, TBQ also produced a 23
± 7 % decrease of the peak Ca current without altering the voltage dependence. SR Ca content was also reduced by 62 ± 4 %.
Interestingly, at higher concentrations (100 μM), TBQ also activated an outward current that demonstrated a current-voltage relationship consistent with a potassium current. This outward current was sensitive to Glibenclamide. Thapsigargin
(another inhibitor of SERCA) did not produce this outward current, suggesting its activation is TBQ dependent, rather than
as a result of SERCA inhibition.
These data show that in rat ventricular myocytes, TBQ can be
used to reversibly inhibit SERCA. However, at concentrations
that decrease SERCA activity, TBQ also decreases the Ca current, and, at higher concentrations, activates an outward current which appears to be an ATP dependent potassium current. We conclude that TBQ cannot be used as a specific
inhibitor of SERCA in rat ventricular myocytes.
O’Neill SC et al. (2004). J Physiol 559, 121-128
requirements.
Figure 1. TT and SS localisation of ICa in Sham and CAL myocytes in the
absence (A) and presence (B) of H-89.
Bryant S et al. (2014). J Mol Cell Cardiol 68, 47-55.
PCA043
Steady-state Ca2+ fluxes and cytosolic Ca2+ buffering in
rabbit atrial and ventricular myocytes
Nikolaev VO et al. (2010). Science 327, 1653-1657.
This work was supported by the British Heart Foundation
requirements.
PCA042
The effects of 2’,5’-di(tert-butyl)-1,4-benzohydroquinone
(TBQ) on calcium handling in rat ventricular myocytes
D.J. Greensmith, L. Miller and D.A. Eisner
Unit of Cardiac Physiology, The University Of Manchester,
Manchester, UK
2’,5’-di(tert-butyl)-1,4-benzohydroquinone (TBQ) is a reversible
inhibitor of SERCA, potentially making it a useful tool to study
the effects of SERCA inhibition. It has been used in a variety
of cell types including ventricular myocytes (1). However, it is
H.C. Gadeberg, C.H. Kong, M.B. Cannell, C.H. Orchard and
A.F. James
School of Physiology & Pharmacology, University of Bristol, Bristol,,
UK
Although the Ca2+ fluxes underlying excitation-contraction
(EC) coupling in ventricular myocytes are well-defined, there
is little information regarding Ca2+ homeostasis in atrial cells.
We have previously shown that the sarcoplasmic reticulum
(SR) plays a greater role in rabbit atrial than ventricular
myocytes (Gadeberg et al., 2013), a finding consistent with
atrial-ventricular differences in rat cardiac cells (Walden et al.,
2009). Here, we compare the steady-state Ca2+ fluxes underlying EC coupling and cytosolic Ca2+ buffering in voltageclamped rabbit atrial and ventricular myocytes.
and conducted according to UK legislation. Rabbit atrial and
ventricular myocytes were isolated by enzymatic digestion.
Resting cytosolic [Ca2+] ([Ca2+]rest) was measured in intact isolated cells loaded with indo-1-AM (atrial 79.0±9.7 nM, n=10;
121P
ventricular 97.7±8.4 nM, n=10, P>0.05). Membrane currents
and intracellular Ca2+ transients ([Ca2+]i) were measured in
separate cells using the whole-cell recording technique (holding potential=-80 mV) and a nominally Ca2+-free, K+-rich pipette
solution (pH 7.2) containing 100 μM fluo-4. Cells were superfused with Tyrode’s solution containing 1 mM Ca2+ (pH 7.4,
22 °C). [Ca2+]i was calculated from the normalised fluo-4 fluorescence (F/F0) using the mean [Ca2+]rest obtained for the cell
type as measured using indo-1 (Cannell et al., 1994). Ca2+ currents (ICa) were activated by voltage-clamp pulses (100 ms) to
0 mV following a 50 ms pre-pulse to inactivate Na+ current.
Steady-state was achieved by a series of 10 command pulses
(1 Hz). Unloading of SR Ca2+ was achieved by rapid application
of 10 mM caffeine. Na+/Ca2+ exchange current (INCX) was measured as the Ni2+-sensitive component of the caffeine-induced
current. SR Ca2+ content was measured as the integral of INCX.
The buffering power was calculated as the ratio of the SR Ca2+
content to the amplitude of the caffeine-induced [Ca2+]i transient. Mean data were compared by Student’s unpaired t-test
and P<0.05 was considered significant.
Although mean ICa density was greater in atrial (6.1±0.4 pA/pF,
n=4) than in ventricular myocytes (3.8±0.8 pA/pF, n=3, P>0.05),
the ICa-activated [Ca2+]i transient was smaller (192±29 vs.
293±24 nM, P<0.05). This was not due to a difference in the
steady state SR Ca2+ content (atrial : 62.4±13.1 μM, ventricular: 58.1±7.2 μM, ns; in contrast to the rat, Walden et al., 2009),
however buffering power was greater in atrial cells (238±15 vs
139±13, P<0.01). There was no significant difference in EC coupling gain but fractional release was greater in ventricular
(44.9±4.6 %) than in atrial myocytes (31.7±2.0 %, P<0.05).
These data demonstrate atrial-ventricular differences in Ca2+
homeostasis in the rabbit heart and that these differ from those
previously reported in the rat.
Cannell MB, Cheng H & Lederer WJ. (1994). Spatial non-uniformities
in [Ca2+]i during excitation-contraction coupling in cardiac myocytes.
Biophys J 67, 1942-1956.
Gadeberg HC, Kong CHT, Cannell MB & James AF. (2013). Differences
in intracellular calcium handling in rabbit atrial and ventricular
myocytes. In 37th Congress of IUPS, PCB029. Physiological Society,
Birmingham, UK.
Walden AP, Dibb KM & Trafford AW. (2009). Differences in intracellular calcium homeostasis between atrial and ventricular myocytes. J
Mol Cell Cardiol 46, 463-473.
requirements.
a biophysically detailed computational model of the human
atria and torso, to investigate the correlation between PWM
and origins of atrial ectopic activity.
Methods: We apply a recently developed 3D human atrial
model (3) to simulate electrical activity during normal sinus
rhythm and ectopic pacing. The atrial model is placed into a
newly developed torso model, taken from the visible human
dataset with consideration for the lungs, liver and spinal cord.
A boundary element method is used to compute the body-surface potential (BSP) resulting from atrial excitation. Elements
of the torso mesh corresponding to the locations of the electrode placement are selected to simulate 12 and 64 ECG systems. PWM associated with ectopic activity from a variety of
regions throughout the atria were analysed. An algorithm to
obtain the location of the stimulus from a 64 lead ECG system was developed.
Results: During sinus rhythm, the simulated P-waves of 12 and
64 leads ECG and BSP dipole direction show strong agreement
with experimental data. Marked changes in PWM are associated with ectopic atrial activity, with some areas of the torso
being more sensitive to specific activity than others. The success rate of the algorithm was 93%.
Conclusion: Our simulation data suggested that atrial ectopic
activity can be reflected in changes of PWM. This study established a correlation between PWM and ectopic activity. An
algorithm to identify the location of this ectopic activity has
been developed, with 93% accuracy.
Benjamin EJ, et al. Impact of atrial fibrillation on the risk of death: the
Framingham Heart Study. Circulation. 1998 Sep 8;98(10):946–52.
Giacopelli D, et al. P. Spatial pattern of P waves in paroxysmal atrial
fibrillation patients in sinus rhythm and controls. Pacing Clin Electrophysiol PACE. 2012 Jul;35(7):819–26
Aslanidi OV, et al. 3D virtual human atria: A computational platform
for studying clinical atrial fibrillation. Prog Biophys Mol Biol. 2011
Oct;107(1):156–68.
CONACYT
requirements.
PCA045
Inhibition of PKCα and β prevents harmful effects of
hyperglycaemia on cardiomyocyte function and reveals a
metabolism-dependent cardioprotection
S. Brennan and R.D. Rainbow
PCA044
Cardiovascular Sciences, University of Leicester, Leicester, UK
New algorithm to diagnose atrial ectopic origin from 64
lead ECG – insights from 3D virtual human atria and torso
E. Perez Alday2, M. Coman2, P. Langley3, D. Giacopelli3,
S.R. Kharche1 and H. Zhang2
1CEMPS, University of Exeter, Exeter, UK, 2University of Manchester,
Manchester, UK and 3University of Hull, Hull, UK
Backgrounds and aims: Rapid atrial arrhythmias such as atrial
tachycardia and atrial fibrillation (AF) predispose to ventricular arrhythmias, sudden cardiac death and stroke (1). Associated with ectopic and re-entrant activity, the presence of such
arrhythmias is reflected in alteration to the P-wave morphology (PWM). Identifying the origin of ectopic atrial activity from
a more complete electrocardiogram (ECG) lead configuration
can help to diagnose the early onset of AF in a cost effective
manner (2). In this study we developed a new algorithm, from
122P
There is increasing evidence that hyperglycaemia at the time
of acute myocardial infarction (MI) increases the probability
of mortality(1). The degree of glycaemia, rather than diabetes,
has been postulated to be the cause of deleterious effects(1),
including pro-arrhythmic action potential prolongation(2, 3).
Our previous study identified that inhibition of PKCα and β
attenuated the damaging effects of hyperglycaemia on cardiomyocytes(3). Here we present data demonstrating that inhibition of PKCα and β reveals a metabolite concentrationdependent cardioprotection.
Cardiomyocytes isolated from adult male Wistar rats were subjected to a chemically mimicked ischaemia/reperfusion injury
(I/R) model. Briefly, with 1 Hz of electric field stimulation cells
were paced at 32±2 °C and perfused with normal Tyrode’s (NT)
solution for 2 mins, followed by 7 mins with substrate-free
metabolic inhibition Tyrode’s (SFT) containing 2 mM cyanide
and 1 mM iodoacetic acid. Cells were then ‘reperfused’ with
NT for 10 mins and the contractile recovery assessed as a measure of cardioprotection.
In control rat cardiomyocytes in 5 mM glucose NT, 29±2% of
cardiomyocytes recovered contractile function and 25±3%
entered a hypercontracted state. In 20 mM glucose, contractile recovery was 27±3% (P>0.05) whilst hypercontracture was
increased to 39±4% (P<0.01). After PKCα and β inhibition with
300 nM Gö6976 contractile recovery in 20 mM glucose NT was
increased to 59±2% (P<0.001), with an EC50 glucose concentration of 12.7±1.4 mM, and hypercontracture reduced to
10±1% (P<0.01). Inhibition with LY379196 (PKCβ-specific
inhibitor) also increased contractile recovery (56±3%, P<0.001)
and reduced hypercontracture (14±3%, P<0.01). Replacing glucose with fructose also increased contractile recovery at 20
mM (62±2%, P<0.001, EC50 10.8±1.8 mM) and reduced hypercontracture (9±2%, P<0.001) after treatment with 300 nM
Gö6976. Similar results were obtained using cell permeant
(Tat-peptide linked) PKCα and β inhibitor peptides. The cardioprotection-like phenotype afforded by elevated glucose was
unaffected by inhibition of PKCε using 100 nM Tat-PKCε
inhibitor peptide (contractile recovery 58±3%, P<0.001). Similar results were found in guinea pig cardiomyocytes. All data
tested using a one-way ANOVA and Bonferroni’s post-hoc test,
n≥6 experiments, ≥93 cardiomyocytes from ≥3 animals.
These data suggests that, as well as reducing pro-arrhythmic
action potential prolongation; inhibition of PKCα and β in
hyperglycaemic conditions may impart protection to cardiomyocytes during ischaemia. This metabolism-induced,
PKCε-independent, cardioprotection may provide a useful clinical adjunct therapy for reducing the deleterious effects of
hyperglycaemia during MI.
Squire I, Nelson C, Ng L, Jones D, Woods K & Lambert P (2010). Prognostic value of admission blood glucose concentration and diabetes
diagnosis on survival after acute myocardial infarction: results from
4702 index cases in routine practice. Clin Sci 118,527–535
Hreiche R, Plante I, David L-P, Simard C, Turgeon J & Drolet B (2009).
Impact of glucose concentration on cardiac ventricular repolarization under I Kr/I Ks blocking agents. J Mol Cell Cardiol 47, 210–220
missing. Hence, we aimed to analyse IK1-changes in the presence of ethanol in enzymatically isolated rat atrial myocytes
and respective changes of the current in human Kir2.3 channels transiently expressed in Chinese Hamster Ovary (CHO)
cells, for comparison. Myocytes were isolated from the atria
of adult male Wistar rats (300 ± 10 g) anaesthetised by intramuscular administration of a mixture of tiletamin and
zolazepam (65 mg/kg; Zoletil® 100 inj.), and xylazine (20
mg/kg; Rometar® inj.). To digest the atrial tissue, the heart
was retrogradely perfused via aorta with nominally Ca2+-free
Tyrode solution containing collagenase (type S, Yakult Pharmaceuticals; 0.2 mg/ml), protease (type XIV, Sigma-Aldrich;
0.053 mg/ml) and EGTA (Sigma-Aldrich; 34 μM) in the first step
(2.75 min), and, subsequently, with the same solution but without protease (16 min). Experiments were performed by the
whole cell patch clamp technique at 23 ± 1°C. In rat atrial cells,
a transient inhibition of IK1 by 10.5 ± 2.0 % (13 cells) was
observed at the beginning of application of ethanol in a clinically relevant concentration of 80 mM (~3.7‰). The inhibition
declined with a delay and, in about half of the cells, it was followed by an activation of the current, at the quasi steady-state
by 9.0 ± 1.9 % compared to the control current (7 cells; a representative record is shown in Fig. 1). In our preliminary experiments on the expressed human Kir2.3 channels, 80 mM
ethanol induced only an inhibition of the current by 11.5 ± 2.4
% under the same experimental conditions (5 cells; a representative record is shown in Fig. 2). In conclusion, this is the
first study demonstrating an effect of ethanol on the atrial
IK1. The ethanol effect was apparent in a clinically relevant concentration and partly differed in rat atrial myocytes and in the
expressed human Kir2.3 channels. The decline of IK1-inhibition
during application of ethanol observed in rat atrial cells along
with the following increase of IK1 in a part of these cells may
be caused by an unknown mechanism which is not present in
the human Kir2.3 channels expressed in CHO cells. Our experimental data suggest that ethanol-induced changes of the atrial
IK1 might participate in the reported alterations of atrial electrophysiology under alcohol consumption.
Rainbow RD, Sims MW, Norman RI & Squire IB (2012), PKC-mediated
toxic effects of acute elevations of extracellular glucose concentration
on cardiomyocyte contractile function. (Proc Physiol Soc 27)
requirements.
PCA046
Ethanol affects inward rectifier potassium current in rat
atrial myocytes and in expressed human Kir2.3 channels
M. Bébarová1, P. Matejovič1, J. Hošek2, M. Šimurdová1 and
J. Šimurda1
1Department
of Physiology, Masaryk University, Faculty of
Medicine, Brno, Czech Republic and 2Department of Natural Drugs,
University of Veterinary and Pharmaceutical Sciences Brno, Faculty
of Pharmacy, Brno, Czech Republic
Figure 1: Effect of 80 mM ethanol on rat atrial IK1.
Alcohol intoxication may induce electrocardiographic changes
and arrhythmias, most frequently the atrial fibrillation (AF; e.g.
Kodama et al., 2011). Modifications of inward rectifier potassium currents including IK1 have been implicated in the pathogenesis of AF (e.g. Ehrlich, 2008; Wakili et al., 2011). Recently,
we have reported that ethanol significantly affects IK1 in rat
ventricular myocytes (Bébarová et al., 2013). Nevertheless,
data describing the effect of ethanol on the atrial IK1 are still
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Figure 2: Effect of 80 mM ethanol on human Kir2.3 channels.
Bébarová M et al. (2013). Biomed Pap Med Fac Univ Palacky Olomouc
Czech Repub 157, S1
Ehrlich JR (2008). J Cardiovasc Pharmacol 52, 129-135
Kodama S et al. (2011). J Am Coll Cardiol 57, 427-436
Wakili R et al. (2011). J Clin Invest 121, 2955-2968
vical, 3.80 ± 0.42 for thoracic and 4.47 ± 0.59 for lumbar sections). In lamina X GFP was co-localised with ChAT in 9.04%
(121/1338) of GAD and ChAT neurones in the cervical sections,
8.14% (114/1400) in the thoracic sections and 9.76%
(134/1373) in the lumbar sections. A limited number of colocalised neurones were also observed in the dorsal horn (colocalised neurones per section 1.13 ± 0.35 (mean ± SEM) for
cervical, 0.43 ± 0.03 for thoracic and 0.67 ± 0.07 for lumbar
sections) and lamina VII (co-localised neurones per section
0.20 ± 0.12 (mean ± SEM) for cervical and 0.10 ± 0.06 for lumbar sections). The co-localised neurones were negative for Fluorogold indicating that they are not motor or preganglionic
neurones. In addition, based on a reconstruction of the thoracic cord serial sections, the majority of co-localised neurones
were located ventral and ventrolateral to the central canal.
Since the projections and functions of these co-localised neurones in lamina X have not been reported, it is worth studying further how they are involved in neuronal circuits.
Kosaka T, Tauchi M & Dahl JL (1988). Exp Brain Res 70(3), 605-17.
Tamamaki N et al. (2003). J Comp Neurol 467(1), 60-79.
requirements.
This work was supported by the grant project NT143013/2013.
requirements.
PCA048
Cardiac effects of brief seizures in a chronic model of
temporal lobe epilepsy
A. Ashby-Lumsden1, W. Chang1, T. Lovick2 and J. Jefferys1
PCA047
Co-localisation of the enzymes synthesising GABA and
acetylcholine in the mouse spinal cord
J. Gotts, I.J. Edwards, S.A. Deuchars and J. Deuchars
School of Biomedical Sciences, University of Leeds, Leeds, UK
The spinal cord contains neuronal networks that are involved
in various important functions; including sensory, motor and
autonomic controls. In 1988 Kosaka et al. reported on the colocalisation of choline acetyltransferase (ChAT) and glutamic
acid decarboxylase (GAD) immunoreactivity within the cervical regions of the rat spinal cord. The present study examines
the distribution of ChAT immunoreactivity and co-localisation
with green fluorescent protein (GFP) in the cervical, thoracic
and lumbar cord sections of transgenic adult mice expressing
GFP under control of the GAD67 promoter (GAD67-GFP mice)
(Tamamaki et al., 2003).
GAD67-GFP mice (4-6 weeks, n = 3) were injected intraperitoneally with 0.1 ml of 1 % Fluorogold. After 24-48 hours the
mice were anaesthetised intraperitoneally with 60 mg/kg
sodium pentobarbitone and when the pinch reflex was absent,
they were perfused transcardially with 4 % paraformaldehyde.
The spinal cords were postfixed with 4 % paraformaldehyde
for 24 hours. The tissues were sectioned at 50 μm on a vibrating microtome and the sections were processed utilising double labelling immunofluorescence (IHC) for ChAT and GFP. Colocalisation of staining was determined for neurones per
section and percentage co-localisation was determined from
10 sections from each animal for each spinal region.
Neurones immunoreactive (IR) for ChAT or GAD67-GFP were
found throughout the spinal cord. Co-localisation of GFP-IR and
ChAT-IR was observed predominantly in lamina X (co-localised
neurones per section being 4.03 ± 0.29 (mean ± SEM) for cer-
124P
1School
of Clinical and Experimental Medicine, University of
Birmingham, Birmingham, UK and 2School of Physiology and
Pharmacology, University of Bristol, Bristol, UK
Sudden unexplained death in epilepsy (SUDEP), where the
death of a patient cannot be attributed to any other cause, is
a relatively poorly understood phenomenon. Sudden death is
over 20 times more prevalent in people with epilepsy than in
the general population and is of considerable concern to those
at risk and their families. The mechanism of SUDEP is unknown,
but ictal respiratory depression, autonomic dysfunction and
fatal arrhythmia are all hypothesised to have a role. Using the
Tetanus Neuro-Toxin (TeNT) model of temporal lobe epilepsy,
we examined the affect of chronic epileptic seizures on the
electrocardiogram (ECG) of freely moving rats.
Adult male Wistar rats were injected with 2.5ng TeNT in 1μl
phosphate buffered saline with bovine serum albumin at
200nl.min-1 (toxin omitted in controls) into the ventral hippocampus. Electrocorticogram and ECG were continuously
recorded via dual-biopotential Telemetry Research radiotelemeters (Millar Instruments, US) implanted ~2 weeks before injection. Both surgeries were performed under isoflurane anaesthesia (5% induction, 2-3% maintenance). Animals were housed
with naïve companion rats and video-recorded for seizure classification. Seizure-related changes in heart rate and the incidence of arrhythmias were analysed. Data are given as
mean±SEM.
Seizures lasted 75.3±2.9 s. They induced dramatic changes in
heart rate: ictal bradycardia occurred in 89% of seizures with
the heart rate as low as 70 bpm, along with ictal tachycardia
(447.5±4.7 bpm) that persisted 863.5±86.7s post-ictally.
Seizure-induced arrhythmias occurred in 71% of seizures, with
missed beats (59%), ventricular premature depolarisations
(22%) and ventricular fibrillation (17%) seen both in isolation
and in concert. The ECG waveform also changed, with the Pwave absent for many heartbeats in the majority of seizures.
Our results demonstrate that repeated brief epileptic seizures
have substantial effects on heart rate, with arrhythmias present during the majority of seizures. Similar cardiac consequences of seizures have been seen in human patients. We
conclude that the TeNT model provides a valuable tool to investigate autonomic mechanisms implicated in SUDEP.
Thank you to John Coote for his continuing input.
ergic neurons contained dual-labelled neurons. We propose
these observations highlight a common pattern of organization of the CNS, where neurons with axon collateral project
to different output pathways having complementary functions.
These neurons provide the anatomical framework for the fine
control of organ function required for homeostatic regulation
and the coordination of organ responses to enable complex
behaviours.
Banfield BW et al, 2003 J Virol. 77: 10106-10112
Jansen AS et al., 1992 Brain Res. 572: 253-260
Alexander Ashby-Lumsden was supported by the BBSRC
throughout this research.
requirements.
Strack AM et al., 1989 Brain Res. 491: 156-162
Supported by the Australian NHMRC
requirements.
PCA049
PCA050
Identification of CNS neurons with axonal collaterals
projecting to both the sympathetic and parasympathetic
innervation of the submandibular gland
N.S. Hettigoda1, A.Y. Fong1, E. Badoer2, M.J. McKinley3,1,
B.J. Oldfield4 and A.M. Allen1,3
1Physiology, The University of Melbourne, Parkville, VIC, Australia,
2School
of Medical Sciences, RMIT University, Bundoora, VIC,
Australia, 3Florey Neurosciences Institute, The University of
Melbourne, Parkville, VIC, Australia and 4Physiology, Monash
University, Clayton, VIC, Australia
Coordinated modulation of sympathetic and parasympathetic
nervous activity is required for physiological regulation of tissue function. Anatomically, whilst the peripheral sympathetic
and parasympathetic pathways are separate, the distribution
of premotor neurons in the higher brain regions often overlaps. This co-distribution would enable coordinated regulation
and might suggest individual premotor neurons could project
to both sympathetic and parasympathetic outflows. To investigate this we exploited the anatomical organization of the
innervation of the submandibular gland in Sprague Dawley
rats. Under anaesthesia by inhalation of isoflurane, with surgical anaesthesia assessed by loss of the pedal withdrawal and
corneal reflexes, the submandibular gland on one side was
sympathectomized. One strain of pseudorabies virus (1),
expressing a red fluorescent protein (PRV614), was injected
into the cut sympathetic nerve. An isogenic strain of pseudorabies, expressing a green fluorescent protein (PRV152), was
directly injected into the sympathectomized submandibular
gland. The rat’s wound was sutured and they were left to
recover in their home cage. After 52 – 96 hours the rats were
deeply anaesthetized (sodium pentobarbitone, 100 mg/kg i.p.)
and perfused transcardially with saline followed by 4%
paraformaldehyde. Brains, spinal cords, peripheral ganglia and
the submandibular gland were removed and prepared for standard immunohistochemical examination of the distribution of
the fluorophores. Independent labeling of the sympathetic
and parasympathetic pre- and post-ganglionic neurons was a
strict requirement for inclusion in further analysis. In 4 animals
the distribution of fluorescently-labelled neurons was examined throughout the neuraxis. The distribution showed a high
correlation with previous studies that separately examined the
sympathetic or parasympathetic innervation of the submandibular gland (2,3). Dual-labeled neurons were observed
in many CNS regions known to be involved in regulating salivary function. Triple-labelling immunohistochemistry showed
that specific populations of serotonergic and catecholamin-
Functional and proteomic changes which underlie
differences in neuronal function in the rostral ventrolateral
medulla (RVLM) of Spontaneously Hypertensive rats (SHR)
compared to normotensive rats
A.K. Goodchild, V.J. Tallapragada, L.M. Parker, N.N. Kumar,
P.G. Burke, P.A. Haynes and M. Mirzaei
The Australian School of Advanced Medicine, Macquarie University,
Sydney, NSW, Australia
Activity of presympathetic neurons in the RVLM contribute
to sympathetic hyperactivity seen in hypertension. Voltage
gated calcium channels (VGCC) underpin essential neuronal
functions including excitability, neurotransmitter release and
synaptic plasticity. LVA currents activate at hyperpolarized
potentials and inactivate rapidly so can generate spontaneous
neuronal firing and pacemaking activity, “burst firing” patterns and window currents. VGCC conductances are critical
determinants of the ongoing activity of RVLM neurons. For
example, Ni2+-sensitive Ca2+ currents drive bursting of RVLM
neurons in response to focal hypoxia and in the absence of
synaptic inputs in neonatal brain slice. We have previously
shown that LVA, but not HVA conductance, in adult RVLM, is
critical to maintaining sympathetic vasomotor tone in normotensive Sprague Dawley (SD) rats. The first aim was to determine functionally the role of the LVA conductance in the RVLM
of SHR and normotensive Wistar Kyoto (WKY). Urethane anaesthetised (1.3 g/kg ip) ventilated animals were used. Bilateral
microinjection of NiCl2 (50 nl, 50mM) were made into the
RVLM of SD, WKY and SHR. In both SD (n=4) and WKY (n=5)
SNA decreased immediately (peak change -56 ± 8% and -63 ±
4.5 %) as did BP (peak change -31 ± 5 and -39 ± 2 and mmHg)
and HR (-33 ± 13 bpm and -57 ± 6 bpm). Vehicle had no effect.
Ni2+ evoked effects were not significantly different between
the two strains. In contrast injections of NiCl2 in SHR (n=6)
elicited sustained increases in SNA (+54 ± 6 %), AP (+42 ± 8
mmHg) and HR (+41 ± 9 bpm). The quandary is to explain how
Ni2+ could evoke this response in SHR. T-type channels can be
modulated by various endogenous ligands and a number of
functional and chemical differences have been individually
described in the RVLM of SHR compared to WKY. In order to
identify all possible candidates that could modify T-type channels we next sought to compare the protein profile in the RVLM
of SHR (n=3) and WKY (n=3) by shot gun proteomics. Brains
were rapidly removed under pentobarbitone anaesthesia
(100mg/kg ip) and frozen before RVLM region dissection
125P
(~1mm x 1.5mm). Conventional protein preparation and digestion strategies preceded LC-MS/MS. In total 1438 proteins were
identified with 168 significantly differentially expressed:70 up
regulated and 98 down regulated in SHR vs WKY. Key pathways affected include: increased glutamatergic signalling
(VGLUT2, EAAT, GNAO); synaptic vesicle related activity (SYN1,
SYN2, SYT, SNAP25, SYNGR3, NRXN3, FLOT1, FLOT2), remodelling (MAP6, CAPZB, SEPT6, GSN, TUBB3, TUBB4, TUBB5,
TUBB4B, EMS1), decreased GABAergic signalling (GAD, ABAT,
GPHN), oxidative phosphorylation (ND1, NDUF6, CYTB, CYTC,
ATP5G1) and altered glutathione signalling (GSS, GSTT1).
These results suggest that although 1400W may alleviate pain
by iNOS inhibition, its role as a potential anti-inflammatory
agent is more complex. Analysis of blood plasma revealed that
1400W can successfully upregulate some anti-nociceptive
mediators, such as IL-1α and IL-10, in contrast to its ability to
increase levels of IL-1β, TNFα and CCL5. It is well known that
several pro-inflammatory mediators are elevated in pain states
but this study suggests that 1400W may function by increasing anti-nociceptive/pro-healing mediators (IL-1α and IL-10) to
counteract and balance the neuroimmune response and in
turn reduce neuronal hypersensitivity.
requirements.
Koch A, Zacharowski K, Boehm O, Stevens M, Lipfert P, von Giesen
HJ, et al. Nitric oxide and pro-inflammatory cytokines correlate with
pain intensity in chronic pain patients. Inflammation research : official journal of the European Histamine Research Society [et al].
[Research Support, Non-U.S. Gov’t]. 2007 Jan;56(1):32-7.
PCA051
Leung L, Cahill CM. TNF-alpha and neuropathic pain - a review. J Neuroinflamm. 2010 Apr 16;7.
1400W alleviates pain and increases the expression levels
of anti-nociceptive and neuromodulatory mediators
Marchand F, Perretti M, McMahon SB. Role of the Immune system in
chronic pain. Nature Reviews Neuroscience. [10.1038/nrn1700].
2005;6(7):521-32.
C.A. Staunton1,
T. Thippeswamy3
Staunton C, Djouhri L, Barrett-Jolley R, Thippeswamy T, editors. Chronic
neuropathic pain is alleviated by the highly specific iNOS inhibitor,
1400W. IUPS Proc 37th 2013; Birmingham, UK.
L.
Djouhri2,
R.
Barrett-Jolley1
and
1Institute of Ageing and Chronic Disease, University of Liverpool,
Liverpool, UK, 2Biomedical Sciences, King Faisal University, AlAhssa, United Arab Emirates and 3Biomedical Sciences, Iowa State
University, Ames, IA, USA
Chronic neuropathic pain (NP) results from nerve dysfunction/lesion and affects approximately 20% of European adults.
Due to its complexity NP is often misdiagnosed and inadequately treated. Nerve injury induces several changes including synthesis of neurotoxic mediators and prolonged transcription of the neurotoxic iNOS. These mediators are believed
to contribute to peripheral and central neuronal sensitizations,
and their levels have been shown to correlate with pain severity, suggesting their importance in nociception(1-3).
We have previously shown that 1400W dihydrochloride, a
highly specific iNOS inhibitor, reduced behavioural nociceptive responses in a NP model(4). The aim of the present study
was to determine whether 1400W has an effect on inflammatory mediators, as well as having anti-nociceptive effects.
Using the L5-Spinal Nerve Axotomy rodent model of NP in Wistar rats(5), carried out under anaesthesia (2% isoflurane),
1400W (20mg/kg) or its vehicle were administered to rats that
were euthanized 10 days later. We investigate the effect of
1400W treatment on the expression of several inflammatory
mediators, including TNF-α, MCP1, IL-1β, in injured L5 dorsal
root ganglion (DRG) neurons.
DRG immunohistochemical staining results showed no increase
in TNF-α expression 10days post injury in any DRG cell population. MCP1 immunoreactivity was present in all DRG subpopulations, with the majority of small sized (<23μm) neurons
being MCP1 positive. MCP1 levels increased with 1400W in
both small and medium sized (23-32μm) DRG neurons (n=4;
p<0.05, Fishers exact test used throughout). Small sized DRG
neurons had the most intense IL-1β staining, and 1400W treatment resulted in an increase in the percentages of IL-1β positive small and medium sized neurons (n=4; p<0.05).
Using a Multiplex ELISA, blood plasma obtained from all animals was analysed. Twelve inflammatory cytokines were simultaneously examined under uniform conditions. Plasma
obtained from 1400W treated rats had increased levels of 4
of the cytokines analyzed; IL-1α (0.10±0.02 vs 0.16±0.01), IL1β (0.35±0.04 vs 0.54±0.04), IL-10 (0.10±0.02 vs 0.15±0.01)
and CCL5 (2.94±0.02 vs 3.02±0.02) (n=6, p<0.05, General linear model and Tukey posthoc test).
126P
Li YB, Dorsi MJ, Meyer RA, Belzberg AJ. Mechanical hyperalgesia after
an L5 spinal nerve lesion in the rat is not dependent on input from
injured nerve fibers. Pain. 2000 Apr;85(3):493-502.
requirements.
PCA052
Study of the level of troponin T in sera of acute stroke
patients
L. Sultan1, W. El Gendy2, G. Achmawi1 and K. Elleboudy1
1neurology, faculty of medicine,Alexandria university, Alexandria,
Egypt and 2clinical and chemical pathology, faculty of
medicine,Alexandria university, Alexandria, Egypt
Background: Levels of the cardiac muscle regulatory protein
troponin-T (cTnT) are frequently elevated in patients with acute
stroke and elevated cTnT predicts poor outcome and mortality. The pathomechanism of troponin release may relate to comorbid coronary artery disease and myocardial ischemia or,
alternatively, to neurogenic cardiac damage due to autonomic
activation after acute stroke.
The aim of the work: Was to assess the level of troponin-T in
the sera of acute stroke patients and its effect on stroke severity.
Patients and methods: Fifty patients with acute cerebrovascular stroke (25 with intracerebral hemorrhage, 25 with acute
ischemic stroke) diagnosed by brain CT scan. Serum cardiac
troponin-T (cTnT) and creatine kinase–myocardial band (CKMB) levels were measured by immunoassay in all patients at
the time of admission and five days later. Twelve lead electrocardiograms (ECGs) were done at the time of admission and
12 hours later. The stroke severity was assessed by Scandinavian stroke scale(SSS).
Results: Serum cardiac troponin-T was elevated in 17 patients,
(8 patients with hemorrhage (32%), and 9 with infarction
(36%)). Abnormal ECGs findings were observed in 27 cases,
(13 patients with hemorrhage, and 14 with infarction). All
patients with elevated serum troponin showed abnormal ECG
(100%). ECG changes were more in patients with elevated cTnT
than in patients with elevated CK-MB .There is a positive correlation between Elevated cTnT sreum levels and Scandinavian
stroke scale and mortality. CK-MB was not elevated in most
patients with elevated cTnT, the non parallel increase of CKMB with cTnT may signify the non cardiac source of CKMB .
Patients with a left parietal lobe stroke compared with those
with stroke in other brain locations were at higher risk of
myocardial injury.
Conclusion: Measurement of the serum level of cardiac troponin-T provides a useful measure in assessment of the prognosis of the stroke in clinical practice. Serum cardiac troponinT is a sensitive marker in detecting myocardial injury after
cerebral stroke .
Key Words: Cardiac troponin-T; creatine kinase-MB; stroke;
myocardial injury.
Abbreviations: Cardiac troponin-T (cTnT), creatine
kinase–myocardial band (CK-MB), electrocardiogram (ECG),
Scandinavian stroke scale (SSS).
requirements.
PCA053
Genetic polymorphism in IL-18137G/C: Relation to subtypes
and severity of acute ischemic stroke
W. El neanaey2, H. SaadAllah1 and G. Achmawi1
1neurology, faculty of medicine,Alexandria university, Alexandria,
Egypt and 2clinical pathology, faculty of medicine,Alexandria
university, Alexandria, Egypt
Stroke remains the leading cause of severe disability and the
third leading cause of death after heart disease and cancer.
Stroke is a complex multifactorial disorder that is thought to
result from an interaction between a person`s genetic background and various environmental factors . This study aimed
to evaluate the association of IL-18137G polymorphism and
high sensitivity (hs) C-reactive protein ( CRP) with risk in acute
ischemic stroke (AIS )and their relationship with subtypes and
severity of stroke. 106 patients with acute ischemic stroke in
addition to 97 age and sex matched healthy controls were evaluated for genetic polymorphism of IL-18137G/C by PCR
–sequence specific primer and hs CRP by nephelometry using
BN prospec system . Results showed significant association of
137/CC genotype with increased risk of acute ischemic stroke
( OR= 2.131, 95%CI=2.559). Similar results were observed in
large artery atherosclerosis(LAA) and cardioembolic (CE) subtypes
(OR=3.226,95%CI=2.408-4.321
and
OR=8.667,95%CI=4.688-16.022,respectively) and severity of
stroke (p=0.004). CRP levels on admission were significantly
higher in AIS patients than controls (p=<0.001). Also as they
were associated with increased risk of AIS (or=4.880,
95%CI=3.440-6.922),all subtypes (or=8.462,95%CI=5.o7914.097 for LAA, or=10.700,95%CI=5.931-19.305 for small vessel disease (SVD) and or=49.500,95%CI=12.55-195.157 for
CE subtype and severity (p=0.013). Conclusion IL-18137G/C
polymorphism and CRP acting together as inflammatory and
atherosclerotic players in the progression of cerebral tissue
injury.
requirements.
PCA054
Inhibition of neuronal and heterologously expressed small
conductance calcium-activated potassium (SK) channels by
benzamil
M. Sampedro Castaneda, R. Tonini, C.D. Richards, P. Pedarzani
and M. Stocker
UCL, London, UK
Small conductance Ca2+-activated K+ (SK) channels are
expressed throughout the soma and dendrites of hippocampal pyramidal neurons, where they participate in the local regulation of membrane excitability and synaptic signals. Using
selective SK channel inhibitors and enhancers, we recently
described an inter-play between SK and L-type Ca2+ channels
in the regulation of Ca2+ influx triggered in the proximal dendrites by back-propagating action potentials (bAP) (1). Besides
affecting the amplitude of Ca2+ transients, SK channel inhibition also prolonged their duration, but the role of Ca2+ clearance mechanisms and their link to SK channel activity was not
established. In this work, we used the Na+/Ca2+ exchanger
(NCX) inhibitor benzamil to examine the interaction between
Ca2+ extrusion and SK channels in the regulation of dendritic
Ca2+ signals. Trains of four suprathreshold depolarizing steps
(20 Hz) were delivered in the whole-cell patch clamp configuration to the somata of Fluo-4 loaded rat pyramidal neurons
in dissociated cultures, prepared in accordance with UK Home
Office regulations. This stimulus triggered bAPs and voltagedriven Ca2+ transients monitored by 2-photon Ca2+ imaging in
the initial 50 μm of the apical dendrite. Consistent with an
NCX-dependent Ca2+ clearance from the dendrites in these
neurons, benzamil application prolonged the duration of the
Fluo-4 signals (160.1 ± 18.8% of control; n=6, p=0.044). However, a significant increase in the amplitude of Ca2+ transients
(117.6 ± 6.4% of control; n=6, p=0.017) was also detected, similar to what we had observed with SK channel blockers (1), suggesting that benzamil might act as a SK channel inhibitor. This
hypothesis was tested on both neuronal SK currents and heterologously expressed SK channels. The neuronal IAHP was fully
suppressed upon application of benzamil 30 μM (104 ± 3% inhibition, n=11), whilst recombinant SK(1-3) currents in human
embryonic kidney (HEK) cells were inhibited by 45%, 26% and
34% (n=6, 10 and 7) respectively at the same concentration
under symmetric [K+] conditions (IC50 35-67 μM). The inhibition of SK channels in HEK cells persisted in a physiological K+
concentration gradient, and was stronger at negative voltages.
Whilst the different extent of inhibition in the two systems
hints at additional actions of the drug in neurons, the suppression of SK channels by benzamil corroborates our findings
on the modulation of Ca2+ signals by SK channels and points
at this and other amiloride analogues as potential lead compounds for the development of selective and reversible SK
channel inhibitors. The results also prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native
systems, given the spectrum of ion channels and exchangers
this compound targets within a similar range of concentrations.
(1) Tonini R, Ferraro T, Sampedro-Castañeda M, Cavaccini A, Stocker
M, Richards CD, Pedarzani P. (2013) Small-conductance Ca2+-activated
K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons. J. Neurophysiol. 109: 1514-24.
requirements.
127P
PCA055
Role of calcium-induced calcium release and type 3
ryanodine receptors in the regulation of the slow
afterhyperpolarising current,sI(AHP), and its potentiation
in hippocampal pyramidal neurones
Van de Vrede Y, Fossier P, Baux G, Joels M, Chameau P. (2007) Pflugers
Arch. 455: 297-308.
Zhang L, Pennefather P, Velumian A, Tymianski M, Charlton M, Carlen
PL. (1995) J. Neurophysiol. 74: 2225-41.
requirements.
A. Tedoldi1, P. Ludwig1, G. Fulgenzi1, N. Dalgleish1,
H. Takeshima2, P. Pedarzani1 and M. Stocker1
1Research
Department of Neuroscience, Physiology and
Pharmacology, University College London, London, UK and
2Department of Biological Chemistry, Graduate School of
Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
In cortical pyramidal neurones, the slow afterhyperpolarising
current (sI(AHP)) plays an important role in the late phase of
spike frequency adaptation. sI(AHP) is a Ca 2+-dependent
K+ current: in hippocampal pyramidal neurones it is activated
2+channels, while the role of calby voltage-gated Ca
cium from ryanodine-sensitive intracellular stores, released by
calcium-induced calcium release (CICR), is controversial (1-4,
but see 5). All three types of ryanodine recptors (RyR1-3) are
expressed in the hippocampal formation, with RyR3 showing
a predominant expression in CA1 neurones. We investigated
the specific role of RyR3-mediated CICR in the regulation of
the sI(AHP) amplitude and time course and its activity-dependent potentiation (run-up) (3,5).
Whole-cell patch clamp recordings of the sI(AHP) were performed on rat CA1 and CA3 and mouse CA1 pyramidal neurones in hippocampal slices, prepared in accordance with UK
Home Office regulations. Ryanodine (10 μM) caused a
23.3±3.7% (n=5, p=0.04) and a 22.6±4.4% (n=6; p=0.01) reduction in the sI(AHP) amplitude in rat CA1 and CA3 neurones
when applied once the current had reached stable conditions.
Caffeine, known to enhance ryanodine-dependent CICR at low
concentrations (0.5 mM), had a biphasic effect on the sI(AHP)
in rat CA1 neurones, causing first an increase in the current
amplitude (25.3±2.9%; n=22) followed by a decrease
(24.5±3.6%; n=16). The caffeine-induced reduction of sI(AHP)
was abolished by protein kinase A inhibitors. Application of
ryanodine (10 μM) from the beginning of the recording and
throughout the run-up phase of the sI(AHP) reduced the potentiation of the current measured at 15 min in rat CA1 neurones
(control sI(AHP)=79.0±7.7 pA, n=5; sI(AHP) in ryanodine=51.3±7.8 pA, n=7; p=0.03). Similar results on the sI(AHP)
run-up were obtained with cyclopiazonic acid (CPA; 50 μM),
2+-ATPase inhibitor. In
an endoplasmic reticulum Ca
CA1 neurones from mice lacking RyR3, ryanodine (10 μM)
applied to the sI(AHP) after it reached stable conditions displayed a similar effect (34.5±11.7% inhibition, n=4) as in RyR3
wildtype littermates (41.1±9.8% inhibition, n=5; p=0.7). The
overall potentiation of sI(AHP) was not different in RyR3 knockout (n=12) compared with wildtype mice (n=9), as judged by
the final amplitude of the sI(AHP) reached at 15 min. However,
the time course of sI(AHP) run-up is different in RyR3 knockout compared with their wildtype littermates. The results suggest that ryanodine-mediated CICR plays a role in the regulation of the sI(AHP), particularly in its potentiation. RyR3 does
not seem to be necessary for the current generation or overall potentiation, but its absence affects the time course of the
sI(AHP) run-up.
Tanabe M, Gähwiler BH, Gerber U. (1998) J. Neurophysiol. 80: 226873.
Shah M, Haylett DG. (2000) J. Neurophysiol. 83: 2554-61.
Borde M, Bonansco C, Fernandez de Sevilla D, Le Ray D, Buño W. (2000)
Hippocampus 10: 198-206.
128P
PCA056
The implication of melatonin in the improvement of
neuronal mitochondria function in ageing
A.J. Idowu and I.I. Olatunji-Bello
Physiology, Lagos State University College of Medicine, Ikeja,
Nigeria
The changes in mitochondria bioenergetics play a critical role
in neuronal function with age. Mitochondria from synaptosomes in the cerebral cortex of young (6 months), n=6, middle-aged (13 months), n=6 and old (26months), n=6, mice
were isolated and transferred to mitochondria DNA-less LL/2m21 cell line (rho-zero) to generate young, middle-aged and
old cybrids. The cybrids show a significant reduction in mitochondria membrane potential (MMP) and ATP levels with age.
Melatonin has been reported to be a potent therapeutic for
age related cell dysfunction. Therefore we investigated how
melatonin can influence mitochondrial function during ageing. Melatonin treatment in young mitochondria bearing
cybrids, S-Y-24 lowered MMP from 130496.50±5118.75 to
84062±2633.43 (P<0.05), n=6 and increased MMP in middleaged mitochondrial bearing cybrid S-M-29 from 10399.43
±177.11 to 12862.79±186.51 (P<0.05), n=6. In the old cybrids,
melatonin treatment raised MMP from 24151.46±1025.93 to
29824.68±563.96, P<0.05, n=4 in galactose medium. In the
young cybrids, there was no significant difference in ATP levels between the melatonin treated cybrids - 4169.23±83.37
and controls - 4108.24±114.63, P>0.05, n=6. The young mitochondria bearing cybrids were able to produce ATP optimally
in the galactose medium showing they have functional mitochondria. ATP level was significantly increased in the middleaged mitochondria cybrid, S-M-29 from 3080.28±55.63 to
8542.85±130.38, P<0.05, n=6 and in old mitochondria bearing cybrids, S-O-48 from 2456.65±46.13 to 12415.50±265.16,
P<0.05,n=6 with melatonin treatment. The unpaired t-test was
used to analyze all the data presented in this study and the
level of significance placed at P<0.05. These results establish
a strong relationship between melatonin and mitochondria
bioenergetics. Melatonin may be a potential therapeutic mitochondria target in neuronal dysfunction observed in ageing.
Fernandez-Vizarra., Ferrin G., Perez-Martos A., Fernandez-Silva P.,
Zeviani M., Enriquez J.A. (2010). Isolation of mitochondria for biogenetical studies: An update.Mitochondrion 10;253-262.
Gaines, G., Attardi, G., (1984). Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and
in vivo-like processing patterns. Mol. Cell Biol. 4, 1605–1617.
Reiter RJ, Tan DX, Burkhardt S. (2002). Reactive oxygen and nitrogen
species and cellular and organismal decline: amelioration with melatonin. Mechanisms of Ageing and Development. (123) 1007–1019.
I acknowledge the funding provided by the US government
through the Fulbright Scholarship Program to conduct research
at the University of Texas Health Science Center, San Antonio,
Texas,United States.
requirements.
requirements.
PCA057
PCA058
Grey matter demyelination in cuprizone mouse model
causes spontaneous network excitability and anomalous
ectopic spike generation
The distribution of neuronal nitric oxide synthase (NOS) and
action of nitric oxide (NO) on neuronal excitability in
auditory cortex
M.S. Hamada and M. Kole
B.M. Pigott, J.A. Roberts and I.D. Forsythe
Axonal Signalling Group, Netherlands Institute for Neuriscience,
Cell Physiology and Pharmacology, University of Leicester, Leicester,
UK
Disruption of sodium (Na+) channels at the nodes of Ranvier
and increased lateral expression in internodes are major hallmarks of myelin loss, causing impairment of action potential
(AP) conduction and predisposing axons to injury. While most
previous studies on demyelination focused on white matter
fibre tracts (e.g. optic nerve and corpus callosum), the impact
of demyelination on the ion channel composition of axons in
the grey matter and the axon initial segment (AIS), the primary site for AP initiation, remain elusive. Here, we used the
well-established cuprizone toxin mouse model for grey matter demyelination (Matsushima & Morell 2001). Six weeks old
male mice (C57BL/6, cuprizone n = 124, control n = 79) were
put on 0.2% or 0.3% cuprizone mixed with powder food for 5
or 9 weeks, respectively. The weight loss of cuprizone-fed mice
was closely monitored and ranged between 20 – 25%. After
treatment mice were decapitated under deep isoflurane anaesthesia (3% v/v) and 300 μm parasagittal brain slices were made
according to routine methods. Immuno-labelling of myelin
basic protein (MBP) in the somatosensory cortex revealed that
myelin loss was most prominent in the layers 5 and 6. In vitro
somatic current-clamp whole-cell recordings from visually identified thick-tufted layer 5 neurons showed a significant ~3-fold
increase in spontaneously active cells (30%, n = 53 of 179) compared to control animals (11%, n = 5 of 46; Chi-square analysis P = 0.004). These spontaneous depolarisations were driven
by increased network excitability, the appearance of ectopic
APs (n = 27 of 179) and a large number of high-frequency burst
firing neurons (41%, Chi-square analysis P = 0.0001). Myelin
loss was furthermore associated with a shift of the AIS domain
to more proximal locations near the soma (2.45 ± 0.1 μm, n =
40; Control 4.5 ± 0.5 μm, n = 14; Student’s t-test P = 0.001)
and accompanied by diffuse lateral expression of Na+ channels (Nav1.6) around branch points of identified layer 5 axons
(3.84 ± 0.3 μm, n = 24; Control 2.5 ± 0.1 μm, n = 13; Student’s
t-test P = 0.0002). This study reveals for the first time that
demyelination of grey matter circuits affects the intrinsic neuronal excitability and induces spontaneous aberrant network
excitation, which may significantly deteriorate the spatio-temporal structure of information encoding, providing possibly
new insights into the cognitive impairment in multiple sclerosis.
Changes in the distribution, complement and/or activity of
voltage-gated K+ channels can influence the stability of neural networks through homeostatic effects on neuronal
excitability and may be neuroprotective during hyperexcitability (1). In auditory brainstem, NO generated by neuronal
NOS in response to synaptic stimulation switches postsynaptic action potential repolarisation from Kv3 to Kv2 K+ channel
dominance (2, 3). This switch decreases neuronal excitability
and increases transmission fidelity, thereby adapting neuronal
output to synaptic input. To determine whether similar modulation occurs at other levels of the auditory system, wholecell patch recordings were made from morphologically identified pyramidal neurones in slices of mouse auditory cortex.
Studies focused in cortical layer 2/3 since immunofluorescence
for neuronal NOS was predominant in this area (n = 3 mice).
Data are means ± SEM. Under current clamp, and consistent
with past reports (4), layer 2/3 neurones fired action potentials with a rheobase around 50 pA and a peak frequency of
39 ± 1 Hz (n = 12 cells).The NOS antagonist L-nitroarginine (LNNA, 100 μM) significantly reduced the peak frequency (29 ±
3 Hz; n = 14; unpaired t test, p < 0.05). Under voltage clamp,
NOS inhibition by L-NNA or 7-nitroindazole (10 μM) significantly
increased the amplitude of the whole-cell outward current
(at 50 mV, currents ± 1 nA were 11 nA, 19 nA and 16 nA for
control, L-NNA and 7-nitroindazole, respectively; n = 5-20 cells.
ANOVA with Tukey test, control vs. either inhibitor, p < 0.05;
L-NNA vs. 7-nitroindazole, p > 0.05). Immunofluorohistochemistry revealed protein for Kv2 K+ channel subunits
throughout auditory cortex, including in micron-sized clusters
in layer 2/3 pyramidal neuron soma and proximal dendrites
(n = 3 mice). Under voltage clamp the Kv2 channel gating modifier guangxitoxin-1E (100 nM) reversed the effect of NOS inhibition (100 μM L-NNA) on the amplitude of the whole-cell outward current (currents ± 1 nA were 18 nA, 14 nA and 12 nA at
50 mV for L-NNA, L-NNA plus guangxitoxin-1E, and control
respectively. ANOVA with Tukey test, L-NNA vs. L-NNA plus
toxin, p < 0.05; L-NNA plus toxin vs. control, p > 0.05; n = 6-7
cells), but had no effect on the amplitude of the control current (control ± toxin, p > 0.05; n = 6-7 cells). These data suggest that NO, produced tonically or in response to spontaneous
synaptic activity, persistently augments pyramidal neuron
excitability in layer 2/3 of the auditory cortex, possibly by
inhibiting Kv2-mediated voltage-gated K+ currents. NO therefore appears to control cell excitability at multiple levels of the
auditory system. Important differences exist in the effect of
NO between auditory cortex and brainstem (3), but Kv2 K+
channels appear to be a common downstream target.
Matsushima, G.K. & Morell, P., 2001. The neurotoxicant, cuprizone,
as a model to study demyelination and remyelination in the central
nervous system. Brain pathology (Zurich, Switzerland), 11(1),
pp.107–116.
This investigation was supported by grants from the National
Multiple Sclerosis Society (RG 4924A1/1) and European
Research Council under the European Community’s Seventh
Framework Programme (FP7), ERC-StG #261114.
We thank Sharon De Vries for her technical assistance.
Misonou (2010). Neuroscientist 16(1), 51-64.
Steinert et al. (2008). Neuron 60(4), 642-656.
Steinert et al. (2011). Neuron 71(2), 291-305.
Oswald and Reyes (2008). J Neurophysiol 99(6), 2998-3008.
129P
Supported by the MRC, UK.
requirements.
PCA059
Developing the non-toxic subunits of cholera toxin and heatlabile enterotoxin to transport therapeutic proteins into
neurones in mice
J.
Haigh1,
B.
Turnbull2,
S.A.
Deuchars1
and J.
Deuchars1
Havton, L.A. and Broman, J. (2005) Systemic administration of cholera
toxin B subunit conjugated to horseradish peroxidase in the adult rat
labels preganglionic autonomic neurons, motoneurons, and select primary afferents for light and electron microscopic studies. J Neurosci
Methods 149, 101-109
Beers, D.R., Ho, B.K., Siklos, L., Alexianu, M.E., Mosier, D.R., Mohamed,
A.H., Otsuka, Y., Kozovska, M.E., McAlhany, R.E., Smith, R.G., and Appel,
S.H. (2001) Parvalbumin overexpression alters immune-mediated
increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis. J Neurochem 79,
499-509
requirements.
2School
of Chemistry, University of Leeds, Leeds, UK
Cholera toxin B (CTB) the non-toxic subunit of the toxin produced by Vibrio cholerae labels motor, sensory and autonomic
preganglionic neurones in the CNS when administered systemically1. Heat-labile enterotoxin B (LTB) is a homologous
protein produced by Escherichia coli with similar binding affinity. This work aims to develop these toxin subunits as a means
to deliver other proteins into these neurones to influence their
function. The labelling of the CNS by LTB was evaluated via two
routes of systemic administration. Unanaesthetised GAD67GFP mice were injected I.P. with 2000 μg (n=4), 1000 μg (n=4),
or 500 μg (n=4) of LTB dissolved in 100 μl of 0.1 M PBS. For
I.V. injections mice were anaesthetised with isoflurane and
500 μg (n=2), 250 μg (n=3), or 125 μg (n=3) of LTB dissolved
in 50 μl of 0.1 M PBS was administered to the right jugular vein.
Either 3 days (I.P.) or 5 days (I.V.) following injections, mice
were anaesthetised with 80 mg/kg pentibarbitol I.P. and transcardially perfused with 4% paraformaldehyde. Brain and spinal
cord were sectioned at 50 μm on a vibrating microtome. LTB
immunoreactivity was detected in motor neurones in the brainstem and ventral horn of the spinal cord in all treatments.
The highest doses for both routes also labelled autonomic preganglionic neurones and primary afferents in the brainstem
and dorsal horn of the spinal cord. These results provided
rationale for delivery of proteins to these neurones via conjugation to CTB/LTB. However, to enable smaller amounts to be
used for test purposes it was determined that intramuscular
tongue injections of 50 μg (n=2) of LTB dissolved in 5 μl of 0.1
M PBS effectively labelled the hypoglossal nucleus. Parvalbumin is a calcium buffer that is not expressed in most motor
neurones. It is hypothesised that introducing parvalbumin into
the cells could provide protection against excitotoxicity involving increases in calcium, for example as may occur in amyotrophic lateral sclerosis2 . A fusion protein of CTB and parvalbumin (CTBparv) was created to be expressed using a
bacterial system. Tongue injections of 100 μg (n=2) of CTBparv in 5 μl 50 mM HEPES successfully labelled the hypoglossal nucleus as revealed by immunoreactivity to both CTB and
parvalbumin. Control mice injected with 100 μg CTB in 5 μl
of 0.1 M PBS had CTB labelled hypoglossal neurones with no
parvalbumin immunoreactivity. These data indicate that
CTB/LTB can be used to target motor neurones, as well as autonomic preganglionic fibres and primary afferents when administered I.P. or I.V. It is possible to conjugate a protein of interest to the toxin subunit, with the fusion protein maintaining
its binding affinity to GM1 to allow entrance into neurones.
Further experiments will seek to determine the sub-cellular
localisation and functionality of the parvalbumin within the
neurones.
130P
PCA060
Purinergic signalling through P2X2–containing receptors
in cells of the central canal area of the spinal cord
C. MacLean1, J. Deuchars1, N. Cohen2 and S.A. Deuchars1
2School
of Computing, University of Leeds, Leeds, UK
The central canal region of the mammalian spinal cord displays
increases in proliferation after injury and suggests neurogenic
potential [1, 2]. Ependymal cells and Cerebrospinal fluid-contacting cells (CSFcCs) are involved in these functions. The specific role of the CSFcCs is not known, nor how they communicate with the ependymal cells. The presence of purinergic
P2X2 receptors [3] indicates a role for ATP signalling. The P2X
receptors in this region could be a damage recognition system, as in cochlea [4], initiating cell proliferation. This study
aims to investigate the response to ATP and analogues in the
cell types in this area.
Spinal cord slices were obtained from Wistar rats (8 days)
anaesthetised with either pentobarbital 120 mg kg-1 or urethane 2 g kg-1 I.P. and transcardially perfused with sucrose
ACSF. The whole cell patch clamp method was used to obtain
single cell recordings from CSFcCs or ependymal cells. GABA
(100 μM) was applied in the bath solution; ATP (300 μM) was
applied using a puff electrode and a PicoPump (World Precision Instruments). A fluorescent dye was included in the intracellular solution allowing post-recording visualisation. Data are
presented as mean ± standard error of the mean and analysed
with T-tests, using a 5 % level of significance.
Cells were characterised by their responses to hyperpolarising
and depolarising current pulses. Results show significantly different responses between the different cell types to bath application of GABA in terms of the input resistance (IR). Ependymal cells show a small increase in IR (6.00 ± 3.29 MΩ; n=16),
whilst CSFcCs show a consistent decrease in IR (-284.29 ± 85.63
MΩ; n=7). No significant difference was found with respect
to changes in membrane potential.
Local application of ATP elicited fast depolarisations (63.01 ±
13.87 ms from onset of pressure pulse to start of response;
n=12) in CSFcC type 1, type 2 and ependymal cells; however
the majority of responses were seen in CSFcCs. Bath application of suramin (50 μM) significantly antagonised the responses
to ATP, reducing the magnitude from 21.50 ± 5.16 mV to 9.73
± 3.03 mV (n=7). The time to response was also significantly
longer after suramin application (71.50 ± 20.95 ms in control
and 154.43 ± 24.30 ms with suramin; n=7).
This data shows clear evidence for a role for purinergic signalling via P2X receptors in the central canal area of the spinal
cord. The differences in GABA responses between ependymal
cells and CSFcFs allowed for differentiation between cell types
and verification of cell viability. Suramin is a broad-spectrum
antagonist that blocks both P2X and P2Y receptors so could
be acting on multiple receptor types and subtypes. The next
steps for this study are to repeat the experiments using more
specific antagonists for P2X receptors and for specific P2X subtypes.
1. Cizkova, D., M. Nagyova, L. Slovinska, I. Novotna, J. Radonak, M.
Cizek, E. Mechirova, Z. Tomori, J. Hlucilova, J. Motlik, I. Sulla, Jr., and
I. Vanicky, Response of Ependymal Progenitors to Spinal Cord Injury
or Enhanced Physical Activity in Adult Rat. Cellular and Molecular Neurobiology, 2009. 29(6-7): p. 999-1013.
2. Shihabuddin, L.S., P.J. Horner, J. Ray, and F.H. Gage, Adult Spinal
Cord Stem Cells Generate Neurons after Transplantation in the Adult
Dentate Gyrus. The Journal of Neuroscience, 2000. 20(23): p. 87278735.
3. Kanjhan, R., G.D. Housley, L.D. Burton, D.L. Christie, A. Kippenberger,
P.R. Thorne, L. Luo, and A.F. Ryan, Distribution of the P2X2 receptor
subunit of the ATP-gated ion channels in the rat central nervous system. The Journal of Comparative Neurology, 1999. 407(1): p. 11-32.
4. Lahne, M. and J.E. Gale, Damage-induced cell-cell communication
in different cochlear cell types via two distinct ATP-dependent Ca
waves. Purinergic signalling, 2010. 6(2): p. 189-200.
requirements.
PCA061
Investigating the roles of synaptic vesicle dynamics in
epileptic high frequency activity
E.M. Fox, E.J. Rawlins, G. Morris and A.D. Powell
School of Clinical & Experimental Medicine, University of
Birmingham, Birmingham, UK
Temporal lobe epilepsy is the most common form of epilepsy,
a neurological disorder with increased tendency for repeated,
unprovoked seizures. A biomarker for epileptic tissue is the
presence of high frequency activity (HFA), which is defined as
neuronal oscillations above 100Hz[1]. Although the mechanisms behind HFA are unclear, experimental evidence using
Levetiracetam, an anti-epileptic drug targeting synaptic vesicle protein 2, suggests synaptic vesicle dynamics are
involved[2]. The synaptic vesicle cycle, and endocytosis in particular, ensures the maintenance of the readily releasable pool
of synaptic vesicles at the pre-synaptic terminal. Dynamin is
one of the major proteins involved in the scission of the budding vesicles in the endocytotic process. Dynasore and Dyngo4a are selective dynamin inhibitors which have been shown to
stop endocytosis[3,4]. Through the use of Dynasore and
Dyngo-4a, the role of synaptic vesicle dynamics on HFA will be
investigated.
400μm horizontal brain slices were prepared from VGAT-Venus
A rats, anaesthetised with 0.24mg/kg medotomidine and
58.2mg/kg ketamine via I.P. injection, and transcardially perfused with sucrose ACSF. Extracellular recordings were made
from stratum pyramidale in hippocampal region CA3b. HFA
was evoked by perfusion of slices with high potassium (8-9mM)
ACSF. After 20 minutes of HFA activity, Dynasore (80μM) or
Dyngo-4a (30μM) were applied to the slices via bath application, and recording was continued for 20 minutes. ShapiroWilks was used to for normality; significance was determined
using the Wilcoxon Signed Rank Sum test (α=0.025). Data is
given as the median ± IQR.
HFA were generated through application of high potassium
(frequency= 242±31 Hz, power= 350±1900 μV2; n=26). The
application of Dynasore decreased the frequency (243±95 Hz
to 220±60 Hz, p=0.007; n=12) and the summated power
(710±2500 to 240±2000 μV2, p=0.024; n=12) of HFA. Application of Dyngo-4a did not change the frequency (p=0.62; n=8)
or summated power (p=0.73; n=8).
These results show that the inhibition of dynamin using Dynasore modulates HFA, suggesting that the peak frequency and
power of HFA is dependent on synaptic vesicle endocytosis.
Results from Dyngo-4a suggest that further investigation is
required on the possible non-specific interactions the drugs
may have on other synaptic proteins.
1. Jefferys JGR, Menendez de la L, Wendling F, Bragin A, Avoli M, et al.
Mechanisms of physiological and epileptic HFO generation. Prog Neurobiol, 2012; 98: 250-264.
2. Lynch BA, Lambenng N, Nocka K, Kensel_Hammes P, Bajjalieh SM,
et al. The synaptic vesicle protein SV2A is the binding site for the
antiepileptic drug levetiracetam. Proc Natl Acad Sci USA, 2003;
101(26): 9861-9866
3. Newton AJ, Kirchhausen T, Murthy VN. Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis. Proc Natl
Acad Sci USA, 2006; 103 (43): 17955-17960.
4. McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, et al. Building a better Dynasore: the Dyngo compounds potently inhibit dynamin
and endocytosis. Traffic, 2013; 14: 1272-1289.
This work was supported by a Epilepsy Research UK grant to
ADP and University of Birmingham Studentships to GM, EMF
and EJR. VGAT-Venus transgenic rats were generated by Drs.
Y. Yanagawa, M. Hirabayashi and Y. Kawaguchi in National
requirements.
PCA062
Understanding the dorso-ventral profile of epileptic high
frequency activity
E.J. Rawlins, S. Hannan, E.M. Fox, G. Morris and A.D. Powell
School of Clinical & Experimental Medicine, University of
Birmingham, Birmingham, UK
Temporal Lobe Epilepsy is the most common form of epilepsy
– a neurological disorder characterised by abnormal synchronous neuronal activity. High frequency activity (HFA) can be
characterised into ripples and fast ripples. Ripples are physiological and have a frequency around 100-200Hz; fast ripples
are pathological have a frequency of around >250Hz[1]. As
fast ripples are a biomarker for the epileptogenic zone[2] and
the ventral hippocampus is more susceptible to epileptic activity[3], we hypothesised that changes in HFA could explain the
increased epileptogenicity. Previous studies have demonstrated that the ventral and dorsal regions of the hippocampus differ intrinsically[4]; with an increase in functional Ih channels[5]. We therefore examined the dorso-ventral profile of
HFA in the hippocampus.
Adult VGAT-Venus A rats were anaesthetised with 3mg/kg
medotomidine and 300mg/kg ketamine using I.P. injection.
After cervical dislocation or intracardial perfusion with sucrose
aCSF, 400 μm sections containing the dorsal and ventral hippocampus were prepared. Extracellular field potentials were
recorded from the stratum pyramidale of CA3b. Seizure-like
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discharges with HFA were induced by increasing the potassium
concentration to 8-12mM. Data is given as the median±IQR.
The high potassium model generated HFA which was seen
superimposed on sharp waves. The average K+ concentration
required to generate HFA was not different in the ventral and
dorsal hippocampus (8±2 mM and 8.25±1.5 mM, respectively;
p=0.81, Mood’s Median Test). The average frequency and
power of the HFA was analysed for 9 ventral slices and 8 dorsal slices (from 9 animals). The frequency of HFA was lower in
dorsal slices than in ventral slices (187±38 Hz and 246±53 Hz,
respectively; p=0.002, Mann-Whitney U test). The power of
the HFA was also reduced (0.15±0.78 mV2 and 4.57±7.90 mV2,
respectively; p=0.01, Mann-Whitney U test). The Ih channel
blocker, ZD7288 (10μM), significantly reduced the average frequency of HFA (Control - 219.5±77.2, ZD7288 - 214.1±76.0
Hz; p=0.04, Wilcoxon signed rank test). The HFA power was
not affected by ZD7288.
These data show that HFA is more pronounced in the ventral
hippocampus than in the dorsal, which supports previous
observations of increased excitability and susceptibility to
epileptic activity in the ventral hippocampus. The underlying
mechanisms that contribute to this increased excitability are
yet to be determined and the contribution of Ih channels
remains unclear.
Bragin A, Wilson C.L., Almajano J et al. (2004) High-frequency Oscillations after Status Epilepticus: Epileptogenesis and Seizure Genesis.
Epilepsia, 45: 1017-1023.
Jiruska P, Finnerty G.T., Powell A.D. et al. (2010) Epileptic high-frequency network activity in a model of non-lesional temporal lobe
epilepsy. Brain, 133: 1380-1390.
Gilbert M, Racine R.J., Smith G.K. (1985) Epileptiform burst responses
in ventral vs dorsal hippocampal slices. Brain Research, 361: 389-391.
Dougherty K.A., Islam T, Johnston D. (2012) Intrinsic excitability of
CA1 pyramidal neurones from the rat dorsal and ventral hippocampus. The Journal of Physiology, 590: 5707-22.
Dougherty K.A., Nicholson D.A., Diaz L et al. (2013) Differential expression of HCN subunits alters voltage-dependent gating of h-channels
in CA1 pyramidal neurons from dorsal and ventral hippocampus. Journal of Neurophysiology, 109: 1940-53.
This work was supported by a Epilepsy Research UK grant to
ADP and University of Birmingham Studentships to GM, EJR,
EMF and SH. VGAT-Venus transgenic rats were generated by
Drs. Y. Yanagawa, M. Hirabayashi and Y. Kawaguchi in National
requirements.
PCA063
Delayed neurovascular coupling after transient global
cerebral ischemia in rat
A. Zagrean1, C. Acatrinei1, A. Calin2, D. Zahiu1, A. Paslaru1,
A. Stoian1, M. Stancu1, L. Zagrean1 and M. Moldovan1,3
1Physiology and Neuroscience, Carol Davila University of Medicine
and Pharmacy, Bucharest, Romania, 2Oxford University, Oxford,
UK and 3Neuroscience and Pharmacology, University of
Copenhagen, Copenhagen, Denmark
A transient increase in cortical neuronal activity is followed
by a transient increase in cerebral blood flow. This physiological process of neurovascular coupling, measured by its hemodynamic response function (HRF), is used to infer neuronal
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activity in modern vascular-based brain imaging techniques.
Nevertheless, brain pathology may alter the HRF confounding
the interpretation of imaging studies. Our aim was to investigate the extent of HRF alterations after a minimally injuring
transient global cerebral ischemia (GCI) in adult male Wistar
rats (n=11). We introduced a new technique of measuring HRF
by taking advantage of the whole-brain discontinuous EEG
burst-suppression (BS) state induced by a chloral-hydrate overdose (400 mg/kg BW i.p.). We tested the changes in HRF at 48
hours after a 5-minute GCI induced using a variation of the
“4-vessel occlusion model”. Simultaneous cortical electroencephalographic (EEG) activity and Laser Doppler (LD) signal
were recorded from the left hemisphere. The rectified EEG was
convoluted with a prototype HRF to obtain a synthetic LD signal. Two parameters of the prototype HRF (peak response
latency and response width) were then programmatically optimized to attain a maximum correlation between the recorded
and the synthetic LD signals within the 0.1-0.4 Hz band. The
HRF was found to depend on the depth of BS anaesthesia. With
decreasing bursting frequency there was a progressive increase
of HRF latency (time to HRF peak) that could be reasonably
described by a linear regression. The slope of the relationship
was similar prior to and after GCI. Nevertheless, the Y-intercept was about double after GCI (F=81, P<0.01). The delayed
hemodynamic response after GCI could not be attributed
either to cardio-vascular changes (heart rate remained normal) or to changes in electrical activity patterns (intra-burst
EEG was similar to that during anaesthetic coma). Our data
suggest that the process of neurovascular coupling itself
remains delayed for days after a brief global cerebral ischemia.
This work was supported by grants of the Romanian National
Authority for Scientific Research, CNCS-UEFISCDI, project PNII-ID-PCE-2011-3-0847 and PN-II-PT-PCCA-2011-3.2-1290.
requirements.
PCA064
Co-modulation as a means of enhancing signal detection
and object formation in mouse primary auditory cortex
J. Sollini, A. Morris and P. Chadderton
Department of Bioengineering, Imperial College London, London,
UK
In the natural world, individual sensory signals rarely occur in
isolation. In audition, salient signals commonly occur within
constantly fluctuating, complex soundscapes. A key problem
for the auditory system is therefore the differentiation of temporally and spectrally overlapping signals. The mammalian
auditory system is excellent at grouping signals into separate
objects and can do so using a small number of cues (Bregman,
1990), but the neural mechanisms that underlie these
processes are poorly understood. One phenomenon that uses
these grouping processes is co-modulation masking release
(CMR, Verhey et al., 2012). Coherent amplitude modulation
of sound (co-modulation) across a broad range of frequencies (in the form of a broadband signal) can increase the
detectability of concurrent, narrow band signals (such as a
pure tones, Hall et al., 1984). Here we investigate how neuronal populations in primary auditory cortex (A1) represent
distinct overlapping objects, by recording activity evoked by
narrowband signals in the presence of a co-modulated broadband signal. High density, multi-site extracellular recordings,
and whole cell patch clamp recordings were made from neurons in primary auditory cortex (A1) of anaesthetised mice
(female, NMRI, 5-10 weeks). Functional responses to pure tones
were used to confirm probe location within A1. Cell responses
(defined as changes in firing rate or subthreshold Vm for extraand intracellularly recorded neurons respectively) could be categorised according to their responses to either the broadband
or narrowband signals. While the majority of cells responded
only to the onset and/or offset of the broadband signal (~52%),
the firing rate of a subpopulation locked to the phase of the
modulation (~13%). Also a small percentage of cells represented the tone signal (~13%): either by suppressing their
phase locking (~3%), or by sharply increasing their firing rates
at the onset (~5%) or offset of the tone (~5%). Surprisingly,
tone offset-evoked responses were more sensitive to sound
levels than the onset-evoked responses. These results suggest
that features of both narrow- and broadband sounds are reliably encoded in two separable, yet overlapping, populations
of A1 neurons. Sufficiently increasing the strength of one signal, relative to the other, allows that signal to suppress the
response to the other and enhance its own representation
within A1. We demonstrate that when using cues of object
formation, A1 is able to encode both signals as separately represented objects, even at low signal levels. This mechanism
could be used in the selective perception of low level signals
in noisy environments.
Bregman (1994). Auditory Scene Analysis: The Perceptual Organization of Sound. London: MIT Press. 1-46.
Verhey, J.L., Ernst, S. and Yasin, I. (2012). Effects of sequential streaming on auditory masking using psychoacoustics and auditory evoked
potentials. Hearing Research. 285, 77-85.
Hall, J. W., Haggard, M. P., and Fernandes, M. A. (1984). Detection in
noise by spectro-temporal pattern analysis. The Journal of the Acoustical Society of America, 76, 50-56.
Supported by a MRC Career Development Award and a HFSP
Young Investigator Award.
requirements.
PCA065
stancy of action potential conduction in the central nervous
system (CNS) relies on uniform axon diameter, coupled with
fidelity of the overlying myelin providing high resistance, low
capacitance insulation. Whereas the effects of demyelination
on conduction have been extensively studied/modeled, equivalent studies on the repercussions for conduction of axon
swelling, a common early pathological feature of (potentially
reversible) axonal injury, are lacking. The recent description
of experimentally acquired morphological and electrical properties of small CNS axons and oligodendrocytes prompted us
to incorporate these data into a computer model, with the aim
of simulating the effects of focal axon swelling on action potential conduction. Demyelination of complete internodal regions
caused decreased conduction velocity the degree of attenuation commensurate with degree and extent of demyelination.
A single swelling on an otherwise intact axon, as occurs in optic
nerve axons of Cnp1 null mice caused a small decrease in conduction velocity. The presence of single swellings on multiple
contiguous internodal regions (INR), as likely occurs in
advanced disease, caused qualitatively similar results, except
the dimensions of the swellings required to produce equivalent attenuation of conduction were significantly decreased.
Our simulations of the consequences of metabolic insult to
axons, namely the appearance of multiple swollen regions,
accompanied by perturbation of overlying myelin and
increased axolemmal permeability, contained within a single
INR, revealed that conduction block occurred when the dimensions of the simulated swellings were within the limits of those
measured experimentally, suggesting multiple swellings on a
single axon could contribute to axonal dysfunction, and that
increased axolemmal permeability is the decisive factor that
promotes conduction block.
Keywords: axonal spheroid, myelin, aglycemia, permeability
requirements.
PCA066
The transmembrane AMPA-receptor regulatory protein γ-2
differentially regulates excitatory inputs of the spinal cord
superficial dorsal horn
Conduction velocity is attenuated by focal axonal swellings
and associated ultra-structural changes attenuate in central
nervous system axons: a computater modeling study
S.J. Sullivan, M. Farrant and S.G. Cull-Candy
A. Brown1,2 and K.K. Kolaric1
AMPA receptors (AMPARs) mediate fast excitatory synaptic
currents in the superficial dorsal horn (SDH) of the spinal cord.
Pain-processing neurons in lamina II of the SDH receive excitatory input from peripheral fibres (mainly C-fibres) as well as
local input from excitatory interneurons. Transmembrane
AMPAR regulatory proteins (TARPs) such as γ-2 (stargazin) are
critical in determining the trafficking and gating of AMPARs
in many brain regions (1), but their role in the spinal cord is
less clear. TARPs have been implicated in pain (2), yet there is
no direct evidence to demonstrate that TARPs regulate AMPARmediated currents in the SDH. Furthermore, there is little information about the distribution of TARP subtypes at specific lamina II inputs.
To gain insight into TARP modulation of AMPARs in the SDH,
we made whole-cell patch-clamp recordings from neurons in
lamina II of acute spinal slices from stargazer (stg/stg) mice
that lack the prototypical TARP γ-2. CNQX, a partial agonist at
AMPARs associated with Type-I TARPs (γ-2, -3, -4, -8) (3,4),
induced whole-cell currents in wild-type cells (9/12 cells;
1University
of Nottingham, Nottingham, Nottingham, UK and
2Department of Neurology, University of Washington, Seatlle, WA,
USA
One of the key pathologies associated with central white matter axons is disruption of the myelin sheath that surrounds
axons, which can manifest either as demyelination, where existing myelin breaks down, or dysmyelination, where myelin formation is abnormally affected. Myelin pathologies affecting
the central nervous system include the inflammatory condition multiple sclerosis resulting in focal lesions, genetic abnormalities resulting in more generalized hypomyelination such
as the leukodystrophies and developmental pathologies such
as periventricular leukomalacia. Focal demyelination can also
result from swelling of the axon underlying the myelin, where
the physical expansion of the axon disrupts the integrity of the
myelin sheath, or as a consequence of axonal demise. Pathology of myelin is an important clinical condition as the con-
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–39.4±8.2 pA) but not in stg/stg cells (0/8), suggesting that γ2 may be the only Type-I TARP present. The amplitude of focally
evoked quantal events, recorded in the presence of extracellular strontium, was also reduced in stg/stg (–13.8±0.7 pA,
n=9) compared with wild-type controls (–21.9±1.7 pA, n=8).
To promote the selective release of glutamate from TRPV1
expressing C-fibres, we applied the TRPV1 agonist capsaicin
(5). Capsaicin increased the frequency of miniature EPSCs
(mEPSCs) in approximately half of the lamina II cells examined
(5.60 ± 2.83–fold in 13 cells; no response in 15 cells). Unexpectedly, the mean amplitude of capsaicin evoked mEPSCs
was similar between wild-type (–21.9±1.5 pA, n=13) and
stg/stg cells (–23.5±2.6pA, n=9), suggesting that γ-2 is not
associated with AMPARs at TRPV1-expressing C-fibre synapses.
In contrast, the amplitude of mEPSCs recorded in control conditions, prior to the addition of capsaicin in this subset of cells,
was reduced in stg/stg compared to wild-type (–15.7±0.7 versus –21.5±1.7 pA, n=9 and 8) suggesting γ-2 is normally present at other synapses. Interestingly, the amplitude of baseline
mEPSCs in cells that were insensitive to capsaicin did not differ (–19.1±2.2 pA and –17.5±1.0 pA, n=11 and 15).
Collectively, these data suggest that γ-2 is the major Type-I
TARP of lamina II neurons and that it is important in regulating the excitatory drive between lamina II interneurons. However, it appears to play a lesser role at synapses receiving input
from TRPV1-expressing C-fibres.
1. A.C. Jackson, R.A. Nicoll, Neuron. 70, 178-99 (2011).
2. F. Tao, J. Skinner, Q. Su, R.A. Johns, J. Neurosci. Res. 84, 867-73
(2006).
3. K. Menuz, R.M. Stroud, R.A. Nicoll, F.A. Hays, Science. 318, 815-7
(2007).
4. C. Bats, D. Soto, D. Studniarczyk, M. Farrant M, S.G. Cull-Candy, Nat.
Neurosci. 15, 853-61 (2012).
5. M.L. Baccei, R. Bardoni, M. Fitzgerald, J. Physiol. 549, 231-42 (2003).
SS is grateful to the National Science Foundation (USA) for a
Postdoctoral Fellowship (NSF-IRFP 1158971). This work was
supported by the MRC and the Wellcome Trust.
requirements.
PCA067
Do Kv7 and HCN channels play a role in a rat model of
diabetic neuropathic pain?
membrane potential are likely to be involved. Thus expression
of Kv channel subunits including Kv1.4, Kv3.4, Kv4.2, and Kv4.3
was reduced in DRG neurons in a rat model of DNP (Cao et al.
2010). Expression of Kv7.2 has also been shown to decrease
after nerve injury (Rose et al. 2011).
Here we used: a) immunofluorescence to examine whether
expression of Kv7 and HCN channel subunits changes in a rat
model of DNP, and, b) behavioural testing to examine whether
activation of Kv7 channels with retigabine alleviates pain hypersensitivity in this model. All in vivo procedures were regulated
under the Animals (Scientific Procedures) Act, 1986.
Diabetes was induced in adult male Sprague Dawley rats
(around 200g) by an injection of streptozotocin (STZ) (60
mg/kg, i.p.). Using anti-Kv7.2, Kv7.5, HCN1, HCN2 and HCN3
antibodies, we performed immunofluorescence on L4/5 DRG
from diabetic and control rats (n=4). We also examined the
effects of the Kv7 channel activator, retigabine (7.5 mg/kg,
i.p.) and the Kv7 channel blocker XE991 (3 mg/kg, i.p.) on
evoked pain behaviours (heat and mechanical hypersensitivity) in diabetic rats 5 weeks post STZ (n=7-10). Data were presented as mean±SEM. An unpaired t test was used to analyse
immunofluorescence data, and two-way repeated measures
ANOVA was used to analyse behavioural data.
Comparison of normalised densitometric data from control
and STZ DRG showed no significant change in Kv7.2 or Kv7.5
immunoreactivity between the two groups. Similar analysis
of HCN staining showed no significant change between control and STZ groups for HCN1 or HCN3, but HCN2 immunoreactivity was significantly increased in the STZ group compared
to controls (P<0.001). Behavioural studies showed retigabine
caused a non-significant reversal in mechanical hypersensitivity associated with DNP. This analgesic effect of retigabine
was completely reversed by the Kv7 antagonist XE991
(P<0.05). Whether blocking HCN channels also reduces pain
hypersensitivity associated with DNP remains to be determined. Taken together these findings suggest that modulation of neuronal Kv7 and HCN channels may have therapeutic
potential for the treatment of DNP.
Cao et al. (2010). Reduction in voltage-gated K+ channel activity in
primary sensory neurons in painful diabetic neuropathy: role of brainderived neurotrophic factor. J Neurochem. 114: 1460-1475.
Rose et al. (2011). Transcriptional repression of the M channel subunit Kv7.2 in chronic nerve injury. Pain. 152: 742-754.
This work was supported by a studentship from the IMB/BBSRC.
requirements.
A.L. Dermody1, T.M. Smith2, T. Kamishima1, J. Sathish2,
J.M. Quayle1 and L. Djouhri3
1Cellular
and Molecular Physiology, University of Liverpool,
Liverpool, UK, 2Molecular and Clinical Pharmacology, University
of Liverpool, Liverpool, UK and 3Biomedical Sciences, King Faisal
University, Al-Ahsa, Saudi Arabia
Persistent pain is a prominent symptom of diabetic neuropathy (DN), the most common complication of diabetes mellitus. Around 50% of the diabetic population have some degree
of diabetic neuropathy (American Diabetes Association). The
pathophysiology of diabetic neuropathic pain (DNP) is poorly
understood. DNP is believed to be partly due to abnormal
hyperexcitability of dorsal root ganglion (DRG) neurons.
Although, the underlying cellular and molecular mechanisms
of this hyperexcitability are unknown, certain voltage-gated
potassium (Kv) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that normally stabilize the resting
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PCA068
The relationship between spontaneous activity patterns
and light responsiveness of SCN cells recorded from male
rats
T. Tsuji, G. Leng and M. Ludwig
Centre for Integrative Physiology, The University of Edinburgh,
Edinburgh, UK
The suprachiasmatic nucleus (SCN) of the hypothalamus has
an essential role in orchestrating circadian rhythms of behavior and physiology. In the present study, we categorized SCN
neurons by statistical features of their electrical activity, and
by their responses to light, and examined how recordings made
in the light phase differ from recordings made in the dark
phase. Male Sprague-Dawley rats (250-450 g) anesthesized by
urethane (ethyl carbamate, 1.3 g/kg i.p.) were tracheotomized
and the optic chiasm just below the SCN region was exposed
by ventral surgery (Leng & Dyball, 1991). We recorded from
671 SCN cells (375 cells, mean rate 7.30 ± 0.34 Hz in rats maintained on a normal light cycle, 296 cells, mean rate 10.1 ± 0.47
Hz in rats on a reversed light cycle). We subdivided cells into
three groups according to their light-responsiveness: light-on
cells that increased their firing frequency in response to light;
light-off cells that decreased their firing frequency; and nonresponsive cells. In rats that had been maintained on a reversed
light cycle, light-on cells fired at a higher mean rate than in rats
that had been maintained on a normal light cycle, and their
responsiveness to light was stronger. Neuronal firing patterns
in conditions of maintained room light on and room light off
were analysed by constructing hazard functions from interspike interval data (Leng et al. 1995; these functions display
how the excitability of a cell changes with time since the last
spike). For most light-responsive cells, the hazard functions
showed a multimodal distribution, with a harmonic sequence
of modes, indicating that the neuronal discharge was driven
by an oscillatory input; this oscillatory pattern was rarely seen
in non-responsive SCN cells. The “oscillatory” cells comprised
two distinct populations: about 40% of the light-on cells (46
in normal light cycle and 53 in reversed light cycle) fired with
a first mode at 33.6 ± 0.8 ms reflecting an oscillatory drive at
~ 30 Hz, and about 60% (74 in normal light cycle and 73 in
reversed light cycle) fired with a first mode at 88.7 ± 4.2 ms
reflecting an oscillatory drive at ~ 15 Hz. By contrast, only
about of 10% of light-off cells (2 in normal light cycle and 9 in
reversed light cycle), fired with an early first mode compared
to 90% (48 in normal light cycle and 45 in reversed light cycle)
with a late first mode. These data suggest that light-responsive cells are organized into local networks that generate strong
rhythms of activity, that these rhythms differ between lightoff cells and light-on cells, and that these rhythms differ according to the stage of the light cycle.
glomeruli, axons of ORN synapse onto the apical dendrites of
mitral and tufted cells in the main olfactory bulb. The information is then passed on to the primary olfactory cortex including the anterior olfactory nucleus (AON), the piriform cortex,
amygdala and the entorhinal cortex. However, how the information is transduced and processed in each brain region is not
well known.
Previously, we reported that the rat AON contains many
interneurons which express vasopressin, and that these show
an increase in the expression of the immediate early gene product, EGR-1, when exposed to social, but not predator odour,
suggesting that these vasopressin neurons may be selectively
involved in the coding of social odour information (1). To better understand the processing of information in the AON, we
recorded spontaneous firing activity from single AON neurons
by in vivo extracellular electrophysiology. All experiments were
performed on adult male Sprague-Dawley rats (~ 450 g) under
urethane anaesthesia (ethyl carbamate, 1.3 g/kg i.p.). We studied their discharge patterning by constructing hazard functions, which show how the excitability changes with time after
a spike. Almost all AON cells fired in repeated clusters of spikes
separated by very short intervals (< 33 ms), and this was
reflected in hazard functions with a pronounced early peak.
These cells could be subdivided into 3 classes: in one class, the
hazard function was uniformly flat after the early peak, reflecting random occurrence of clusters; another class showed a second peak hazard at ~600 ms reflecting clusters occurring at
regular intervals with a frequency of ~ 2 Hz, a third class
showed peaks at both 600 ms and 1200 ms, indicating an oscillatory drive to these cells at ~ 2 Hz. The origin of this rhythm
is unclear, as it is substantially faster than the respiratory
rhythm that drives mitral cells in the olfactory bulb. The firing
rate for the 3 classes are 4.3 ± 0.5 Hz (n = 71), 9.1 ± 0.7 Hz (n
= 69), 2.6 ± 0.2 Hz (n = 51) respectively. We are now testing
how these 3 distinct subpopulations differ in their processing
of social odour information.
Leng G & Dyball REJ (1991) In Neuroendocrine Research Methods, ed.
Greenstein B, pp769-791. Chur; Harwood. Academic Publishers GmbH,
Switzerland.
Wacker DW et al.(2010). J Physiol 588, 4705-4717.
Leng G et al. (1995). In Neurohypophysis. Recent Progress of Vasopressin and Oxytocin Research, ed. Saito T, Kurokawa K & Yoshida S,
pp225-235. Elsevier Science B.V., The Netherlands
This study was supported by the Overseas Extension Program
of Multidisciplinary Research on Social Cognition and Autism
Spectrum Disease of Kanazawa University (TT) and a BBSRC
grant (ML) .
requirements.
PCA069
CT is JSPS Research Fellow and this study was supported by a
BBSRC grant (ML) .
requirements.
PCA070
Post activation depression of the Ia EPSP in motoneurones
is reduced in both aged mice and in the G127X SOD1 model
of Amyotrophic lateral sclerosis
A. Hedegaard1,2, J. Lehnhoff2, M. Moldovan2, L. Groendahl2
and C.F. Meehan2
1Department
Electrophysiological characteristic of the anterior olfactory
nucleus
C. Tsuji, T. Tsuji, G. Leng and M. Ludwig
Centre for Integrative Physiology, University of Edinburgh,
Edinburgh, UK
The olfactory system influences a wide range of fundamental
behaviors including food finding, reproduction, fear response
and social communication. These are regulated by the mixtures of odorant molecules which stimulate olfactory receptor neurons (ORN) and axons of these neurons project into the
glomerular layer of the main olfactory bulb. Within the
of Experimental Psychology, University of Oxford,
Oxford, Oxfordshire, UK and 2Department of Neuroscience and
Pharmacology, University of Copenhagen, Copenhagen, Denmark
Post Activation Depression (PActD) is a long lasting depression
of Ia afferent EPSPs in response to repetitive activation. This
is of clinical relevance given its consistent reduction across a
range of spastic disorders. We used in vivo intracellular recording in mice to explore changes in PActD in both normal aging
and in the neurodegenerative disease Amyotrophic Lateral
Sclerosis (ALS). We used both wild type (WT) C57BL/6J mice
and the G127X SOD1 transgenic model of ALS (Jonsson et al
2004)
135P
Mice were anaesthetized with Hypnorm (0.315mg/mL fentanyl-citrate +10mg/mL fluanisone), Midazolam (5mg/mL) and
water, mixed 1:1:2 (induction: 0.15mL/25g, maintenance:
0.05mL/20min, SC). Anaesthesia was assessed by the lack of
reflexes to a short noxious pinch on the hind foot. All mice
received Atropine (0.02mg, SC). Prior to recording, mice were
ventilated and paralysed with Pancuronium Bromide
(0.01mg/h, IP). Anaesthesia was then maintained using the
above doses and monitored by heart rate. Intracellular recordings were made with sharp microelectrodes (for detailed methods see Meehan et al 2010). Mean values are given with SD.
Motoneurones were antidromically identified by stimulation
of the common peroneal or tibial nerves. The stimulation was
then reduced to below spike threshold to visualise the monosynaptic EPSP. A 20Hz train of 4 EPSPs was followed by a test
EPSP with a varied time delay with respect to the train. Depression was expressed as the size of the test EPSP with respect to
the first EPSP of the train.
PActD in mice was similar to that observed in larger animals
by Hultborn et al. (1996) with respect to both the magnitude
and the time course (~79% at the 0.5sec time interval gradually diminishing to 100% by the 4sec interval). The following
values are means±SD at the 0.5sec interval.
PActD was significantly reduced in aged mice (~580days old)
compared to adult (~100-200day old) mice (Adult: 79.12%
±4.993, n= 74 cells, 12 mice. Aged: 83.31% ±7.271, n= 31 cells,
4 mice. Two-tailed T-test P=0.0009).
Age-matched WT (~200days) were compared to both presymptomatic (PS) G127X mice and symptomatic (S) G127X
mice. A significant effect was found (WT mice: 77.57% ±5.312,
n = 33 cells, 7 mice. PS G127X: 84.05% ±6.487, n=28 cells, 5
mice. S G127X: 82.71 ±4.624, n=18 cells, 2 mice, Kruskall Wallis, P=0.0002). Dunns Multiple comparisons test revealed significant differences between WT and both PS G127X (P<0.0001)
and S G127X (P<0.05) mice but no significant difference
between PS and S G127X mice.
Our result validate the use of mice models to study PActD and
show that it is reduced in both normal aging (without spasticity) and in ALS (a disorder with spasticity) questioning a
direct causal role in the development of spasticity.
PCA071
The transmembrane AMPA receptor regulatory protein γ-5
is constitutively trafficked to degradative pathways
I.D. Coombs, D. Studniarczyk, M. Zonouzi, M. Farrant and
S. Cull-Candy
Neuroscience, Physiology and Pharmacology, UCL, London, UK
K Graffmo & S Marklund for the G127X SOD-1 mice
AMPA-type glutamate receptors (AMPARs) are the principal
receptors that mediate fast excitatory synaptic transmission
in the brain. Their trafficking and functional properties depend
critically on their association with transmembrane AMPAR regulatory proteins (TARPs). Six functional TARP family members
have been identified and classified as type I (γ-2, -3, -4 and -8)
or type II (γ-5 and -7). Type I TARPs promote AMPAR surface
expression and synaptic trafficking (Tomita et al, 2003). The
properties of type II TARPs are less clear but they regulate
predominantly calcium-permeable AMPARs (CP-AMPARs) (Soto
et al, 2009; Studniarczyk et al, 2013). In addition, γ-5 appears
unique amongst TARPs, in that it differentially modifies the
channel properties of AMPAR subtypes based on the length of
their C-tail and reduces AMPAR surface trafficking (Soto et al,
2009).
We used confocal fluorescence imaging to investigate the subcellular distribution of YFP-γ-5 expressed in HEK293 cells and
cultured cerebellar granule cells from C57BL/6 mice. Unlike γ2 and γ-7, the majority of γ-5 did not localize in the plasma
membrane, but rather in intracellular puncta (observed in >20
cells in each of 4 cultures). γ-5 colocalization with CD-63 indicated that the puncta were late endosomes and lysosomes,
and hence that γ-5 is trafficked to the cell’s degradative pathways (>30 cells from 10 cultures). By examining fluorescent
chimeric constructs of γ-5 and γ-7 we found that, as with the
type I TARPs (Tomita et al, 2005), its intracellular C-tail region
dictated the trafficking. Specifically the distal C-terminus was
critical in determining γ-5 distribution (>20 cells from 3 cultures). Experiments using point mutations ruled out a role for
monoubiquitination (>50 cells from 2 cultures) or for the YXXM
AP-3 trafficking motif (>50 cells from 2 cultures). While ablation of γ-5’s PDZ-binding domain did not affect lysosomal trafficking, replacement of its PDZ-binding domain (SSPC) by one
that corresponded to the conventional class 1 PDZ-binding
domain of type I TARPs (TTPV), led to increased surface trafficking (>50 cells from 3 cultures). Finally, mutation of Ser271
(a site adjacent to the PDZ binding domain) increased the trafficking of γ-5 to the cell surface (>40 cells from 3 cultures).
Our data are consistent with a model whereby γ-5-associated
AMPARs are constitutively trafficked to degradation pathways.
This behaviour appears to be determined, at least in part, by
the C-terminal PDZ-binding domain of the TARP.
Funding by the Lundbeck Foundation
Soto D et al. (2009). Nat Neurosci 12, 277-85.
requirements.
Studniarczyk D et al. (2013). Nat Neurosci 16, 1266-74.
Jonsson PA, Ernhill K, Andersen PM, Bergemalm D, Brannstrom T,
Gredal O, Nilsson P, & Marklund SL (2004). Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis. Brain 127, 73-88.
Meehan CF, Sukiasyan N, Zhang M, Nielsen JB, Hultborn H. Intrinsic
properties of mouse lumbar motoneurons revealed by intracellular
recording in vivo. J Neurophysiol. 2010 May;103(5):2599-610.
Hultborn H, Illert M, Nielsen J, Paul A, Ballegaard M, & Wiese H (1996).
On the mechanism of the post-activation depression of the H-reflex
in human subjects. Exp Brain Res 108, 450-462.
Tomita S et al. (2003). J Cell Biol 161, 805-16.
Tomita S et al. (2005). Nature 435, 1052-8.
This work was supported by Programme Grants from the
Wellcome Trust and the Medical Research Council (S.G.C.-C.
and M.F.).
requirements.
136P
PCA072
requirements.
Nitric oxide modulation of synaptic release parameters
S. Bradley, S. Robinson and J.R. Steinert
PCA073
MRC Toxicology Unit, Leicester, UK
Abnormal nitric oxide (NO) signalling is implicated in several
neurodegenerative diseases (Steinert et al., 2010) but its exact
contribution to neuronal death remains elusive due to great
complexity of downstream nitrergic targets. Several NO-mediated mechanisms are associated with neuronal degeneration,
including mitochondrial and synaptic dysfunction or activation of cell death pathways. Elevated NO can lead to formation
of cytotoxic peroxynitrite which in turn can modulate a wide
range of protein functions via nitration of tyrosine residues (3Nitrotyrosination [3-NT]). Toxic NO signalling can further alter
protein functioning in a process known as S-nitrosylation. To
date, little is known as to what extent NO-mediated post-translational modifications contribute to or exacerbate neuronal
dysfunction (Nakamura et al., 2013). We use glutamatergic
synapses as a model system to identify novel nitrergic signalling
pathways to correlate protein modifications with functional
changes.
Here we investigate the effects of NO on synaptic function
which may involve S-nitrosylation or 3-NT signalling. The
Drosophila neuromuscular junction was used as a model to
characterise NO-mediated effects at the synapse employing
immunohistochemical and two-electrode-voltage-clamp
(TEVC) analyses. Electrophysiological recordings were carried
out in HL-3 solution in 1.5mM Ca2+ using sharp electrodes (2030 MΩ). Data denote mean±SEM (n-number) with *p<0.05
indicating statistical significance using unpaired Student’s ttest. Confocal analysis showed elevated levels of S-nitroso-cysteine and 3-Nitrotyrosine at synapses following NO-donor exposure (GSNO, SNAP, 500μM each for 1h, 3 NMJs each). TEVC
data showed little NO effects on miniature excitatory junctional current (mEJCs) amplitudes or frequencies but induced
a reduction in mEJC decay kinetics (τ; Ctrl: 6.1±0.1ms (49) vs
NO: 5.5±0.2ms* (24)). Furthermore, evoked EJC (eEJC) amplitudes (Ctrl: 101±5nA (18) vs NO: 48±6nA* (5)) and quantal
content (QC; Ctrl: 119±6 (18) vs NO: 75±7* (5)) were strongly
reduced following NO exposure for >35min indicative for a
reduction in release probability (pvr). An increased paired-pulse
ratio at various interspike-intervals under NO conditions confirmed a reduction in pvr (20ms: 1.1±0.0 vs 1.3±0.1*; 40ms:
1.0±0.0 vs 1.3±0.1* (n=7-11)). Cumulative postsynaptic current analysis (500ms 50Hz train) further showed a reduced
number of release-ready vesicles following NO exposure
(382±45 (7) vs 154±44* (5)) which was also confirmed by fluctuation analysis (Silver, 2003).
Together, our data suggest that NO can induce 3-NT or S-nitrosylation of proteins involved in synaptic signalling possibly
leading to protein modifications as detected by changes in
synaptic physiology. This data extends our understanding of
NO signalling, potentially leading to the identification of putative targets for therapeutic intervention(s) in disease.
Nakamura T, Tu S, Akhtar MW, Sunico CR, Okamoto S & Lipton SA.
(2013). Aberrant protein s-nitrosylation in neurodegenerative diseases.
Neuron 78, 596-614.
Silver RA. (2003). Estimation of nonuniform quantal parameters with
multiple-probability fluctuation analysis: theory, application and limitations. Journal of Neuroscience Methods 130, 127-141.
Steinert JR, Chernova T & Forsythe ID. (2010). Nitric oxide signaling
in brain function, dysfunction, and dementia. The Neuroscientist 16,
435-452.
Synchronization of epileptiform activity between CA1 and
CA3 rat hippocampus in nonsynaptic models of epilepsy
O. Netsyk1, O. Zapukhliak1, A. Romanov1, E. Isaeva1,
O. Krishtal1,2 and D. Isaev1,2
1Cellular Membranology, Bogomoletz Institute of Physiology, Kyiv,
Ukraine and 2State Key Laboratory for Molecular and Cellular
Biology, Kyiv, Ukraine
The different mechanisms of synchronization of epileptic bursts
(synaptic) & of the population spikes within them (nonsynaptic) have practical implications for estimating the severity of
epileptic responses. Synaptic mechanisms clearly are the key
to the functioning of the nervous system. Nevertheless, nonsynaptic interactions between neurons have significant functional & practical consequences that need to be understood.
This is particularly important with intense neuronal activity,
for instance, during epileptic & other synchronous discharges
(Jefferys, 1995). The aim of this work was to evaluate the manifestations & synchronization level of epileptiform events in 3
nonsynaptic epilepsy models.
All experiments were performed in accordance with the guidelines set by the National Institutes of Health for the humane
treatment of animals and the Animal Care Committee of Bogomoletz Institute of Physiology. The Wistar rats (P12) were
deeply anesthetized using sevoflurane & decapitated. Extracellular field potentials were recorded from the CA1 & CA3
pyramidal cell layers of hippocampal slices (500 μm) with glass
microelectrodes. Values are means ± SEM, compared by Student’s t-test.
For generation of nonsynaptic epileptiform events we perfused
slices with ACSF solution contained: a) low Ca2+ or b) Cd2+
(15μM) or c) synaptic transmission blockers (CNQX, 10μM;
MK-801, 1μM; bicuculline, 10μM). After application of
proepileptic solutions the delay time for epileptiform activity
was: a) CA1 – 16±2 min & CA3 – 11±1 min (n=24, p<0.05); b)
CA1 – 9±2 min & CA3 – 8±1 min (n=13); c) 9±1 min (n=15) in
CA1 & CA3 zones. In response to inhibition of synaptic transmission we observed next manifestations: sigle-, multi-spikes,
slow waves with spikes and bursts, burst- and clonic-like activity in CA1 as well as in CA3 area of hippocampus. Worth to
note that delay time before epileptic activity is shorter in CA3
area for low Ca2+ & Cd2+ models of epilepsy, while blockade
of postsynaptic receptors leads to synchronous start of
epileptoform net activity. Reducing the osmolarity of extracellular solution decreases delay time in response to the synaptic blockade.
These data suggest that synaptic transmission blockade caused
the higher level of epileptiform activity synchronization
between CA1 & CA3 comparing with low Ca2+ & Cd2+ models.
Thus, other than glutamatergic & gabaergic synaptic transmission systems are involved in epileptiform activity synchronization.
Jefferys JGR (1995). Physiol Reviews 75/4, 689-723.
requirements.
137P
PCA074
PCA075
Kv1.2-containing heteromers regulate the intrinsic
excitability of spiral ganglion neurons in mouse cochlea
An oligodendrocyte-astrocyte microcircuit siphons [K+]o at
action potential resolution
K.E. Smith, L. Browne, D. McAlpine and D. Jagger
A. Battefeld, J. Klooster and M. Kole
Ear Institute, University College London, London, UK
Axonal Signaling Group, Netherlands Institute for Neuroscience,
Spiral Ganglion Neurons (SGNs) are the first primary afferent
neurons of the auditory pathway. Numerous SGNs innervate
each sensory inner hair cell of the cochlea and transmit action
potentials to the auditory brainstem. In vivo recordings show
a complexity in the firing properties between individual SGNs,
a feature attributed to variations in pre- and post-synaptic
mechanisms. In SGNs cultured from pre-hearing mice, the population shows a heterogeneous distribution of adaptation rates
(Mo and Davis, 1997). Rapidly-adapting neurons have a prominent Dendrotoxin-K-sensitive low voltage-activated (LVA) K+
current, implicating voltage-gated K+ (Kv) subunits of the Kv1.1
subtype (Mo et al., 2002). The exact composition of the underlying channel subunits remains undetermined, as does the
physiological relevance of these channels in mature hearing.
Here, we used a pharmacological approach to investigate the
potential contribution of Kv1.2 subunits to the LVA current in
post-hearing mice. Primary SGN cultures, prepared from
C57BL/6 mice, were maintained for 2-3 days in vitro for wholecell patch clamp electrophysiology. Two age groups, P12-14
and P20-21 were used to compare the properties of SGNs
around hearing onset and one week post-hearing, respectively.
Current-clamp recordings revealed the presence of slowlyadapting and rapidly-adapting neurons in both age groups,
irrespective of cochlear tonotopic location. Rapidly-adapting
neurons were associated with a distinct LVA K+ current. The
application of 100 nM Tityustoxin-Kα (TsTx) which blocks channels containing Kv1.2 subunits caused a substantial reduction
of the conductance in the LVA region in both age groups (mean
± SEM: P12-14, 66.0 ± 6.5%, n=6; P20-21, 72.2 ± 4.3%, n=6).
Similarly, the application of another structurally unrelated
Kv1.2-specific blocker, κM-Conotoxin RIIIJ, blocked the LVA conductance in P12-14 SGNs (70.8 ± 7.8%, n=5). The co-application of Dendrotoxin-K, which blocks channels containing Kv1.1
subunits, did not substantially enhance the block by TsTx (P1214, 66.8 ± 8.9%, n=6; P20-21, 80.2 ± 5.7%, n=6), suggesting
that Kv1.1 and Kv1.2 subunits do not exist as two homomeric
populations. Consistent with the electrophysiological findings,
immunofluorescence localised Kv1.1 and Kv1.2 subunits to
the soma and proximal regions of neurites in SGNs in vivo.
Together, these findings suggest that heteromeric channels
comprising Kv1.1 and Kv1.2 subunits underlie the LVA current
in SGNs thereby contributing to rapid adaptation. The intrinsic properties of SGN membrane physiology may therefore
contribute to the complexity of observed input/output functions of auditory nerve fibres in vivo.
Mo ZL and Davis RL (1997). J. Neurophysiol. 77:1294-1305.
Mo ZL et al. (2002). J. Physiol. 542.3: 763-778.
Supported by Action on Hearing Loss
requirements.
138P
The myelin sheath produced by oligodendrocytes ensures high
velocity action potential conduction along CNS axons. Besides
the white matter a great number of oligodendrocytes can be
found in the neocortical grey matter where a population of
oligodendrocytes is in close anatomical proximity to the soma
of pyramidal neurons. These satellite oligodendrocytes are
thought to be non-myelinating in nature and provide metabolic support for neurons, but their functional physiological
properties remain unknown. In this study we examined the
intrinsic properties of satellite oligodendrocytes and investigated to which extent there might be functional coupling with
neurons. Adult male and female mice (22.8±0.5 g) were anaesthetised with isoflurane (3% v/v) and subsequently decapitated. Neocortical acute brain slices were prepared using standard methods and we subsequently performed simultaneous
whole-cell recordings from neocortical layer 5 neurons and
identified satellite oligodendrocytes. All results are mean ±
SEM. Satellite oligodendrocytes were resting at −85.9±0.6 mV
(n=101), had a low membrane resistance (13.4±1.9 MΩ, n=63)
and were characterized by a linear current-voltage relationship
in the steady state. Live confocal images revealed that satellite oligodendrocytes (n=31) had on average 32±2 myelinating internodal processes that were oriented in all angles. Action
potentials generated in pyramidal neurons evoked in adjacent
voltage-clamped oligodendrocytes temporally correlated
inward currents (n=20). These currents were highly sensitive
to 0.1 mM barium application (81±2.5% block, n=4, paired ttest, p=0.01) and were also reduced by carbenoxolone (58±4%,
n=4). Subsequently, immunohistochemistry labelling indicated
that the soma of satellite oligodendrocytes expressed Kir4.1
channels (n=3), a member of the barium-sensitive inward rectifying potassium channel family that is implicated in K+ buffering. Consistent with the presence of these channels, fast application of 1-30 mM [K+] revealed that the Kir4.1 mediated
currents were highly sensitive to [K+]o and reversed at 3 mM
[K+]o at a voltage of –85.4±1.1 mV (n=6). Furthermore, dual
whole-cell recordings revealed that satellite oligodendrocytes
are reciprocally gap junction coupled to astrocytes in a distance dependent manner (coupling ratio 2.3±0.7%, n=5). Lastly,
Ca2+ imaging of satellite oligodendrocytes displayed no Ca2+
change in the cell body (p=0.7) in response to action potentials. In summary our data suggest that satellite oligodendrocytes in conjunction with neighbouring astrocytes co-regulate
the [K+]o homeostasis near the somatic and proximal axon initial segment domains of pyramidal neurons via Kir4.1 channels and a gap-junction coupled microcircuit. [K+]o flux is thus
efficiently siphoned at the temporal resolution of single action
potentials.
We thank S de Vries for excellent technical assistance.
requirements.
PCA076
PCA077
The role of mitochondrial permeability transition pore in
the development of Alzheimer’s disease
The identification of neurones in the spinal cord of the
mouse innervating the external urethral sphincter: a
confocal microscopic immunofluorescent study of their
presynaptic inputs
I. Kravenska, V. Chopovska and O. Lukyanetz
O.O. Bogomoletz Institute of Physiology of the NAS of Ukraine,
Kiev, Ukraine
It is known that the typical feature of Alzheimer’s disease is a
gradual progression of memory impairment and higher brain
functions until the complete disorder of intelligence and mental activity. Significant degeneration of hippocampal neurons,
the senile plaques (the main component of which is β-amyloid) and the intracellular neurofibrillary tangles in the brains
of patients are the main indicators of the pathology. There is
evidence that β-amyloid can directly affect the mitochondria,
causing the mitochondrial permeability transition pore opening. Consequently, the damaged dendritic tree, apoptosis and
necrosis of neurons appear. However, the role of mitochondrial permeability transition pore in the development of
Alzheimer’s disease is not still clear. Therefore, the aim of our
study was to establish the influence of cyclosporine A, mitochondrial pore blocker on calcium homeostasis and viability
of hippocampal neurons at the modeling of Alzheimer’s disease.
The studies were performed on hippocampal neuronal culture
from Wistar neonatal rats. The animals were anesthetized by
inhalation with diethyl ether and decapitated. Intracellular calcium concentration was determined by fluorescent microscopy
by using the calcium-sensitive dye fura-2/AM (5 μM, 30 min),
whereby the ratio of fluorescence signals at two wavelengths
R = F1/F2 was measured (1). We compared the amplitude of
calcium transients caused by membrane depolarization with
KCl (50 mM, 5 sec) in neurons after incubation with β1–42-amyloid (Aβ; the modeling of Alzheimer’s disease) (2 μM, 24 h)
and after following incubation with cyclosporine A (1.25 μM,
30 min) and Aβ (2 μM, 24 h). The viability of neurons was evaluated by confocal microscopy by using Hoechst 33258 (1
μg/ml, 20 min), the indicator of alive cells and Propidium Iodide
(2 μg/ml, 10 min), the indicator of necrotic cells. We compared
the fluorescence ratio of each of these stains relatively to all
fluorescent neurons after incubation with Aβ (2 μM, 24 h) and
after following incubation with cyclosporine A (1.25 μM, 30
min) and Aβ (2 μM, 24 h).
We have found that after incubation with Aβ the amplitude
of calcium transients in neurons was equal to 3.565 ± 0.133
(n = 80). This value was decreased to 54 ± 4 % (P ≤ 0.001) and
accounted for 1.912 ± 0.149 (n = 23) after cyclosporine A treatment. We have found 59 ± 4 % (P ≤ 0.001, n = 1601) of necrotic
neurons relatively to all cells after incubation with Aβ. This
value was decreased to 42 ± 6 % (P ≤ 0.001, n = 1134) after
cyclosporine A treatment. Thus, we supposed that the mitochondrial permeability transition pore is directly involved in
the redistribution of calcium in hippocampal neurons, and consequently can participate in necrotic death of these cells during the development of Alzheimer’s disease.
Grynkiewicz G et al. (1985). J Biol Chem 260, 3440-3450.
requirements.
Y. Merican, R.M. Ichiyama, S.A. Deuchars and J. Deuchars
In the elderly, involuntary passing of urine or incontinence is
a common and distressing problem. The external urethral
sphincter of the bladder acts as the muscle of continence, and
is controlled by the pudendal nerve which carries signals from
motoneurones in the Onuf’s nucleus in spinal cord. We hypothesised that in ageing, alteration in the balance between excitatory and inhibitory influence to the motoneurons affects the
sphincteric control, especially since there is evidence that the
urethral sphincter architecture and volume is unaltered in ageing (Russell et. al., 1996). In this study, we investigated the
Onuf’s nucleus homologue known as the dorsolateral nucleus
(DLN) in young 3 month old mice, as well as the types and numbers of presynaptic terminals to the DLN motoneurones. Nine
wild type female C57BL/6 mice (3 month) were used. In 3 mice,
motoneurones innervating the external urethral sphincter were
first identified by injection of cholera-toxin B chain (2μl of 1%
in saline) under Fluothane anaesthesia. Following 3-5 days
recovery animals were anaesthetised with 80mg/kg pentobarbitone IP and perfused with 4% paraformaldehyde (PFA),
spinal cord sectioned at 50μm using a vibrating microtome
and processed for anti-cholera toxin B chain immunohistochemistry. The location of neurones projecting to the external urethral sphincter was verified using retrograde tracing
with cholera-toxin B chain as the DLN in the ventral horn. Six
mice were anaesthetised and perfused as above. The sections
from 3 mice were stained using choline acetyl transferase
(ChAT) and processed for peroxidase immunohistochemistry.
The location of ChAT immunoreactive motoneurones was sufficient to distinguish the DLN in the ventral horn, at the level
of the sixth lumbar to the first sacral segments of the spinal
cord. The mean length of the nucleus was 0.65 ± 0.2 mm, and
the mean number of dorsolateral motoneurones was 38.5 ±
1.5 per spinal cord. Spinal cords from 3 mice were processed
for triple labelling immunofluorescence for ChAT, glutamic
acid decarboxylase (GAD67) and glycine transporter 2 (GlyT2).
Quantitative analysis of the immunofluorescence staining by
confocal microscopy (Chang and Martin, 2009) revealed that
there were 2.0 (± 0.7), 4.4 (± 0.6) and 3.2 ± (0.8) of ChAT-,
GAD67- and GlyT2-immunopositive boutons respectively per
100 μm membrane perimeter of motoneurones in the DLN.
These initial results will be compared with the findings in aged
mice to determine if there are age-related changes on the
presynaptic inputs to dorsolateral motoneurones.
Russell B et al (1996). International Urogynecology Journal 7, 30-36
Chang Q & Martin LJ (2009). The American Journal of Pathology 174,
574-585
requirements.
139P
PCA078
requirements.
PIP2 determines excitability of Spiral Ganglion Neurons via
effects on Kv1-containing heteromeric channels
L.P. Browne1,2, K.E. Smith1, D. McAlpine1, D. Selwood2 and
D. Jagger1
1Ear Institute, Univerity Colege London, London, Non-US/Canada,
UK and 2Wolfson Institute of Biomedical Research, Univerity Colege
London, London, Non-US/Canada, UK
Type I Spiral Ganglion Neurons (SGN) synapse onto cochlear
inner hair cells, and constitute the majority of afferent fibres
in the auditory nerve. In the absence of functional hair cells,
SGN can be stimulated by cochlear implant electrodes to provide “electrical” hearing. Better characterisation of their biophysical properties may identify therapeutic targets for optimising auditory nerve sensitivity. SGN excitability has been
shown to be set by a Dendrotoxin-K-sensitive low voltage-activating K+ current (1). Activity of Kv channels can be sensitive
to binding of Phosphatidylinositol 4,5-bisphosphate, known
as “PIP2” (2), and consequently PIP2 can be an important determinant of neuronal excitability. Here we have assessed the
contribution of PIP2 signalling to SGN function. SGN from juvenile C57BL/6 mice (P12-P21) were cultured for 2-3 days in the
presence of Brain Derived Neurotrophic Factor. Under control
conditions ~70% (8/11) of SGN fired rapidly-adapting (“phasic”) action potentials under current clamp, and the remaining cells were slowly-adapting or non-adapting (“tonic” firing).
~20 % (2/11) exhibited spontaneous firing at the resting membrane potential. Pre-incubation for 1 hr at 37oC with 10 μM
Wortmannin, an enzyme inhibitor of PIP2 production (3),
reduced the prevalence of phasic firing to ~50% (12/23) and
increased the prevalence of spontaneous activity to ~40%
(10/23). The effects of Wortmannin treatment could be partially rescued by the intracellular application of diC8-PIP2, a
non-metabolisable PIP2analogue (n=10). In separate experiments, SGN were depleted of membrane-bound PIP2 by transient exposure to a membrane-targeting palmitoylated peptide (“PalPeptide”) based on the putative PIP2 binding domain
of the Kv7.2 channel (4). Bath application of 1-3 μM Pal-Peptide slowed adaptation in all cases (9/9). Under voltage clamp,
1 μM PalPeptide inhibited a low voltage-activated K+ current
(77 ± 4% (mean ± SEM) measured at 2 minutes, n=6). This effect
could be reduced significantly by intracellular application of
diC8-PIP2 (41 ± 5%, n=7, P<0.001). This PalPeptide-sensitive
current was also blocked by Dendrotoxin-K, identifying Kv1.1
as an important contributor. Other work from our lab has suggested Kv1.1 forms heteromeric channels with Kv1.2 in these
cells (Smith et al, this meeting). PalPeptide inhibited membrane currents in HEK-293 cells expressing Kv1.1/Kv1.2 channels (55 ± 14%, n=4), and this effect was reduced by intracellular application of diC8-PIP2 (23 ± 7%, n=4). We suggest PIP2
binding may provide an adjustable brake on the output of the
auditory nerve via its binding to Kv1-containing channels, and
our observations identify phosphoinositide signalling as a novel
therapeutic target in the cochlea.
Mo & Davis. (2002) J Physiol 542, 763-78
Kruse et al. (2012) J Gen Physiol 140, 189-205
Suh & Hille. (2005) Curr Opin Neurobiol 15, 370-8
Robbins et al. (2006) J Neurosci 26, 7950-61
This project was funded by a UCL Crucible Foundation
Studentship, and a grant from Action on Hearing Loss.
140P
PCA079
In vivo intracellular recordings from spinal lumbar
motoneurones in P0-deficient mice indicate an activitydependent axonal conduction failure in otherwise
functional motoneurones
J. Lehnhoff1, M. Moldovan1, A. Hedegaard2, L. Grøndahl1 and
C.F. Meehan1
1Department
of Neuroscience and Pharmacology, University of
Copenhagen, Copenhagen, Denmark and 2University of Oxford,
Oxford, UK
Mice deficient for the peripheral myelin binding protein zero
(P0-/-) show a progressive dysmyelinating neuropathy phenotypically resembling severe forms of Charcot-Marie-Tooth
(CMT) disease. Traditionally, the progression of the disease
was attributed to axonal loss, but the effect of chronic dysmyelination remains poorly understood. In this study, in vivo
electrophysiological recordings were used to assess the function of both central and axonal components of spinal lumbar
motoneurones in adult P0-/- mice.
Three month old P0-/- mice (n=7) and wild type (WT) littermate
controls (n=5) were anaesthetized with Hypnorm (0.315
mg/mL fentanyl-citrate + 10 mg/mL fluanisone), Midazolam
(5 mg/mL), and sterile water, mixed in the ratio 1:1:2 (induction: 0.15mL/25g, maintenance: 0.05 mL/20 minutes, S.C.).
Anaesthesia during surgery was assessed by the lack of reflexes
to a short noxious pinch on the hind foot. Prior to recording,
mice were ventilated before being paralysed with Pancuronium Bromide (0.01mg/h, I.P.). Anaesthesia after this was
maintained using the same doses as previously necessary during the surgical procedures (ensuring a deep anesthesia) and
its adequacy confirmed by the continuous monitoring of the
heart rate. Intracellular recordings with sharp microelectrodes
were made from identified spinal motoneurones (for detailed
methods see Meehan et al. 2010). After end experiments, animals received an overdose of anesthesia and were perfused
intracardially with 4% paraformaldehyde. Mean values are given
with SD.
Antidromic action potentials (APs) generated in the motor
axons often failed to reach the soma in P0-/- (34/62 cells). Intracellular current injection into the soma of these cells, however,
always resulted in somatic APs and repetitive firing was
observed at high frequencies. Furthermore, rheobase, voltage
threshold, input resistance, and AP amplitude and width were
not significantly different (all P>0.05, Mann Whitney). Further
investigation (including intra-axonal recordings) suggested
that the axonal conduction failure could be aggravated by prolonged stimulation, consistent with axonal dysfunction rather
than axonal loss. Recordings from motoneurone cell bodies of
P0-/- mice did show some abnormalities: The amplitude of the
post-spike after-hyperpolarization was increased (WT:
2.07±1.02mV (n=25), P0-/-: 2.50±0.65mV (n=22), Mann-Whitney: P=0.0311). There was also an earlier onset of the secondary range on I-f slopes (WT: 157±16Hz (n=11), P0-/-:
121±13Hz (n=18), Mann-Whitney: P<0.0001) and an increased
ΔI (de-recruitment minus recruitment current) following triangular current injections (WT: -1.10±2.69nA (n=25), P0-/-: 0.19±2.44nA (n=35), Mann-Whitney: P= 0.0148), indicating
increased activation of persistent inward currents.
Our identification of a functional, thus potentially reversible,
conduction failure in otherwise healthy motoneurones in P0/- mice raises the hope that symptomatic treatments could be
developed to improve motor function in CMT disease.
Meehan et al. (2010). J Neurophysiol. 103(5):2599-610
brain endothelial cells suggesting a possible role in vascular
dementia.
requirements.
This work was funded by a project grant from the Lundbeck
Foundation.
PCA081
requirements.
Effects of enriched environment on the improvement of
motor learning and the changes of striatal dopamine and
N-methyl-D-aspartate receptors in 6-hydroxydopamineinduced Parkinson’s Rat model
PCA080
Y. Huang, Y. Yang and R. Wang
The role of p17 protein from HIV in early onset of
neurodegenerative disease
Physical Therapy and Assistive Technology, National Yang-Ming
University, Taipei, Taiwan
Y. Zeinolabediny, J. Krupinski and M. Slevin
Parkinson’s disease (PD) is a neurodegenerative disease that
leads to impairments of motor control and learning. Degeneration of nigrostriatal dopaminergic neurons causes endogenous dopamine (DA) loss in the striatum of PD patients, affecting the functions of the circuit system. Previous studies have
found that dopamine D2 receptor and N-methyl-D-aspartate
(NMDA) receptor subtype, NR1 and NR2A receptors in the striatum participate in the mechanisms of motor skilled learning.
Motor skilled learning performance in 6-hydroxydopamine (6OHDA) rat model using rotarod test is vulnerable. Whether the
expressions of receptors that involved in motor skilled learning are affected by the inadequate striatal dopamine level is
not well known. Enriched environment (EE) is a housing condition that facilitates enhancement of sensory, cognitive and
motor stimulation. It has been reported that EE prevents
dopamine neuronal death, improves motor performance of 6OHDA rats and increases the expressions of NMDA receptors
in normal mice. We are looking forward to investigate whether
EE can influence the motor learning performance and the
expressions of striatal dopamine D2 and NMDA subtype NR1
and NR2A receptors in the Parkinson’s rat model. Eight-week
old male Sprague-Dawley (SD) rats (250-350g, n=48) were randomly assigned into three groups: control group, standard
environment group (SE) and enriched environment group (EE).
Each group was divided into two subgroups for the behavioral
and protein analysis. Rats in the SE and EE groups received unilateral (right substantia nigra pars compacta, SNpc) intracranial administration of 6-OHDA under pentobarital (50mg/kg)
anesthesia. Rats in the control group received intracranial
administration of saline as injection control. Rats in the control and SE groups received standard environment housing
condition (1 rat/cage) and rats in the EE group received
enriched environment housing condition (4 rats/cage) for four
weeks after surgery. Motor learning behavior was measured
by accelerating rotarod test at the first, third and eighth day
after the four-week intervention. The expressions of striatal
dopamine D2, NR1 and NR2A receptors were measured by
western blotting after the four-week intervention. Data were
presented as mean ± S.E.M. and compared by ANOVA. Motor
learning behavior of 6-OHDA rats showed a significant decrease
when compared with control group. However, rats received
EE intervention reserved the learning ability especially at the
first day of rotarod test and the learning rate showed similar
to the control group. The expressions of dopamine D2 and
NR1 receptors in the ipsilateral lesioned striatum showed no
significant difference among groups. However, the expression
of NR2A receptor in EE group showed a significant increase
when compared with SE group. Our results suggested that
enriched environment might improve the motor skilled learn-
Manchester Metropolitan university, Manchester, Lancashire, UK
HIV had evolved in a way that can protect itself from the human
immune system, however scientists have been researching to
achieve new findings to either stop or treat AIDS. The p17
matrix protein, which is one of the products of the HIV gene,
serves a structural function inside mature HIV particles. This
protein might play a key role in the complex network of hostand virus-derived stimulatory factors contributing to create a
favourable environment for HIV-1 infection and replication.
Patients with HIV may show signs of neuroinflammation and
are known to be at an increased risk of developing neurodegenerative diseases such as Alzheimer’s disease. Neurodegenerative disorders are believed to share common cellular
and molecular mechanisms and P17 is now thought to be a
key molecule responsible for modulating these processes. In
this project the role of p17 in promoting Alzheimer-like plaque
formation, neuronal damage, tau phosphorylation, Amyloidbeta cleavage, neurofibrillary tangle formation and endothelial dysfunction associated with vascular dementia is investigated by using the following methods: Immunofluorescence,
Immunohistochemistry, Cell culture including assays to examine neuronal health and endothelial angiogenesis and Western blotting to investigate signalling pathways linked to p17.
(Slevin et al, 2013)
Results showed co-staining of amyloid-β and p17 in human HIV
patient neurons and plaques associated with the progression
of Alzheimer’s disease. Its also showed Immune cell staining
of p17 co-staining with amyloid-β. p17 was expressed in blood
vessels, plaques and dying neurons. In mouse brain injected
with p17, p17 was identified in cortical microvessels co-staining with CD105 and hippocampal neurons co-staining with pTau.
In endothelial cells, the scratch wound migration assay showed
that both p17 and S75 increased significantly in number of
cells and distance migrated. Tube like structure formation
showed p17 and S75 increased the number of tubes compared
to control with those of S75X appearing to be thinner. A spheroid assay demonstrated cells and sprouts were more strongly
chemo-attractive in S75X compared to p17. Western blot
demonstrated that both p17 and S75X induced phosphorylation of proteins ERK, AKT and EGFR which are known to be
involved in angiogenesis.
In conclusion, p17 is found in regions of human brain tissue
showing evidence of neurodegenration. p17 injected to mouse
hippocampus induces Alzheimer’s like symptoms and pathology. p17 and its variant S75X strongly induce angiogenesis in
141P
ing ability, and enhance the expression of NMDA subtype NR2A
receptor in 6-OHDA-indused Parkinson’s rat model.
Edwards, C. M., S. Abusnana, et al. (1999). “The effect of the orexins
on food intake: comparison with neuropeptide Y, melanin-concentrating hormone and galanin.” J Endocrinol 160(3): R7-12.
requirements.
1. We are grateful to Kasturba Medical College, Mangalore
(Manipal University), for the facilities provided for this study.
PCA082
Role of orexin B in basolateral amygdala mediated
regulation of feeding behaviour
2. We are grateful to Department of Biotechnology, Ministry
of Science and Technology, Government of India for the
financial assistance for this study.
requirements.
G. Bolumbu, R. Ks, S. Mayannavar and K. Deshpande
Physiology, Kasturba Medical College (Manipal University),
Mangalore, Karnataka, India
Basolateral amygdala (BLA) has been implicated in the regulation of food intake apart from hypothalamic centres(1). In
the present study, we hypothesized that the Orexin B, a
polypeptide discovered in the lateral hypothalamic region,
may be involved in modification of role of amygdaloid centres(2). Therefore we studied the effect of bilateral infusion
of Orexin B and its antagonist (TCS-OX2-29) into BLA to study
the food and water consumption.
Adult male Wistar albino rats (n=54) were selected and
grouped into (i) Orexin infusion – Low dose; (ii). High dose (3
ng/kg, 30 ng/kg, on the basis of previous studies(3,4) (iii). TCSOX2-29 infused group. Each group was subdivided into control, sham operated control and experimental subgroups (n=6
each). Fasted animals were infused with Saline Or Orexin B OR
TCS-OX2-29 (as the case may be) into BLA bilaterally by
implanting a cannula using stereotaxic method. Harvard Pico
Plus infusion pump was used for microinfusion of these chemical agents. Following infusion, sequential Food intake and
water intake were measured in 1, 2, 4, 6 & 12 hours and intake
for the day was also recorded in all groups and the results
(mean±SD) were statistically analysed by Kruskal Walli’s test
and p<0.05 was considered significant. All experiments were
conducted adhering to strict ethical standards after getting
the approval from the institutional committee.
The food intake (g) and water intake (ml) were increased in
the experimental groups in first hour, 4th hour, 6th hour and
total intake for the day. In the high dose (p<0.01) group
increase was statistically significant. The consumption for the
whole day was also increased significantly (p<0.01) in the high
dose group. Injection of Orexin B antagonist decreased the
food and water intake significantly in the period following
the infusion (p<0.05).
There was an increase in feeding behaviour on infusion of
Orexin B and decrease after the infusion of its antagonist into
BLA. Form the results it appears that Orexin plays a role in the
Basolateral amygdaloid nucleus mediated modulation of feeding behaviour. Role of orexin in ingestive behaviour is further
confirmed by the action of Antagonist injection. One of the
centres which mediated the orexin effects on the feeding
behaviour could be BLA. Therefore Orexin B could be considered as a candidate neuroactive substance in feeding behaviuor. Further studies are being conducted in our laboratory for
elucidating the role of Orexin on ingestive behaviour.
Ganaraja, B. and P. S. Jeganathan (1999). “Increased sweet taste preference following the lesion of basolateral nucleus of amygdala (BLA)
in rat.” Indian J Physiol Pharmacol 43(4): 443-8.
Arch, J. (2000). “Orexins, feeding and the big picture.” Br J Nutr 84(4):
401-3.
Sakurai, T., A. Amemiya, et al. (1998). “Orexins and orexin receptors:
a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior.” Cell 92(4): 573-85.
142P
PCA083
Unexpected anaesthesia by combination of alkaline and
acidic gases in Drosophila assayed by behavioural
geotropism
L. Orchard, A.H. Schwiening and C.J. Schwiening
Department of Physiology, Development and Neuroscience,
University of Cambridge, Cambridge, Cambridgeshire, UK
It is well known that changes in CO2 concentration alter neuronal excitability. Whilst Hickman’s early attempts to promote
CO2 anaesthesia met resistance (Antiquack, 1826) it is now
routinely used on insects and small mammals. Despite this
its mode of action remains obscure (Badre et al., 2005). Since
CO2 reacts with water, catalyzed by carbonic anhydrase, there
appear to be three possible active
agents: molecular CO2, pH and [HCO3-]. To distinguish between
these we have combined CO2 with NH3. If the anaesthetic effect
derives from the lipid soluble form of the gas (molecular CO2
or NH3) then the effect of the application of the two gases
should be arithmetically additive. However, If the anaesthetic
effect is due to acidosis produced by CO2 then NH3, which is
well known to alkalinize, should be antagonistic. Finally, if the
anaesthetic effect is due to [HCO3-] then NH3 should be synergistic with CO2 since NH4+ formation ‘mops-up’ H+ allowing
a given CO2 concentration to produce more [HCO3-].
We have used image analysis software to quantify the percentage of Drosophila remaining at the bottom of jars after
being tapped-down following exposure to various gas concentrations. Within each experiment we averaged the response
to 10 taps (30 s intervals). Fig 1A shows the percentage of
Drosophila at the bottom of the jar 24 s after tap-down following exposure to NH3 for ~5 mins. Fig 1B shows data for CO2
exposure alone (CO2 KD50 ~60%) and following the addition
of 0.1% NH3 on a CO2-background (CO2 KD50 ~20%). When
applied alone both 40% O2 and 0.1% NH3 have little effect.
However, adding 0.1% NH3 during CO2 exposure (CO2 >20%)
produced a profound anaesthesia. These results are not consistent with either molecular CO2 or CO2-induced acidosis being
the active agents. Indeed, they suggest a role for HCO3-. There
are a number of mechanisms by which HCO3- may alter
excitability. HCO3- may permeate GABAA receptors or it may
act indirectly by altering Cl- or K+ equilibria. Finally, since pH
buffering is proportional to [HCO3-] and [NH4+] we cannot rule
out that these excitability changes occur through the modulation of physiological pH shifts which may drive vesicle-fusion
(Caldwell et al., 2013). Unlike the other HCO3--dependent
mechanisms the pH buffering hypothesis is also consistent
with the sensitivity of vesicle fusion to the presence of other
weak acids even in the absence of CO2 (Drapeau & Nachshen,
1988; Caldwell et al., 2013).
potential, expressing the amplitude of the slowest component
as a fraction of the peak (Fig. 1).
Our data indicate a downward trend in the average slow component amplitude (Fig. 1A), and over the same period a statistically-significant disappearance of the slow component (Fig.
1B), p = 0.0184,(n = 19, 5; day 0 + day 7, day 21, respectively;
Fisher exact test), supporting the hypothesis that in ON, functional loss is likely to be greatest in the population of smallest
axons.
Figure 1 Effect of gaseous NH3 and CO2
(including 20% O2, with N2 as a filler at atmospheric
pressure) on Drosophila negative geotropism assayed at 24 s after
tap-down
as the percentage on the bottom of a clean 10 ml jar (each datum
is the
mean±SEM of 3-5 populations of ~12 naïve Drosophila). A, in NH3
(solubility ~780 times that of CO2) B, in CO2 (open
circles) and after 0.1% NH3 was added during CO2 exposure
Figure 1. Decline in action potential amplitude 0.7 ms following the peak
(mean + s.e.m) (A), and disappearance of the slow component seen at 21
days (B).
(filled circles).
Ganter P et al. (1999). Neuropath and Appl Neurobiol 25, 459-467.
Antiquack (1826). Surgical humbug. Lancet 5, 646–647.
Evangelou N et al. (2001). Brain 124, 1813-1820.
Badre, Martin & Cooper (2005). The physiological and behavioural
effects of carbon dioxide on Drosophila melanogaster larvae. Comp
Biochem Physiol 140, 363-376.
Caldwell et al. (2013). Presynaptic pH and vesicle fusion in Drosophila
larvae neurones. Synapse 67, 729-740.
Drapeau & Nachshen (1988). Effects of Lowering Extracellular and
Cytosolic pH on Calcium Fluxes, Cytosolic Calcium Levels and Transmitter Release in Presynaptic Nerve Terminals Isolated from Rat Brain.
J Gen Physiol 91, 305-315.
Lidster
K
et
al.
(2013).
10.1371/journal.pone.0079188.
Optic nerve functional properties in MOGTCR mice with
induced optic neuritis
A. Anandhakrishnan, S. von Kutzleben, S. Al-izki, G. Giovannoni,
D. Baker and M.D. Baker
Neuroscience and Trauma Centre, Queen Mary University of
London, London, UK
Primary or secondary progression in multiple sclerosis (MS)
leads to irreversible disability. While neuronal and axonal loss
are widely accepted to underlie progression, any axonal subtype selectivity remains equivocal, although human postmortem studies indicate a selective loss of the smallest axons
(e.g. Ganter et al. 1999; Evangelou et al. 2001). One common
early symptom of MS is optic neuritis (ON) and we have studied the functional effects of induced ON in a transgenic mouse
model, with an enhanced immune response to myelin oligodendrocyte glycoprotein (MOGTCR; Lidster et al 2013), in exvivo isolated optic nerve. To induce ON, mice were injected
intraperitoneally with 150 ng Bordetella pertussis toxin on day
0 and day 2, and on day 14 with 0.25 mg MOG-specific Z12
mouse IgG2a monoclonal antibody. All procedures were
approved by the UK Home Office and the local ethics committee. Supramaximal compound action potentials were
recorded that normally comprise more than one component,
the slowest component interpreted as the functional correlate
of the smallest axons. We analyzed the shape of the action
One
8,
e79188:
We acknowledge the support of the British Society for Clinical
Neurophysiology and the intercalated Neuroscience program,
QMUL
requirements.
requirements.
PCA084
PLoS
PCA085
The role of Doc2B in depolarization-evoked and G proteincoupled receptor-modulated exocytosis in mouse
chromaffin cells
C. Bauer1, A. Groffen2, M. Verhage2 and E. Seward1
1Biomedical
Sciences, University of Sheffield, Sheffield, South
Yorkshire, UK and 2Department of Functional Genomics, CNCR,
Neuroscience Campus Amsterdam, VU University and VU Medical
Center, Amsterdam, Netherlands
G protein-coupled receptors (GPCRs) represent the largest
receptor protein family and form part of a complex information processing system used to modulate neurotransmitter
release in the nervous system. Aberrant signalling and excess
neurotransmission in –for example– the peripheral nervous
system is associated with cardiovascular disorders or chronic
and inflammatory pain. Understanding the complex interplay
between GPCR signalling and the exocytotic machinery in
health and disease is therefore pivotal.
Previously we showed that activation of Gq protein-coupled
H1 histamine receptors potentiates stimulus-coupled exocytosis in bovine neuroendocrine chromaffin cells despite inhibiting Ca2+ influx through voltage-gated calcium channels
(VGCCs)[1]. This potentiation is restricted to the immediately
releasable pool (IRP) of vesicles tightly coupled to VGCCs and
requires the SNARE priming protein Munc13-1. Doc2B is a high
affinity calcium-binding protein known to interact with
Munc13-1 to promote exocytosis; whether it plays a role in
agonist-regulated exocytosis is unknown. To address this ques-
143P
tion, we isolated chromaffin cells from adult Doc2B (+/+), (+/) and (-/-) mice and examined the effects of PLC-coupled GqPCR
receptor activation on depolarization-evoked exocytosis, measured as changes in membrane capacitance.
We found that like in neurons, stimulus-coupled synchronous
exocytosis (evoked by a 40 or 200 ms depolarization from -80
to +10 mV) was not significantly different between Doc2B
(+/+), (+/-) and (-/-) cells. In contrast, asynchronous release was
significantly increased in Doc2B (-/-) cells demonstrating a regulatory role of Doc2B in Ca2+-evoked asynchronous release.
Replacing Ca2+ by Ba2+ however showed that Doc2B is not
the sensor for Ba2+-mediated asynchronous release.
Finally, although more than 80 % of mouse chromaffin cells,
independent of their genotype, responded to 100uM histamine with a reduction of Ca2+ influx, potentiation of exocytosis was surprisingly modest (2-fold) and was rarely observed
in all three genotypes ((+/+): 5/17 cells, (+/-): 5/24 cells, (-/-):
2/18). This is in stark contrast to bovine cells, where 80 % of
the cells show a 5-fold potentiation of exocytosis from the
IRP and RRP. Importantly, bypassing GPCRs by using the diacylglycerol analog PMA, mouse chromaffin cells responded
with potentiation of exocytosis from the RRP, but not the IRP;
another difference to their bovine equivalent. Taken together,
the results suggest that there are profound species differences
in the organization of the secretory machinery, VGCCs and its
regulation by GqPCRs in neuroendocrine cells.
lipid membranes, erythrocytes (Holland and Graham, 1955),
oocytes (Burckhardt et al., 1992) and neurons (Krishtal and
Pidoplichko, 1980). Whether excitation of the postsynaptic
cell is due to protons and/or due to intact ACh, is a fundamental
question that has not received any attention up to date.
Figure 1. Hydrolysis of ACh is catalyzed by AChE and liberates acetic acid
as well as choline.
Burckhardt B et al. (1992). Pflügers Arch. 420, 78–82.
Holland WC & Graham JH (1955). Am. J. Physiol. 183, 538–44.
Kaufmann K (1977). Naturwissenschaften 64, 371–376.
Krishtal O & Pidoplichko V (1980). Neuroscience 5, 2325–2327.
Walters D et al. (1961). Nature 189, 381–383.
[1] Bauer C.S., Woolley R. J., Teschemacher A. G., and Seward E.P.
(2007) J. Neurosci. 27: 212-19. Potentiation of exocytosis by PLC-coupled GPCRs requires the priming protein Munc13-1.
We thank K. Kaufmann for advocating the importance of
interfaces in biology and for introducing the analogy of an
enzymatic “proton pistol”. Moreover, we are thankful for his
stimulating lectures and the ensuing discussions on the
thermodynamics of soft interfaces.
requirements.
requirements.
PCA086
PCA087
Does acetylcholinesterase deactivate acetylcholine?
Circulating immune markers of age-related cognitive decline
C. Fillafer and M.F. Schneider
S.L. Bradburn, J.S. McPhee, L.J. Bagley and C.A. Murgatroyd
Department of Mechanical Engineering, Boston University, Boston,
MA, USA
School of Healthcare Science, Manchester Metropolitan University,
Manchester, Lancashire, UK
Acetylcholine (ACh) is one of the most important transmitters
in the nervous system. Presently, it is assumed that the following sequence of events occurs in a cholinergic synapse:
ACh (1) is relased by a nerve cell, (2) binds to receptor proteins
on a neighbouring cell (e.g. muscle, nerve, gland, etc.) and
(3) is rapidly degraded by acetylcholinesterase (AChE). This
theory rests on the fundamental postulate that hydrolysis of
ACh by AChE leads to its deactivation (in the sense of reducing
or eliminating ACh’s excitatory effect).
This widespread assumption was criticised by Kaufmann (Kaufmann, 1977) and is indeed falsified herein. It is demonstrated
that excitable plant cells (Chara australis internodes), which
had previously been unresponsive to ACh, were rendered AChsensitive in the presence of AChE. This was underlined by a
striking difference in cell membrane depolarisation upon exposure to intact ACh (ΔV=-2±5 mV average±StDev (n=6)) and its
hydrolysate respectively (ΔV=81±19 mV (n=14)). This pronounced depolarisation, which also triggered action potentials, was clearly attributed to one of the hydrolysis products:
acetic acid (ΔV=87±9 mV at pH 4.0 (n=5); choline ineffective
in range 1-10 mM (n=3)).
Our findings suggest that AChE does not clean up the synapse,
but actually generates an excitatory agent (acetic acid). Protons excite the plant cells used in this study as well as a large
variety of other systems including gels (Walters et al., 1961),
Investigating the molecular mechanisms underlying the decline
in cognitive abilities during the ageing process is an important
area of research. Peripheral markers of inflammation have
recently been implicated in the regulation of neurogenesis
(Villeda et al, 2011). We investigated the relationship between
circulating cytokines and cognitive abilities in healthy ageing
humans. Young (n = 138, mean age ± SD: 23.4 ± 2.7 yrs, 53%
female) and old (n = 237, mean age ± SD: 74.4 ± 3.3 yrs, 51%
female) participants of the MYOAGE study (McPhee et al, 2013)
gave written informed consent. Neurocognitive performance
was assessed by the Cambridge Neuropsychological Test Automated Battery (CANTAB) and included the Paired Associate
Learning (PAL), Spatial Span (SSP) and One Touch Stockings
(OTS) tests. Fasted plasma samples were used to assess 31
cytokines using multiplex immunoassays (Millipore). Differences between age groups was analysed via the Mann-Whitney U test. Associations between plasma proteins and cognitive performance were analyzed using the Significance Analysis
of Microarray (SAM, v. 4.01 algorithm) software. To visualize
age effects, protein data was further transformed into Z-scores
and unsupervised cluster analysis was performed using the
Gene Cluster (v.3.0) software. The resulting heat map was visualized with Java TreeView (v. 1.1.6). Compared with the young
adults, healthy old showed significantly lower performance
144P
in episodic memory (PAL = P < 0.0005), working memory (SSP
= P < 0.0005) and executive function (OTS = P < 0.0005). Strikingly, sixteen of the cytokines investigated differed significantly
between young and old (P < 0.05). Furthermore cluster analysis demonstrated an inflammatory profile of ageing that associated with cognitive differences in both young and old adults.
In conclusion, we have characterized the inflammatory profile
of healthy ageing and have identified age-related immune
markers that associate with cognition.
McPhee et al. (2013). Biogerontology. 14 (3), 325 – 37.
Villeda et al. (2011). Nature. 477 (7362), 90 – 94.
The authors wish to thank all of the participants for their
involvement in the study as well as the ongoing collaboration
with the MyoAge partners at Paris, France; Leiden, The
Netherlands; Tartu, Estonia; and Jyväskylä, Finland.
thetic neurons. Type 2 PDE2-specific inhibitor Bay 60-7550 (1
μM) restored the capacity for both concentrations of BNP to
reduce intracellular Ca2+ transients in the SHR. Overexpression of PDE2A using a viral vector (Ad.CMV-mCherry.PDE2A)
on the sympathetic neurons abrogated the response to 250
nM BNP in the WKY. This was reversed by PDE2 inhibition.
Conclusion: These data demonstrate that attenuation of
[Ca2+]i and the neuronal calcium current by BNP is impaired
in the SHR, and this may be associated with apparent over
activity of PDE2A. Our results suggest that neuronal PDE2 may
play a potential role as a pharmacological target to restore the
efficacy of BNP to decrease sympathetic neurotransmission.
Key words: B-type natriuretic peptide, Phosphodiesterase 2A,
calcium, sympathetic neuron
Li D et al. Targeted neuronal nitric oxide synthase transgene delivery
into stellate neurons reverses impaired intracellular calcium transients
in prehypertensive rats. Hypertension 2013 Jan;61(1):202-207
The study was funded by the EU FP7 “MyoAge” (223576) grant.
requirements.
PCA088
A novel role for B-type natriuretic peptide and
phosphodiesterase 2A in cardiac sympathetic neurons from
prehypertensive rats
D. Li, L. Woodward, G. Hao, C. Lu, K. Liu and D.J. Paterson
Department of Physiology, Anatomy & Genetics, University of
Oxford, Oxford, UK
Purpose: Natriuretic peptides (NPs) are a family of peptide hormones known to be potent regulators of the cardiovascular
system. They play a pivotal role in the regulation of intravascular volume by modulating blood vessel tone and renal function. Elevated B-type natriuretic peptide (BNP) is regarded as
an early compensatory response to hypertension and heart
failure. However, the use of a recombinant BNP agonist in clinical trials has proved disappointing, with some suggesting it
actually enhances cardiac sympathetic activity. Our previous
data shows that BNP decreases cardiac sympathetic neurotransmission by attenuating activation of neuronal calcium
channels and the intracellular calcium transient via a cGMPPKG pathway. Emerging evidence suggests that overactivity
of phosphodiesterase 2A (PDE2A) may impair the efficacy of
BNP to regulate the intracellular calcium concentration
([Ca2+]i) in CNS neurons. Therefore we tested whether PDE2A
was directly involved in modulating Ca2+ handling in cardiac
sympathetic neurons from pre-hypertensive spontaneously
hypertensive rats (SHRs) that show an enhanced Ca2+ phenotype.
Methods and Results: Rats were humanely killed by an
approved Home Office schedule 1 method and the cardiac stellate ganglia were enzymatically isolated. Calcium current was
measured using the whole cell configuration of the patch-clamp
technique. Currents were evoked by test pulses to -10 mV from
a holding potential of -90 mV. [Ca2+]i transient was measured
by ratiometric fluorescence imaging using fura-2AM in cultured
cardiac sympathetic neurons. The evoked [Ca2+]i transient
was evaluated following 30 sec exposure to 50 mM KCl in the
Tyrode solution. Fura-2/AM was excited alternately at 350 nm
and 380 nm and the emitted fluorescence measured at 510
nm. 100 nM & 250 nM BNP significantly reduced the magnitude of the Ca2+ transients and calcium current in normotensive Wistar-Kyoto (WKY) rats, but not in SHR sympa-
Chan NY et al. An unsuspected property of natriuretic peptides: promotion of calcium-dependent catecholamine release via protein kinase
G-mediated phosphodiesterase type 3 inhibition. Circulation. 2012
Jan;125(2):298-307
requirements.
PCA089
STIM1 controls neuronal Ca2+ signaling and mGluR1/TRPC3dependent synaptic transmission
R.P. Alexander1, J. Hartmann2, R.M. Karl2, K. Sakimura3,
Y. Baba4, T. Kurosaki4 and A. Konnerth2
1Pharmacology and Therapeutics, McGill University, Montreal, QC,
Canada, 2Institute of Neuroscience, Technical University Munich,
Munich, Bavaria, Germany, 3Brain Research Institute, Niigata
University, Niigata, Japan and 4Immunology Frontier Research
Center, Osaka University, Osaka, Japan
In cerebellar Purkinje cells (PCs), the metabotropic glutamate
receptor type 1 (mGluR1) is crucial for cerebellar function.
Synaptic activation of mGluR1 is followed by InsP3 receptordependent Ca2+ release from endoplasmic reticulum (ER) Ca2+
stores and a slow EPSC (sEPSC) mediated by the transient receptor potential channel TRPC3. The intracellular mechanism of
TRPC3 activation is unknown as is how ER Ca2+ content is maintained through many cycles of synaptic activation. In nonexcitable cells TRPC channels are known to interact with the
stromal interaction molecules (STIM1 and STIM2), which are
also critical for Ca2+ store replenishment. Here we tested the
hypothesis that STIM proteins may be involved in TRPC3 activation in central neurons. Values are presented as means ±
SEM, compared by Student’s t-test. Quantitative single cell RTPCR analysis showed that expression of STIM1 in PCs is ten
times greater than that of its homolog STIM2 (103±71 vs. 11±8
copies mRNA/cell of Stim1 and Stim2 from n=23 and 14 cells,
respectively, p<0.01). Using whole-cell recordings and confocal Ca2+ imaging in acute cerebellar slices from newly generated PC-specific STIM1/2 double-knockout (STIM1/2pdko) mice
(C57/BL6 background, anesthetization by CO2 inhalation,
immediate decapitation), we found that mGluR1-mediated
Ca2+ release and the TRPC3-sEPSC evoked by repetitive parallel fibre stimulation (5 x 10V, 10 μM CNQX) are largely abolished in the absence of STIM1 and STIM2 (15.1±3.3 vs
89.1±15.9 pA; ΔF/F =34.1±8.3 vs 162.6±18.0 %, n=68 vs 32
inputs, p<0.01, respectively). Comparison with mice deficient
in STIM1 alone (STIM1pko mice) showed that of the two
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homologs STIM1 has the primary role for mGluR1-dependent
signaling in PCs. Synaptic stimulation-evoked sEPSCs and Ca2+
signals had mean amplitudes of 5±3 pA and ΔF/F = 2±1 %,
respectively (n=21 inputs). We tested the function of ER Ca2+
stores specifically in the absence of STIM1. In control mice,
Ca2+ release signals evoked by local application of the group I
mGluR-specific agonist DHPG were abolished when depleting Ca2+ stores by switching to Ca2+ free extracellular perfusion media and recovered within a few minutes after returning to Ca2+ containing media. In STIM1pko mice, DHPG
applications completely failed to evoke Ca2+ transients before,
during and after the perfusion with Ca2+ free extracellular
media. Interestingly, DHPG applications evoked both Ca2+ signals and TRPC3-mediated inward currents in STIM1pko mice
immediately (4s) following Ca2+ influx through voltage-gated
Ca2+ channels evoked by a short (1s) depolarization but failed
to be effective at later time points. These results demonstrate
that STIM1 is a powerful regulator of the ER store Ca2+ content in mammalian neurons. Furthermore, they identify STIM1
as a messenger that couples mGluR1 and TRPC3 in cerebellar
PCs through the regulation of intracellular Ca2+.
requirements.
PCA090
Isolating a non-canonical metabotropic glutamate receptor
from mechanosensory nerve terminals
K.J. Thompson1, R.W. Banks2 and G.S. Bewick1
1Institute
of Medical Sciences, University of Aberdeen, Aberdeen,
UK and 2School of Biological and Biomedical Sciences, University
of Durham, Durham, UK
There is increasing evidence that the excitatory neurotransmitter glutamate has an important function in the peripheral
nervous system, specifically in modulating mechanosensitivity1, in addition to its well established role as a transmitter in
the central nervous system. We are using stretch-sensitive
annulospiral afferent nerve endings in muscle proprioceptors
(muscle spindles) to explore this system. The endings contain
synaptic levels of glutamate and express glutamate transporters, as well as proteins involved in Ca2+-mediated exocytosis. Exogenous glutamate increases stretch-evoked firing
while glutamate receptor antagonists or blockade of exocytosis inhibit, or even entirely abolish, firing. This suggests the
expression of a glutamate receptor on these nerve endings1.
However, the receptor shows an atypical pharmacological profile2. The drug effects on afferent nerve response most closely
match the pharmacological profile of the phospholipase Dcoupled metabotropic glutamate receptor (mGluR), reported
by a number of groups in the hippocampus 3,4. However, it has
never been isolated, sequenced or characterised. Based on its
pharmacological profile, this atypical mGluR is either an
uncharacterised splice variant, or an entirely novel mGluR.
Pharmacology suggests it is the only mGluR expressed in the
spindle, therefore stretch-sensitive nerve endings are an ideal
source for receptor isolation and characterisation.
The rat deep masseter muscle has been identified as a rich
source of muscle spindles 5, from which we have developed a
method of nerve ending extraction to isolate and characterise
the atypical mGluR. Deep masseter muscles were removed
from adult male Sprague Dawley rats (weight 200 – 450g) and
euthanised by CO2 overdose (in accordance with EU Legislation, 2013). Spindles were dissociated from the tissue by col-
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lagenase digestion and then identified by staining with methylene blue (Figure 1). Screening techniques included Western
blotting, mass spectrometry and immunohistochemistry. Current results suggest that the atypical mGluR may be a splice
variant of mGluR5, based on Western blotting (Figure 2). A
strong band of approximately 102 kDa was observed, whereas
the band in the hippocampus was approximately 150 kDa, consistent with the expected molecular weight. No consistent
labelling was seen for mGluR1, mGluR2, mGluR3, mGluR4,
mGluR6, mGluR7 or mGluR8 in muscle spindles (n=3 for each).
Mass spectrometry has revealed abundant spindle and synaptic proteins in whole tissue homogenates. Candidate channels
thought to play a role in mechanotransduction are also being
sought in our screening process. Progress to date and preliminary findings on these studies will be reported.
Bewick GS, Reid B, Richardson C, Banks RW. Autogenic modulation
of mechanoreceptor excitability by glutamate release from synapticlike vesicles: Evidence from the rat muscle spindle primary sensory
ending. J Physiol. 2005;562(Pt 2):381-394.
Watson S, Zanato C, Dall’Angello S, et al. Development of fluorescent
and biotinylated agonists for a novel glutamate receptor in
mechanosensory terminals. Proc. 37th IUPS. 2013.
Boss V, Nutt KM, Conn PJ. L-cysteine sulfinic acid as an endogenous
agonist of a novel metabotropic receptor coupled to stimulation of
phospholipase D activity. Mol Pharmacol. 1994;45(6):1177-1182.
Albani-Torregrossa S, Attucci S, Marinozzi M, Pellicciari R, Moroni F,
Pellegrini-Giampietro DE. Antagonist pharmacology of metabotropic
glutamate receptors coupled to phospholipase D activation in adult
rat hippocampus: Focus on (2R, 1’S,2’R,3’S)-2-(2’-carboxy-3’-phenylcyclopropyl)glycine versus 3,5- dihydroxyphenylglycine. Mol Pharmacol. 1999;55(4):699-707.
Banks RW, Hulliger M, Saed HH, Stacey MJ. A comparative analysis of
the encapsulated end-organs of mammalian skeletal muscles and of
their sensory nerve endings. J Anat. 2009;214(6):859-887.
We are grateful to Tenovus Scotland and the University of
Aberdeen Development Trust for funding this project. We also
thank the University of Aberdeen Proteomics Facility.
requirements.
vesicles (75.9%, p < 0.0005, n=81) compared to WT, indicating that the vesicles in LRRK2 KO were swollen. Moreover, the
[PolyP] in PINK1 KO non-lysosomal vesicles decreased in
response to FCCP, whereas [PolyP] was observed to increase
in some vesicles for both WT and LRRK1 KO.
This study has shown that PolyP is partially located in lysosomes and partially in unidentified non-lysosomal PolyP vesicles. Lysosomes released PolyP into the cytosol in a calciumdependent manner, while some non-lysosomal PolyP vesicles
increased in [PolyP], likely responding to the increase in
[PolyP]c by taking up PolyP. The increase in [PolyP] in non-lysosomal PolyP vesicles was not observed in PINK1 KO. Furthermore, lysosomes and PolyP vesicles were shown to be swollen
in LRRK2 KO compared to WT, suggesting a novel characteristic for the LRRK2 KO PD model. Considering the significant
redistribution of PolyP and vesicles in astrocytes from PINK1
KO and LRRK2 KO compared to WT, astrocytic PolyP-dependent glial transmission might be altered in these PD models.
Gabel NW, Thomas V. Evidence for the occurrence and distribution of
inorganic polyphosphates in vertebrate tissues. J Neurochem. 1971
Jul;18(7):1229–42.
Kumble KD, Kornberg A. Inorganic polyphosphate in mammalian cells
and tissues. J Biol Chem. 1995;270:5818–22.
Holmström KM, Marina N, Baev AY, Wood NW, Gourine A V, Abramov
AY. Signalling properties of inorganic polyphosphate in the mammalian
brain. Nat Commun. 2013 Jan;4:1362.
PCA091
Abramov AY, Duchen MR. Actions of ionomycin, 4-BrA23187 and a
novel electrogenic Ca2+ ionophore on mitochondria in intact cells.
Cell Calcium. 2003 Feb;33(2):101–12.
Mechanisms and release of a novel glial transmitter –
inorganic polyphosphate
Collins TJ, Lipp P, Berridge MJ, Li W, Bootman MD. Inositol 1,4,5-trisphosphate-induced Ca2+ release is inhibited by mitochondrial depolarization. Biochem J. 2000 Apr 15;347(Pt 2):593–600.
K.Z. Iversen1, P.R. Angelova2 and A.Y. Abramov2
1Deparment
of Natural Sciences, UCL, London, UK and
2Department of Molecular Neuroscience, UCL Institute of
Neurology, London, UK
Inorganic polyphosphate (PolyP) is a polymer of orthophosphate residues that is abundant to all organisms, including the
brain (1,2). Recently, it has been shown that PolyP is released
by astrocytes in a calcium-dependent manner and acts as a
gliotransmitter via P2Y1 purinergic receptors (3). However,
the localisation and mechanisms of release of PolyP from astrocytes are unclear.
Using DAPI and a novel PolyP fluorescent indicators, we studied the location of PolyP in primary astrocytes and the mechanisms of release. PolyP was found to compartmentalise in
vesicle-like structures in primary cultures of astrocytes. Using
Lysotracker Red to visualise lysosomes and doing colocalisation analysis, we identified some of these vesicles as lysosomes
(Mander’s coefficients: Mx=0.49±0.066, n=7; My=0.61±0.046,
n=7). However, PolyP also compartmentalises in non-lysosomal PolyP-vesicles, possibly in ATP-containing vesicles as
demonstrated through intensity profiles.
Release of polyphosphate out of lysosomal vesicles could be
stimulated by the calcium ionophore ferutinin (5 μM) (4) or
the protonophore FCCP (1 μM, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) (5). FCCP furthermore induced
an increase in [PolyP] in some non-lysosomal PolyP vesicles,
indicating a PolyP uptake in response to the increase of cytosolic PolyP due to lysosomal PolyP release.
We also observed a redistribution of the polyphosphate fluorescence signal in astrocytes between different genetic cell
models of PD (PINK1 KO, LRRK2 KO). Using student t-test to
statistically compare the average vesicle area size revealed that
lysosomes were significantly increased in LRRK2 KO astrocytes
(103.6%, p < 0.00005, n=220) as were the non-lysosomal PolyP
requirements.
PCA092
Sulfhydration of P66Shc mediates the anti-oxidant effect of
Hydrogen Sulfide
Z. Xie and J. Bian
Department of Pharmacology, National University of Singapore,
Singapore, Singapore
Mitochondrion is considered as the major source of intracellular reactive oxygen species (ROS). Hydrogen sulfide (H2S) a
newer endogenous gaseous mediator, is reported to be antioxidant(1,2). However, the underlying mechanism remains
largely elusive. P66Shc is an upstream activator of mitochondrial redox signaling (3,4). The aim of this study was to explore
whether the antioxidant effect of H2S is mediated by p66Shc.
We found that pretreatment with NaHS (an H2S donor, 100
μM) for 30 min inhibited H2O2 (50 μM, 2 h) induced mitochondrial ROS generation. Similarly, over-expression of CBS
caused a significant inhibition on H2O2 induced mitochondrial oxidative stress. Both endogenous and exogenous H2S
induced p66Shc sulfhydration and attenuated H2O2-induced
phosphorylation of p66Sh. To identify the sulfhydrated cysteine residue in p66Shc, the conversed cysteine-59 was
mutated to serine (C59S). It was found that C59S markedly
attenuated p66Shc sulfhydration induced by NaHS and the
inhibitory effect of H2S on H2O2-induced p66Shc phospholyration. These data suggest that H2S-induced sulfhydration
may contribute to its inhibitory effect against p66Shc phospholyration. Moreover, C59S also attenuated the protective
147P
effect of NaHS against H2O2-induced mitochonridal ROS production and cell injury. Co-immunoprecipitation and Western
blotting assays showed that NaHS treatment reduced the interaction between PKCβII and p66Shc in HEK293 cells treated
with H2O2. However, this effect was eliminated in C59S, suggesting a key role of Cys-59 in PKCβII mediated p66Shc phosphorylation.
We also evaluated the mitochondrial pool of p66Shc. H2O2
treatment increased the amount of p66Shc within mitochondria in normal SH-SY5Y cells. In contrast, the expressions of
p66Shc were less pronounced in mitochondria isolated from
cells pretreated with 100 μM NaHS for 30 min. Similar effect
was also found in HEK293 cells transfected with WT-p66Shc.
However, no detectable changes were observed in cells transfected with C59S mutants. This was further confirmed by
immunofluorescence assay. Confocal microscopy confirmed a
clear preferential colocalization between p66Shc and mitochondria upon treatment with H2O2 (50 μM) for 30 min and
this effect was abolished by NaHS pretreatment. Our data suggest that H2S may prevent the translocation of p66Shc to the
mitochondria and its contribution to ROS generation. In conclusion, H2S inhibits mitochondrial ROS production via the
sulfhydration of Cys-59 residue, which, in turn, prevents the
phosphorylation of p66Shc.We therefore revealed a novel
mechanism for the anti-oxidant effect of H2S and its role in
the oxidative stress related diseases.
Proposed model for the effect of H2S on p66-Shc mediated mitochondrial
ROS generation. H2S sulfhydrates p66Shc at cysteine-59 in the N-terminal
CH2 domain. This modification disrupts the association between PKCβII
and p66Shc and therefore leads to the inhibition of PKCβII-mediated p66Shc
phosphorylation at Ser36. This, in turn, inhibits p66Shc translocation to
mitochondria, and therefore decreases mitochondrial ROS production.
PCA093
Activation of ATF4 is critical for MCP-1 production in
endothelial cells
J.J. Wang, H. Huang and S.X. Zhang
The State University of New York at Buffalo, Buffalo, NY, USA
Inflammation plays an important role in the pathogenesis of
diabetic retinopathy. Monocyte chemoattractant protein-1
(MCP-1) is a major inducible chemokine that recruits monocytes and T cells to the sites of inflammation. Upregulation of
MCP-1 expression and secretion in the retinal tissue is critical
for leukostasis, breakdown of the blood-retinal barrier, and
neurovascular injury in diabetic retinopathy. The present study
aimed to explore the role of activating transcription factor 4
(ATF4) in MCP-1 regulation in microvascular endothelial cells.
Using mouse brain microvascular endothelial cells and retinal
endothelial cells, we demonstrated that activation of TLR4 signaling by lipopolysaccharide (LPS) resulted in elevated ER stress
and level of ATF4. Overexpression of ATF4 enhanced MCP-1
expression and secretion while inhibition of ATF4 function
reduced but not completely reversed TLR4-induced MCP-1
expression. Intravitreal injection of adenovirus expressing ATF4
dramatically increased retinal MCP-1 level, enhanced CD11b
expression, and increased leukocyte infiltration into vitreous.
In contrast, LPS-induced MCP-1 upregulation in the retina was
markedly attenuated in ATF4 knockout mice. Furthermore,
monocyte/macrophage migration was enhanced by conditioned media from endothelial cells pretreated with Ad-ATF4
or LPS. Condition media from ATF4 knockout mice induced
less monocyte/macrophage migration than that from wild
type mice. Mechanistically, our data suggest that ATF4
increases MCP-1 production through enhancing activation of
MyD88/NF-KB and AP-1 pathways. Simultaneously, ATF4 binds
to the NE-F2 site of MCP-1 promoter. Overexpression of ATF4
up-regulates Nrf2 expression, which in turn suppresses ATF4induced MCP-1 production. Taken together, we identified a
novel regulation and cross-talk loop between ER stress and the
TLR4 pathway, in which ATF4 plays an important role in regulation of MCP-1 production and TLR4-mediated endothelial
inflammation.
requirements.
PCA094
Liu YH et al, (2012) Antioxid Redox Signal 17, 141-185
Hu LF et al, (2011) Antioxid Redox Signal 15, 405-419
Giorgio M, et al, (2005) Cell 122, 221-233
The role of bradykinin and prostaglandins in the
sensitisation of the TRPV1 ion channel
Pinton P et al, (2007) Science 315, 659-663
B.D. Grubb and S. Aldossary
This work was supported by NUSHS bench-to-bedside grant
NUHSRO/2011/012/STB/B2B-08 and National kidney
foundation NKFRC/2011/01/04
University of Leicester, Leicester, UK
requirements.
148P
The transient receptor potential vanilloid subfamily member
1, TRPV1, was the first member of a family of heat sensitive
ion channels to be identified in mammals. Under normal physiological conditions, TRPV1 expressed in dorsal root ganglion
neurons (DRG), is activated by thermal stimuli > 43°C. However, when tissues are damaged thermal allodynia/hyperalgesia occur due to modulation by inflammatory mediators
released following the insult. Capsaicin directly activates TRPV1
and is used as a tool to study the properties of this receptor.
Isolated DRG neurons are a mixed population of sensory neurones, some of which express TRPV1 ion channels, along with
the G-protein coupled receptors for prostaglandins and
bradykinin (BK). The aim of these experiments was to determine 1) which inflammatory mediator pathways converge on
the TRPV1 ion channel using specific antibodies to co-localise
TRPV1 with the EP4 receptor for PGE2, and the B2 receptor for
BK, in tissue sections of rat DRG, and 2) the responses of isolated DRG neurons to capsaicin and their modulation by inflammatory mediators. All rats used in this study were killed under
Schedule 1 of the Animals (Scientific Procedures) Act, 1986.
Analysis of frozen tissue sections (n=12 sections from 3 rats)
labelled with selective antibodies (Chopra et al, 2000) revealed
that 21% of DRG neurons express all three proteins, EP4, B2,
and TRPV1. To examine the sensitising effect of PGE2 on the
TRPV1, adult rat DRG neurone cultures were prepared as previously described (Chopra et al, 2000). DRG neurons were
loaded with fluo-4 and changes in the intracellular calcium concentration were monitored using an Olympus FV300 laser scanning confocal microscope. Capsaicin-evoked (80nM) calcium
responses of cultured DRG neurons desensitised in 88.7% of
capsaicin-sensitive neurons upon repeat application of the agonist. However, in the presence of 500nM PGE2 a significant
sensitization of the capsaicin response was observed in 43.9%
of these neurons (P<0.05, ANOVA) and in 31.7% of neurons
that were initially capsaicin insensitive. Moreover, in presence
of 100nM BK (preincubated with thapsigargin) 61% of capsaicin sensitive neurons and 14% of silent neurons were also
sensitised by this agonist. However, preincubation of the DRG
neurons with 3μM indomethacin (a non-selective cyclooxygenase inhibitors) decreased the proportion of capsaicin-sensitive neurons that were sensitised by bradykinin to 37%, and
significantly decreased the magnitude of the capsaicin-evoked
calcium responses from 2.87+/-0.21 a.u. to 1.93+/-0.11 a.u.
(P<0.05, ANOVA) of the control value. These results suggest a
role for cyclooxygenase enzyme and prostaglandins in the
modulation of bradykinin-induced sensitization of DRG neurone responses to capsaicin.
Chopra B, Giblett S, Little JG, Donaldson LF, Tate S, Evans RJ & Grubb
BD (2000) Eur. J. Neurosci. 12, 911-920.
We acknowledge the support of the King Faisal University who
support SA
requirements.
PCA095
Learning patterns among medical students
S.B. Shetty1, B.A. Shetty2, N. Arunkumar1, S.R. Pai1, R.D. Kini1,
B. Kodavanji1, A. Noojibail1 and S. Kolluru1
collaborative testing and debate. Manipulating models4,and
role playing satisfies kinesthetic and tactile learners. Reading
/writing learners can be approached with information depicted
in words.
Materials and Methods: A cross sectional study of first year
medical students (n= 100) was performed. Clearance was taken
from the college ethical committee. Informed consent was
taken. The validated VARK(version 7.1) questionnaire was used
to categorize the learning styles of students. The questionnaire consists of 16 items which identify four different learning styles: visual, aural, reading/writing and kinesthetic.
Descriptive statistics were used to identify the learning styles
of students.
RESULTS: . The Mean VARK scores (Mean score ±SD) for auditory (4.85 ±2.0) and kinesthetic learners (4.94 ±2.11) were
more than that for reading/writing (3.75±1.82) and visual
(2.67±1.5) learners. Total number of responses in 100 questioniare were 1628 out of which score for kinesthetic component was the highest (510),followed by auditory(470),visual
(270) and read/write(378). The results showed that all of the
students preferred to learn by multiple sensory modalities.
Among the multimodal learning styles the most preferred was
trimodal (5%) and quadrimodal (95%)respectively .Furthur
among them the students who preferred trimodal were VAK
(4%),ARK(1%) and Quadrimodal were VARK (95% ) .
Conclusion:. The students whose learning style preference contained K within one of the multimodal combination (ARK, VAK,
VARK) constituted 32% of all the participants.Similarly, percentage of students preferring A in the modality mix was 28%.
The components V and R were preferred by 17%and 23% of
students.
Our Study showed that the kinesthetic was the most preffered
learning pattern followed by auditory. It furthur showed that
students prefer the multimodal modalities of learning styles
as against the single mode. The results of this study can provide useful information for improving the quality of the teaching and learning experiences of students.
Key words: Learning style, learning preference, the VARK
Fleming ND. VARK: a Guide to Learning Styles 2012 [cited 2012 25
July]; Available from: http://www.varklearn. com/english/index.asp
Lujan H, DiCarlo S. First-year medical students prefer multiple learning styles. AdvPhysiol Educ. 2006;30(1):13-6.
Cortright R, Collins H, DiCarlo S. Peer instruction enhanced meaningful
learning: ability to solve novel problems. AdvPhysiol Educ.
2005;29(2):107-11.
Chan V, Pisegna J, Rosian R, DiCarlo S. Construction of a model demonstrating neural pathways and reflex arcs. AdvPhysiol Educ. 1996;271(6
pt 3):S14-42
requirements.
1Physiology,
Kasturba Medical College, Mangalore, KARNATAKA,
India and 2Medicine, A.J.Institute of Medical Institution, Mangalore,
Karnataka, India
Introduction: The aim of the present study was to evaluate the
common learning pattern among the different styles namely
visual, auditory, reading and kinesthetic in students of first
year MBBS students of Mangalore Population in India. Learning preference of students in VARK questionnaire is widely
used by researchers to identify the learning preference of students1. Visual learners should be stimulated with depictions
of information in charts, flow charts, and all the symbolic
arrows, circles and other devices that instructors use to represent what could have been presented in words2. Auditory
learning is achieved through listening during peer instruction3,
149P
PCA096
requirements.
Impact of an interactive patient-based physiology practical
on student knowledge and engagement in a first year
undergraduate medical course
PCA097
D. Joshi1, A. El-Hilly1, S. Mo1, A. Humphreys1, E.R. Carthy1,
A. Varela-Carver1, P.K. Luther2, K.T. Macleod2, M.J. Morrell2
and S.N. Saleh1
Authentic biology – taking real science into school to model
human diseases
1Faculty of Medicine, Imperial College, London, UK and 2National
P.B. Martin1, C. Hammond1, C.D. Nobes1, H. King1,
B. Lansdowne2 and A. Ellis2
Heart and Lung Institute, Imperial College London, London, UK
1Faculty of Medical and Veterinary Sciences, University of Bristol,
Background: In this study a patient-based physiology practical
(PP) was developed to enhance the 1st year MBBS course at
Imperial College London. It was anticipated that the interactive patient-based PP, would increase understanding and support students to learn more effectively than a traditional practical (TP).
Aim: To objectively evaluate the impact of an interactive PP
compared to a TP on knowledge and engagement within the
1st year undergraduate cohort.
Methods: The PP was developed to teach blood pressure measurement using specialised software focusing on a hypertensive patient’s journey. The session was taught 12 times by the
same tutor; approx. 20 students per session. Prospective evaluation was carried out before and after each session using 6
single best answer (SBA) questions and two visual analogue
scales (VAS). The students answered the SBA questions and
also rated their own engagement with the session; 100% being:
‘this practical was very engaging’ and how much knowledge
they thought they had gained after the practical; 100% being:
‘this practical substantially increased my knowledge’. Data
from the PP was compared to that from a TP on measurement
of the electrocardiogram. The TP was taught 12 times by a different tutor but with the same cohort of students. Two demonstrators assisted with each session and SBAs were prepared by
an author (DJ) who did not take part in the teaching.
Results: 239 students took part in the sessions, before the practical. 99% completed the PP evaluation before compared to
98% after the session. For the TP evaluation 98% completed
the evaluation before and 95% after. The collated data is shown
in Table 1. Before the session, the number of correct answers
was significantly greater for the TP compared to the PP
(p<0.05). The number of SBA questions answered correctly
increased non-significantly after the TP (p>0.05) and significantly increased after the PP (p<0.001). There was also a significant difference in the mean change before and after the practicals between TP and PP (p<0.001). Student VAS scores for
‘knowledge gained’ were significantly higher in PP compared
to TP (p< 0.001) and the ‘engagement’ was also significantly
higher in PP (p< 0.001).
Summary: The interactive patient-based PP significantly
improved student knowledge and engagement when compared to the TP. Further prospective evaluation is needed to
determine the influence of the tutors and the students’ prior
knowledge on the teaching methods.
Table 1: Data from prospective study comparing PP to TP
‘Innovations in Teaching Award’, Faculty of Medicine, Imperial
College London
150P
Bristol, UK and 2Cotham School, Bristol, UK
The Wellcome Trust-supported Authentic Biology Project
brings schools and universities together to help school students learn about cutting-edge progressions made in biological research. As part of the project, the University of Bristol
works closely with Cotham School, allowing students in Year
12 and Year 13 to contribute to the academic studies of a ‘real’
scientist.
Authentic Biology was first conceived 5 years ago by Dr David
Colthurst, a Science Teacher in Kent. The aims were to bridge
the gap between science in schools and universities; to give a
realistic experience of research science in order to motivate
students and also to increase the number of University science
applicants. In 2012, Professors Paul Martin and Catherine
Nobes from the Faculty of Medical and Veterinary Science, University of Bristol, together with Cotham School were invited
to join the project. Professor Martin’s and Dr Chrissy Hammond’s research groups use Zebrafish to look at cancer, heart
disease and osteoarthritis. These researchers along with a
newly-appointed teacher at Cotham School, Beca Lansdowne,
work in collaboration with experts from the School of Social
and Community Medicine at Bristol, to identify novel human
disease genes.
With over 30 students committed to Authentic Biology at
Cotham, the project has increased in size and scope. Students
have engaged in practical sessions, workshops, symposia and
lectures, as well as worked together to keep and maintain their
own Zebrafish in a specially developed aquatic tank. Sessions
cover how Genome Wide Association Studies (GWAS) can aid
finding new human disease genes, as well as how ZFIN software can be used to find the Zebrafish orthologues. Using this
information, the students can observe the developing Zebrafish
embryo and larval stages to look at the potential ‘disease gene’
expression patterns. The importance of fish husbandry skills
is also highlighted, in order to optimise regular egg production to keep the project moving forward.
As well as considering how new therapeutics are researched
and developed, students have also presented at the Authentic Biology Symposium, held at the Wellcome Trust headquarters in London and at a Zebrafish symposium held at the
Watershed, Bristol.
The project is documented both through the Wellcome Trust
and the University of Bristol’s outreach pages, as well as
through
the
content
on
Facebook
(www.facebook.com/pages/Cotham-Biology/182603948594622)
and
Twitter
(https://twitter.com/CothamBio), developed by Cotham students.
Wellcome Trust, UK
requirements.
PCA098
requirements.
Enhancing physiology outreach activities in the Faculty of
Medical and Veterinary Sciences, University of Bristol
PCA099
F.M. MacMillan1, H. King2, D. Davies1 and L.K. Hughes1
1School of physiology and Pharmacology, University of Bristol,
Bristol, UK and 2Faculty of Medical and Veterinary Sciences,
Human-physiology education for non-medical students:
From computer-based to web-based/app-based topicspecific courseware
In January 2013 a generous alumnus donation enabled the
appointment of a designated part-time Outreach Assistant
with the aim of expanding the scope of the outreach work on
offer across our Faculty. Since then we have been able to substantially increase our level of outreach, making visits to schools
and other events, or hosting visits in our teaching labs, more
than once a week. We estimate over 2,000 students and members of the public have accessed the resources and activities
on offer since January 2013.
Previous contacts and practices have also been maintained,
including use of the Mobile Teaching Unit (MTU), a speciallyequipped lorry that expands into a classroom (1)which has
been used more than once a month for the past year. The MTU
enables scientific equipment to be taken to schools, for example a vitalograph to measure lung function, ECG monitors and
pulse oximeters to demonstrate the diving reflex, and reaction timers to compare the speed of visual and auditory reactions. Recently-developed sessions include an exploration of
hearing and deafness and a practical session on size exclusion
chromatography in the context of blood. The scope of visits
to the University has also been widened to include topics such
as electrophoresis. A number of visits have also covered more
general issues such as ethics and careers.
Our outreach work has involved school students and members
of the public in a wide range of contexts including primary
schools, secondary schools, colleges, a Brownies Group, public events, themed days such as Skirting Science (http://skirtingscience.wordpress.com/), Nailsworth Festival, Gloucestershire and University Open Days. It has been possible to provide
free or reduced cost visits to a selected proportion of these
groups through the receipt of Widening Participation (WP)
funding from the University; eligibility for cost reduction is
considered according to WP criteria and on the basis of the
number of students claiming free school meals. This has
allowed the widest range of students possible to access our
resources and information.
In order to disseminate information about the scope of activity on offer, the webpages for the outreach work in the Faculty (www.bris.ac.uk/fmvs/outreach) have been updated and
linked to the Faculty homepage for ease of access. Consideration is also being given to the role of these pages in providing information and advice across the broader context of outreach, including informing potential outreach practitioners
(often undergraduate and postgraduate students).
Similarly, a more rigorous evaluation and monitoring strategy has been developed in order to link our work to Widening Participation impact, to provide formative assessment to
continually improve our work and to provide a record of its
impact. This has included the use of audience response devices
in outreach sessions.
I. Hwang1, M. Tam2, R. Lee3 and C. Tsang4
Hughes LK, Healey K, Hinton RE, Lillie AR, Rickard P, Harris JR (2009)
Final Year Undergraduate Teaching Projects Delivered via a Mobile
Teaching Unit. Proc Physiol Soc 15: PC71
1Teaching
and Learning Unit, School of Biomedical Sciences,
Faculty of Medicine, The Chinese University of Hong Kong, Hong
Kong SAR, China, 2School of Biomedical Sciences, Faculty of
Medicine, The Chinese University of Hong Kong, Hong Kong SAR,
China, 3Academic Support Division, Information Technology
Services Center, The Chinese University of Hong Kong, Hong Kong
SAR, China and 4Department of English, Faculty of Arts, The
Chinese University of Hong Kong, Hong Kong SAR, China
Physiology is part of the core curriculum of all our biomedical
programmes. First-year biomedical and health-care students
are often intimidated by the overwhelming amount of material they are required to master within a short time-frame. Since
2009, we have delivered a number of computer-based animated courseware packages to first-year students to aid their
understanding of physiology. Although these packages depict
complicated physiological mechanisms in clear sequences, students can only access them through the university e-learning
platform. Both flexibility and convenience of use are compromised. We thus began to explore mobile-learning applications
(apps) for our courseware, developing PhysioApp in 2012. This
app (with both iOS and android versions) is specially tailored
for first-year students who have no basic knowledge of physiology. It covers human respiratory and cardiovascular
responses to different levels of exercise and other important
areas such as thermoregulation and hormonal responses. The
students’ main learning objective was to use the app in tandem with the knowledge obtained during their physiology
course to enhance their understanding of how the respiratory
and cardiovascular systems respond to exercise. We measured
the access rates of students from different courses after the
end of term. The average access rates ranged from 33.33 to
88.89%. Students who had not used the app (those studying
human biology) were asked in class to explain their decision
via a student-response system. They all commented that PhysioApp did not include a quiz allowing them to test their knowledge, unlike other, computer-based courseware packages. The
whole class of students was also asked to vote in favour of
using either app-based or computer-based courseware to learn
physiology. Eighty per cent of the students replied that they
had no preference as long as the courseware included an automarking exercise or quiz. In conclusion, we demonstrated the
emergence and growth of new information technologies in
physiology education. We also showed that the development
of apps helps to expand the range of creative teaching and
learning practices. According to our most recent data, however, the students in the human biology programme had no
distinct preference for either computer-based or Webbased/app-based tools for physiology e-learning. Student learning is clearly still driven by assessment, which is reflected in
the students’ request that we add quizzes to our e-learning
packages.
151P
1. The Courseware Development Grant Scheme 2011-2012,
Information Technology Services Center (ITSC) Steering
Committee, The Chinese University of Hong Kong, Hong Kong,
China.
2. Center of Learning Enhancement And Research (CLEAR), The
Chinese University of Hong Kong, Hong Kong, China.
These useful aspects of the NeuroDebian project and benefits from patchclamp integration will be illustrated at the meeting.
requirements.
requirements.
PCA100
PCA101
Effect of amiloride on salt-sensitive and salt-resistant adult
Nigerian subjects with and without β-ENaC polymorphisms
S.O. Elias1, O.A. Sofola2 and S.I. Jaja2
NeuroDebian: an integrated, community-driven, free
software platform for physiology
Y.O. Halchenko2, M. Hanke3 and V. Alexeenko1
1Biochemistry and Physiology, University of Surrey, Guildford, UK,
2Department
of Psychological and Brain Sciences, Dartmouth
College, Hanover, NH, USA and 3Department of Experimental
Psychology, Otto-von-Guericke University, Magdeburg, Germany
Many current physiological techniques require a set of software tools that implement novel techniques ranging from stimulus delivery, data acquisition, real-time or offline analysis and
visualisation of data allowing interaction with the experiment.
Maintenance of a functional research environment that uses
closed-source proprietary solutions may be inefficient and inadequate; it may limit acquisition and analysis and be time-consuming. In consequence laboratories may have restricted
access to adequate facilities and could be reluctant to upgrade
their current tools.
The NeuroDebian project (http://neuro.debian.net) addresses
these problems by integrating free software tools which are
being continually developed by the scientific community. This
project works within the larger Debian project offering universal Linux operating system. NeuroDebian as a result offers
a complete desktop environment and tools for various research
needs in physiology and beyond. These include: EEG/EMG
recording and analysis (e.g., aghermann, OpenMEEG,
sigviewer); generic environments for computational research
(e.g. IPython, Octave, R); analysis and visualisation of neuroimaging data (e.g. AFNI, FSL), simulation and investigation
of magnetic resonance sequences (Odin); delivery of stimuli
for psychophysical research (e.g. PsychoPy, Psychtoolbox-3);
statistical learning analysis using functional magnetic resonance imaging data (e.g. MDP, PyMVPA). In addition, a number of tools are also being developed for later integration into
the NeuroDebian project, that include a specialised patchclamping solution, patchclamp (http://patchclamp.net). The
maturity and reliability of Debian provides a reliable foundation to carry out a wide range of research routines, whilst NeuroDebian tracks and makes available the latest developments
of relevant scientific software.
For more than eight years the NeuroDebian portal, currently
used world-wide, has been providing readily-usable research
software packages for Debian-based operating systems; including Ubuntu, making them easily available for all users of these
systems. For non-Linux systems NeuroDebian provides a convenient virtual appliance that allows researchers to run these
software packages on proprietary systems, such as Windows
and Mac OS X. This software-integration project increases the
overall quality and accessibility of research software by: helping to fix software bugs; permitting its testing on a wide variety of hardware architectures; and simplifying installation and
upgrade procedures.
152P
1Physiology,
Lagos State University College of Medicine, Lagos,
Nigeria and 2Physiology, College of Medicine of The University of
Lagos, Lagos, Nigeria
Selective ENaC blocker, amiloride, is effective in reducing blood
pressure in blacks with T594M polymorphism of ENaC. Every
population is heterogenous for salt-sensitivity. It is thus of interest whether amiloride will block the channel in salt-resistant
individuals as well as salt-sensitive persons with or without βENaC polymorphism especially as amiloride has been suggested as a sole anti-hypertensive agent in blacks [1]. 22 normotensive (NT) and 42 age-matched hypertensive (HT)
subjects earlier divided into salt-sensitive (SS) (NT = 11; HT =
22) and salt-resistant (SR) (NT = 11; HT = 20) subgroups [2]
took part in the study. Blood pressure (BP) was measured
before and after they were salt-loaded with 200mmol Na+/day
for 5 days. After one week wash-out period, subjects ingested
a combination of the salt-load and 5mg amiloride tablets daily
for 5 days. The effect of the combination on blood pressure
was then determined. Blood was also sequenced for β-ENaC
polymorphisms [3]. Ethical clearance for this study was
obtained from the College of Medicine, University of Lagos.
Data are presented as mean ± S.E.M., compared with ANOVA
and appropriate post-hoc tests. Baseline systolic (SBP) and diastolic blood pressures (DBP) were similar (p>0.05) in the SS (SBP
121.5±2.8 mmHg; DBP 80.9±0.8 mmHg) and SR (SBP
120.7±2.4 mmHg; DBP 80.2±1.6 mmHg) NT subjects and
among the SS (SBP 144.7±3.6 mmHg; DBP 92.9±2.4 mmHg)
and SR (142.9±4.8 mmHg; DBP 99.2±2.0 mmHg) HT subjects.
Following the salt-load, BP changed significantly (p<0.01)
(+7.7±2.6% SBP; +11.6±2.1% DBP) among the SS NT subjects
but marginally (p>0.05) (+0.6±1.9% SBP; -0.5±2.6% DBP)
among the SR NT subjects. Similarly, there were significant (p
<0.001) pressor responses to the salt load among the SS
(+7.8±1.7% SBP; +11.6±1.7% DBP) but not among the SR (SBP
+2.1±1.7%; DBP -2.2±1.4%) HT subjects. Amiloride plus saltloading led to significant reduction in SBP among SS (p<0.001)
(-9.9±2.1% NT; -12.9±2.0% HT) and SR subjects (p<0.05) (6.5±2.0% NT; -10.0±1.5% HT) when compared with salt-loading alone. Similarly, the combination significantly reduced DBP
among SS (p<0.001) (-13.9±3.4% NT; -10.4±2.8% HT) subjects
but reduced DBP in SR (-7.1±1.7%, p<0.001) HT subjects only.
7 out of 11 subjects with ENaC polymorphism were HT (4 SS
and 3SR). However there was no relationship between ENaC
status and salt sensitivity, with respect to SBP, as the effect of
ENaC blockade by amiloride are similar in both SS (-12.6±7.7%)
and SR (-16.5±6.2%) HT subjects P=0.59). However in SR HT
subjects, fall in DBP was significantly (P<0.01) less in SR (7.0±3.5%) than in SS (-21.4±1.6%). These preliminary results
suggest that in SR HT, β-ENaC polymorphism may be associated with reduced DBP responsiveness to amiloride.
Baker EH, Duggal A, Dong Y et al., (2002). Hypertension 40, 13-17
Elias SO, Azinge EC, Umoren GA et al., (2011). Nig Qt J Hosp Med 21,
85-91
Elias SO, Sofola OA and Jaja SI (2013). Proceedings of The Physiological Society (The Epithelia & Membrane Transport and Vascular &
Smooth Muscle Physiology) Joint Themed Meeting, Dublin
International Junior Research Grant of The Physiological Society
Research Grant from TETFund, Nigeria
requirements.
PCA102
(P>0.0001, n=4) and Ch – 81.2±3% (P<0.05 n=3). At the same
time, no significant changes were observed upon injection of
CGP-37157 in the mixture with Ch. We suggest that mitochondrial Na+/Ca2+ exchanger is directly involved in the regulation of functioning in the submandibular gland.
requirements.
PCA103
Differential effects of cholesterol-targeting drugs on
functional organization of plasma membrane and actin
cytoskeleton in transformed fibroblasts
Role of mitochondrial Na+/Ca2+ exchanger in the function
of rat submandibular salivary gland
V.I. Chubinskiy-Nadezhdin and E. Morachevskaya
S. Korchynska1, R. Makarovska2 and N. Fedirko1
Russian Academy of Sciences, Institute of Cytology RAS,
St.Petersburg, Russian Federation
1Human
and Animal Physiology, Ivan Franko National University
of L’viv, Lviv, Ukraine and 2Lviv Regional Endocrinological
Dispensary, Lviv, Ukraine
7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin2(3H)-one (CGP-37157) is a specific inhibitor of mitochondrial Na+/Ca2+ exchanger and also inhibits Ca2+-ATPase. As mitochondria controls Ca2+ signaling, mitochondrial Na+/Ca2+
exchanger might play an important role in the secretory
process in salivary glands. Clinical data shows that mouth dryness (xerostomia) can be mediated through the altered
Na+/Ca2+ exchanger function. However, the role of Na+/Ca2+
exchanger in the submandibular salivary gland function
remains unclear. To study the effect of CGP-37157 on salivary
cells function, we analysed in vivo parameters of salivation:
saliva flow rate and electrolyte content (Ca2+, P2+, Na+). Rats
were anesthetised by ketamine (90 mg/kg) and fixed in the
supine position. To collect whole saliva two ducts of submandibular gland were cannulated. Saliva, that secreted over
a period of 5 min, was collected in a tube after the tested substances were injected into gland lobes. Saliva flow rate was
calculated as a volume of saliva secreted per 5 min normalised
to the animal weight/hour. Concentration of electrolytes was
measured: Ca2+ – o-cresol-phtalein method; P2+ – UV detection, and Na+ – flame emission spectroscopy. We showed that
CGP-37157 (5 μM) after an intraglandular injection decreased
saliva flow rate, which reached its lowest level at 25 min after
drug injection (59.9±6.7%, P<0.001, n=7). As expected, administration of carbachol (Ch, 5 μM) increased saliva flow rate
(283.1±65.4%, P<0.05, n= 5). In addition, we established, that
CGP-37157 caused a significant reduction of saliva flow rate
and subsequent injection of Ch (at 20 min of experiment)
increased saliva flow rate and reached a peak at 25 min of the
experiment (46±4.4%, P<0.0001, n=6). However, the stimulation potency of Ch was much less pronounced (compare to
the sole effect of Ch). We further found that application of
CGP-37157, Ch and CGP-37157 in the mixture with Ch significantly changed Ca2+, P2+ and Na+ concentrations in the final
saliva. In particular, the concentration of Ca2+ decreased: CGP37157 – 20.8±0.9% (P<0.0001, n = 3), Ch – 36.7±0.5%
(P<0.0001, n=3), CGP-37157 in the mixture with Ch –
36.1±0.7% (P<0.001, n=3). Similarly, Ch (5 μM) and CGP-37157
in the mixture with Ch decreased of P2+ concentration: Ch –
by 20±3.3% (P<0.001, n=3), CGP-37157 in the mixture with Ch
– by 52.6±4.1% (P<0.001, n=3), but in case of sole CGP-37157,
we observed an increase of P2+ concentration – by 16±8.3%
(P<0.001, n = 3). Contrary to the Ca2+ and P2+, CGP-37157
caused an increase of Na+ concentration – 290.7±13.1%
Cholesterol is the most abundant lipid component of mammalian cells and it plays a crucial role in organization, lateral
heterogeneity and dynamics of plasma membrane. Particularly, the level of cellular cholesterol determines functional
compartmentalization of membrane lipids and proteins into
ordered microdomains (lipid rafts), which may serve as scaffolds for signaling complexes. Cholesterol-enriched rafts are
assumed to be implicated in various cellular reactions involving actin cytoskeleton rearrangements. Changes in cholesterol
metabolism may affect membrane-cytoskeleton interactions
modulating key functions in living cells. Lipid raft-associated
signaling was shown to be crucial for development of a number of pathological processes, including malignant transformation. Nowadays, known cholesterol-lowering agents of
statin family targeting rate-limiting step of mevalonate synthetic pathway are considered as potential instruments in anticancer therapy. The present study focuses on the physiological mechanisms of action of cholesterol-targeting drugs on
membrane organization coupled with cytoskeleton in transformed cells. We compared the effects of selective sterol acceptor methyl-beta-cyclodextrin (MbCD) and HMG-CoA-reductase
inhibitor simvastatin in transformed fibroblasts 3T3B-SV40.
The depletion of membrane cholesterol by MbCD resulted in
a disruption of lipid rafts in plasma membrane as it was
revealed by fluorescent labelling of marker ganglioside GM1.
Alterations of lipid order of cell membranes after MbCD treatment have been confirmed by probing with lipid-sensitive dye
di-4-ANEPPDHQ. In contrast, incubation of the cells with simvastatin caused no changes in raft integrity in plasma membrane. Cholesterol-depleting treatment with MbCD induced
actin assembly and intensive stress fiber formation in transformed fibroblasts whereas simvastatin effects on cytoskeleton were principally different. Rearrangements of actin structures after simvastatin application are likely to be due to the
inhibition of isoprenoid synthesis rather than alterations in the
level of membrane cholesterol. Collectively, our data are consistent with the notion that the disruption of lipid rafts is the
upstream triggering mechanism in cholesterol-dependent filament assembly in transformed cells. Importantly, we show
that the inhibition of Src-kinases did not prevent MbCD-induced
actin polymerization and stress fiber formation in 3T3B-SV40
fibroblasts. These results indicate that Src-kinase-independent
membrane mechanisms are likely to be involved in cholesterolregulated cytoskeleton reorganizations coupled with reversion
of transformed phenotype.
153P
requirements.
PCA104
Mathematical models of signalling through G proteins and
phospholipids
Perez-Mansilla, B., Ha, V. L., Justin, N., Wilkins, A. J., Carpenter, C. L.,
& Thomas, G. M. H. (2006). The differential regulation of phosphatidylinositol 4-phosphate 5-kinases and phospholipase D1 by ADPribosylation factors 1 and 6. Biochimica Et Biophysica Acta, 1761(12):
1429–1442. doi:10.1016/j.bbalip.2006.09.006
requirements.
R. Stanley, K. Bryson and G. Thomas
PCA105
UCL, London, UK
The study of biochemistry has historically included a mathematical approach. From Michaelis-Menten’s original paper (1)
describing a model of a irreversible enzyme onwards, mathematical models have been useful in allowing us to specify the
behaviour of molecular interactions in a precise and communicable form. Modern approaches (2) allow us to easily derive
mathematical models from known biochemical mechanisms.
Subsequently these models can be used as component parts
of models of intracellular processes such as metabolism and
signalling. This approach is particularly helpful when considering complex, highly-interconnected, and cross-regulated systems.
One such signalling pathway is generated by members of the
Arf family of small G proteins. These are capable of acting as
‘molecular switches’ depending on the bound guanine nucleotide - they are ‘on’ when GTP-bound, and ‘off’ when GDPbound. They are activated by guanine nucleotide exchange
factors (GEFs) and deactivated by GTPase-activating proteins
(GAPs). GTP-bound Arf is able to activate the enzymes phospholipase D (PLD) and phosphatidylinositol 4-phosphate 5kinase (PI4P5K) generating the lipids phosphatidic acid (PA)
and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2),
respectively. PA and PI(4,5)P2 are known to have roles in many
fundamental membrane-associated processes (3), and each is
capable of cross-regulating the other enzyme (PA for PI4P5K,
and PI(4,5)P2 for PLD) though by different biochemical mechanisms. The triple of Arf, PLD, and PI4P5K comprises a novel
lipid signalling ‘motif’ – we are interested in the signalling
properties that result from the connections within this motif.
Plausible biochemical mechanisms have been developed, and
models based on these have been constructed, leading to systems of ordinary differential equations (ODEs). Due to the ubiquity of the discussed signalling motif, where possible the models have been analysed without resorting to parameterisation
and experiments designed simply to fit parameters to the models have been avoided. Instead we have derived novel qualitative physiological hypotheses which may then be experimentally verified. For example, from a simplified model we
observe that asymmetry in the mechanisms of PLD and PI4P5K
activation leads to a system that is capable of robust switchlike behaviour. This behaviour is not observed in either of two
symmetric (hypothetical) systems. A model for the activation
of G proteins by GEFs implies the unexpected result that
increasing the amount of GEF decreases the amount of active
G protein unless GAPs are also present.
These results show that mathematical modelling is a useful
tool when attempting to interpret complex signalling pathways, and that it can lead to novel physiologically relevant
hypotheses that may otherwise have gone unnoticed.
Michaelis, L. and Menten, M.L. (1913). Die Kinetik der Invertinwirkung.
Biochem Z 49: 333-369.
Gunawardena, J. (2012). A linear framework for time-scale separation in nonlinear biochemical systems. PloS One, 7(5): e36321.
doi:10.1371/journal.pone.0036321
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Phosphatidylinositol-3,4,5-trisphosphate stimulates distinct
Ca2+ elevation, Akt phosphorylation and forms a major
mechanism of thromboxane A2 formation in human
platelets
K.S. Authi1, N. Kassouf1, A. Ambily1, S. Watson2, S. Hassock1,
H. Authi1 and S. Watson2
1Cardiovascular Division, King’s College London, London, UK and
2Centre
for Cardiovascular Sciences, University of Birmingham,
Birmingham, UK
Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is an important second messenger formed from phosphatidylinositol-4,5,bisphosphate by the action of PI-3kinase. It has been implicated in many cellular functions but its mechanism of action
requires further study. In platelets it has been suggested to be
important in amplifying the activation Rap1b, in integrin activation, PLC activation, enhancement of Ca2+ signalling and
thrombus formation. However the mechanisms associated
with these effects need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-Ophosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3)
or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study effect of PIP3 on
freshly prepared human platelets. We examined the effects of
these analogues on platelet aggregation, Akt phosphorylation,
Ca2+ elevation, PLC and syk activation and thromboxane A2
formation using standard platelet technologies.
Results; Addition of either DiC8- or DiC16-PIP3 analogues
induced platelet aggregation in the presence of extracellular
Ca2+ . This was inhibited by a range of inhibitors including
indomethacin, U73122 and reagents that inhibit Ca2+ elevation such as 2APB. Akt activation was monitored by measuring the phosphorylation of Ser473. DiC16-PIP3 induced Akt
phosphorylation was reduced by Akt inhibitor IV and wortmanin and if EGTA was included in the medium. In Fura2 loaded
platelets DiC8-PIP3 was effective in increasing intracellular Ca2+
in a rapid, distinct and transient manner that was reduced in
the presence of wortmanin, indomethacin, U73122 and 2APB.
It was only marginally affected by the SOC inhibitor BTP2 or
the non-SOCE inhibitor LOE908. DiC8-PIP3 induced the release
of Ca2+ from stores. The presence of bafilomycin A1 did not
affect the Ca2+ transient and the cell permeable analogue of
DiC8-PI-3,5-P2 was not as effective as DiC8-PIP3 in inducing
Ca2+ elevation. DiC8-PIP3 did effect the phosphorylation of
the kinase syk but not of PLCγ2. Importantly DiC8-PIP3 was
potent at inducing the formation of thromboxane B2 that was
reduced by the Akt inhibitor IV.
Conclusion; These studies suggest that PIP3 plays an important role as a stimulus of Ca2+ transients, Akt phosphorylation and thromboxane A2 formation in platelets. The distinct
Ca2+ mobilisation seen by PIP3 may represent an important
mechanism for thromboxane generation in human platelets.
The work was partly supported by the British Heart Foundation.
requirements.
Sun WH, Kreisle RA, Phillips AW, Ershler WB (1992) In vivo and in vitro
characteristics of interleukin-6-transfected B16 melanoma cells. Cancer Res 52:5412–5415
PCA106
Effect of IL-6 and anti-IL6-receptor antibody tocilizumab on
statin induced apoptosis in human melanoma
C.
Minichsdorfer1,
1Oncology,
C.
Wasinger2
and M.
Colombo MP, Maccalli C, Mattei S, Melani C, Radrizzani M, Parmiani
G. Expression of cytokine genes, including IL-6, in human malignant
melanoma cell lines. Melanoma Res. 1992 Sep;2(3):181-9.
Hohenegger2
Medical University of Vienna, Vienna, Austria and
Medical Univerity of Vienna, Vienna, Austria
2Pharmacology,
Introduction: Interleuin-6 (IL-6) is a physiologic cytokine produced by lymphatic cells. Growth of melanocytes and early
stage melanoma can be inhibited, whereas metastatic
melanoma cells are mostly resistant to the anti-proliferative
effects of IL-6. It has been shown that nearly 50% of metastatic melanoma express IL-6 mRNA and secrete IL-6 into the supernatant.(1,2). These stage dependent effects are not fully understood yet.
Statins induce apoptosis in melanoma cells. However,
melanoma cells show different sensitivities to statins (3-5). Our
aim was to investigate the effect of IL-6 in statin induced apoptosis of human melanoma cells.
Methods: The secretion of IL-6 by metastatic melanoma cell
lines (A375, 518A2) and cells that derived from an early lesion
(WM 35) into the cell culture supernatant was investigated
with ELISA. Furthermore we checked the activity of caspase 3
after statin treatment in the presence and absence of IL-6 or
an IL-6 receptor blocking antibody (tocilizumab) by cleavage
of specific fluorescent caspase substrates. Additionaly apoptosis was more closely investigated by using AnnexinV/PI
staining. Phsophorylation of Stat3 and the regulation of pro(Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bxl-XL) was studied by western blot. Proliferation was measured with cell cycle
analysis using PI staining.
Results: Metastatic melanoma cells produce high amounts of
IL-6 in contrast to WM 35. In addition they are highly sensitive
to statin induced apoptosis with EC50 of 0,52μM (518A2) and
1,7μM according to caspase 3 activation. On the contrary WM
35 cells are much more resistant to statins with an calculated
EC50 of 16μM . Interestingly IL-6 signalling is not disrupted in
all cell lines as stimulation with IL-6 led to a strong phosphorylation of Stat-3.
Coincubation of IL-6 led to an increase in the statin induced
apoptosis of WM 35 cells. This effect was not seen in the
metastatic counterparts. Statins can activate the intrinsic pathway of apoptosis which relies on the loss of mitochondrial
membrane potential. We checked for the influence of IL-6 on
the production of anti- and proapoptotic proteins of the Bcl-2
family. Interestingly the amount of the antiapoptotic protein
Bcl-Xl was significantly decreased in WM 35 cells which may
explain the increase in apoptosis upon IL-6 treatment.
Blocking IL-6 signalling with tocilizumab had no effect on the
proliferation of the melanoma cells. However a combined treatment of simvastatin and tocilizumab led to a decrease in caspase 3 activation in A375 and 518A2, compared to the treatment with simvastatin alone.
Conclusion: Taken together our data prove the different sensitivities of melanoma cells to statin induced apoptosis. Moreover we clearly show that IL-6 facilitates apoptosis in early stage
melanoma cells by the down-regulation of anti-apoptotic proteins.
Demierre MF, Higgins PD, Gruber SB, Hawk E, Lippman SM. Statins
and cancer prevention. Nat Rev Cancer. 2005 Dec;5(12):930-42.
Minichsdorfer C, Hohenegger M. (2009) Autocrine amplification loop
in statin-induced apoptosis of human melanoma cells. Br J Pharmacol.
157(7):1278-90.
Werner M, Sacher J, Hohenegger M. Mutual amplification of apoptosis by statin-induced mitochondrial stress and doxorubicin toxicity in
human rhabdomyosarcoma cells. Br J Pharmacol. 2004
Nov;143(6):715-24.
requirements.
PCA107
ATP release from guinea-pig bladder urothelial cells involves
multiple pathways which require vesicle fusion, whereas
acetylcholine release does not
L.M. McLatchie and C.H. Fry
Dept. of Biochemistry and Physiology, University of Surrey,
Guildford, UK
Bladder urothelium acts not only as a barrier to urine but also
as a sensory epithelium releasing and responding to numerous chemical transmitters including ATP and acetylcholine
(ACh). Many lower urinary tract disorders are associated with
a change to this pattern of release and its better characterisation is crucial to develop future treatments. We have investigated pathways of ATP and ACh release, with a novel technique (1) that uses freshly isolated rather than cultured bladder
urothelial cells.
Male, Dunkin-Hartley guinea-pig bladders were excised, the
mucosa removed and incubated with trypsin-EDTA in phosphate-buffered saline, at 37οC for 40 minutes. Urothelial cells
were released by gentle titration, washed with Tyrode’s (no
added Ca), counted and incubated (>90 mins) in 45 μl aliquots
with chemicals to block possible release pathways. Transmitter release was stimulated by pipetting 20 μl of the suspension up and down, 5 (ACh) or 50 (ATP) times. ATP (lucifererinluciferase assay, Sigma) or ACh (Amplex®Red
ACh/ACholinesterase assay kit) was measured in a 20 μl sample. Data are means±SEM. Differences between sets used
paired Student’s t-test, with significance at p<0.05. All agents
were from Sigma except Amplex®Red, Invitrogen.
The role of vesicular transport was tested with N-ethylmaleimide (NEM, 100 μM) which reduced ATP release to
18.0±5.1% of control (p=0.02, n=8) but did not inhibit the more
substantial release of ACh (111.4±1.8%, n=5) (Fig 1). Routes
of ATP release were tested; i) pannexins with 1 mM probenecid
(ATP release reduced to 79.7±7.8%, p=0.012, n=11) or 50 μM
carbenoxolone (70.5±6.5%, p=0.009, n=14): ii) connexins with
1.8 mM CaCl2(67.7±5.1% p<0.001, n=30) or flufenamic acid
(150μM) which blocks connexins in addition to other ion channels (40.1±5.9 %, p=0.002, n=6). Combining carbenoxolone
and calcium was additive (48.2±8.2%, p=0.032, n=7) supporting a role for both pannexin and connexin channels. These
findings identify possible targets to modulate urothelial transmitter release.
155P
Figure 1: ATP and ACh release in the presence and absence of 100 μM NEM.
Mclatchie LM et al. (2014). Br. J. Pharmacology, (in press).
We would like to thank INComb for funding this work as part
of a joint EU FP7 grant.
requirements.
suspension in a Cairn Research spectrofluorimeter with excitation at 340 and 380 nm and emission at 500 nm. 1 mM CaCl2
was added immediately before experiments and the Fura-2
340/380 nm ratio was calibrated in terms of [Ca2+]c using the
method of Grynkiewicz et al (1985). Calcium signals were quantified as the integrals of the rise in [Ca2+]c above basal for 120
s after peroxyinitrite addition.
The addition of peroxynitrite (180 μM, from Merck Millipore)
evoked a rapid rise in [Ca2+]c of 1309 ± 340 nM above basal
levels (n = 4) whereas the vehicle (4.7% NaOH) alone was without effect. Rises in [Ca2+]c evoked by peroxynitrite (180 μM)
were inhibited in a dose-dependent manner by preincubation
of the cells with 5ʹ-iodo-resiniferatoxin or AMG 9810, with
responses being reduced to 43.3 ± 5.0% or 35.6 ± 10.4% of control after preincubation with 30 μM 5ʹ-iodo-resiniferatoxin or
AMG 9810 respectively (both n= 4, Student’s paired t-test P <
0.05).
These data are compatible with peroxynitrite being a potential endogenous activator of TRPV1 in human platelets.
Grynkiewicz G, Poenie M, Tsien RY (1985). J Biol Chem 260, 3440-3450.
Harper AGS, Brownlow SL, Sage SO (2009). J Thromb Haemost 7, 330338.
Ito N, Ruegg UT, Kudo A, Miyagoe-Suzuki Y & Takeda S (2013). Nat
Med 19, 101-108.
Redondo PC, Jardin I, Hernández-Cruz JM, Pariente JA, Salido JM &
Rosado JA (2005). Biochem Biophys Res Commun 333, 794–802.
Savini I, Arnone R, Rossi A, Catani MV, Del Principe D, Avigliano L
(2010). Mol Membr Biol 27,160-169.
PCA108
Peroxynitrite as a potential endogenous activator of TRPV1
in human platelets
requirements.
J.F. Mulvey1, A.G. Harper2 and S. Sage1
1Department
of Physiology, Development & Neuroscience,
University of Cambridge, Cambridge, UK and 2Institute for Science
and Technology in Medicine, Keele University, Stoke-on-Trent, UK
We have previously demonstrated the presence of TRPV1 in
human platelets (Harper et al., 2009). The TRPV1 agonist, capsaicin, was shown to evoke dose-dependent rises in cytosolic
calcium concentration ([Ca2+]c) and capsaicin-evoked
responses were inhibited by the TRPV1 antagonists 5ʹ-iodoresiniferatoxin or AMG 9810. We also demonstrated the presence of TRPV1 by Western blotting, a result since independently confirmed by Savini et al (2010) using a different antibody.
Further, we demonstrated that TRPV1 contributes to platelet
calcium signals evoked by the physiological agonists ADP and
thrombin (Harper et al., 2009). However, the endogenous activators of TRPV1 in platelets are unknown. A recent report has
suggested that skeletal muscle hypertrophy involves the activation of TRPV1 by peroxynitrite (Ito et al., 2013). It has been
suggested that platelets generate peroxynitrite and that this
plays a role in the abnormal Ca2+ signalling observed in
platelets from type II diabetic patients (Redondo et al., 2005).
Here we have investigated whether peroxynitrite generates
calcium signals in human platelets in a TRPV1-dependent manner.
Platelets were isolated from blood obtained by venepuncture
of healthy, drug-free volunteers under informed consent with
local ethical committee approval. Platelet-rich plasma was prepared and platelets loaded with the calcium indicator Fura-2
as previously described (Harper et al., 2009). The cells were
collected by centrifugation and resuspended in a Hepesbuffered saline as previously described except that bovineserum albumin was omitted from the medium. Fura-2 fluorescence was recorded from 1.5 ml stirred aliquots of platelet
156P
PCA109
Using fluoresence resonance energy transfer (FRET) to probe
cystic fibrosis transmembrane conductance regulator
(CFTR)-A2B adenosine receptor interactions
R. Tarran, R. Gilmore and M. Watson
Cell Biology and Physiology, University of North Carolina, Chapel
Hill, NC, USA
We have previously shown that CFTR significantly interacts
with adenosine receptors (A2BR) in the plasma membrane (1).
This interaction leads to a subsequent CFTR-sensitive increase
in adenosine-dependent cAMP signaling in non-cystic fibrosis
cells and suggests that innate defense is impaired by the lack
of CFTR due to reduced cAMP signaling. This enhancement
appears to be specific for A2BR and in contrast, CFTR does
not increase β2 adrenergic receptor (β2AR)-dependent cAMP
production. Little is known regarding how CFTR and A2BR interact to form a macromolecular cluster. Since CFTR and A2BR
are both indirectly connected to the actin cytoskeleton, we
tested the hypothesis that CFTR’s enhancement of A2BR was
dependent on C-terminal PSD-95, Dlg, ZO-1 (PDZ) domain interactions. All experiments were performed in HEK293T cells transiently transfected with cDNA constructs outlined below. FRET
was measured using the acceptor-photobleaching method by
confocal microscopy and cAMP was measured by ELISA as
described (1). Values are means ± S.E.M. and compared by the
Mann-Whitney U test. We used FRET to probe interactions
between CFTRgfp and A2BRmCherry and measured 8.2±1.0%
FRET in the plasma membrane with these constructs (p<0.05;
n =32). CFTR’s C-terminus is involved in CFTR-protein interactions, so we disrupted CFTR’s C-terminal PDZ domain (L1480A)
and looked at the effect on CFTR-A2BR FRET. The L1480A CFTR
mutation significantly reduced CFTR/A2BR FRET to 2.8±0.4%
(n=11), suggesting that PDZ binding is critical for CFTR-A2BR
interactions. These decreases in FRET were mirrored by concomitant reductions in cellular cAMP production (all n=6 and
p<0.05). We then generated new constructs where we
switched the PDZ-binding domains of A2BR and β2AR and
measured FRET and cAMP production. Placing the β2AR C-terminus on A2BR (A2BRDSLL) significantly reduced A2BR FRET
(62% decrease; n=12) and also decreased cAMP production by
a similar value. In contrast, placing A2BR’s C-terminus on β2AR
(β2ARGVGL) significantly increased CFTR-β2AR FRET by 80% and
also increased β2AR-induced cAMP production by 34% (all
n=12). Importantly, these maneuvers reversed interactions
and made A2BRDSLL cAMP production CFTR-insensitive and
made β2ARGVGL cAMP production CFTR-sensitive. We conclude
that PDZ-binding domains play a role in CFTR-A2BR interactions and in differentiating between β2AR and A2BR signaling to coordinate cAMP production.
Watson MJ, Worthington EN, Clunes LA, Rasmussen JE, Jones L, Tarran
R. 2011. Defective adenosine-stimulated cAMP production in cystic
fibrosis airway epithelia: a novel role for CFTR in cell signaling. FASEB
J. 25(9):2996-3003.
Funded by NIH HL108927
requirements.
PCA110
Na+ absorptive function and its regulation by P2Y4
purinergic receptor in the Claudius’ cells of the cochlea
J. Lee1 and W. Chung2
6-azophenyl-2’, 4’-disulfonic acid (PPADS) had any significant
effect on the action of UTP. Isc was reduced from 10.0±1.3μA/cm2 to -7.0±1.3μA/cm2 in the absence of suramin
(n=5), and from -10.0±1.2μA/cm2 to -7.9±1.3μA/cm2 in the
presence of suramin (n=5). Isc was reduced from 9.2±0.7μA/cm2 to -6.1±0.6μA/cm2 in the absence of PPADS,
n=6), and from -8.9±0.9μA/cm2 to -5.5±0.7μA/cm2 in the presence of PPADS (n=6). These results indicate this P2Y receptor
as the P2Y4 subtype. Then, we investigated the role of PLC signaling cascade in the regulation of ENaC activity by UTP. The
inhibition of Isc by UTP was significantly reduced in the presence of PLC inhibitors, U73122 (10μM) and edelfosine (10μM).
Isc was reduced from -10.2±1.0μA/cm2 to -6.0±1.1μA/cm2 in
the absence of U73122 (n=5), and from -9.2±1.1μA/cm2 to 8.8±1.1μA/cm2 in the presence of U73122 (n=5). Isc was
reduced from -9.1±1.2μA/cm2 to -5.7±1.5μA/cm2 in the
absence of edelfosine (n=5), and from -8.3±1.3μA/cm2 to 7.7±1.4μA/cm2 in the presence of edelfosine (n=5). These
results suggest that the physiological role of P2Y4 receptor in
Claudius’ cells likely to regulate Na+ homeostasis in the
endolymph. The acute inhibition of ENaC activity by activation
of P2Y4 receptor is possibly mediated by decrease of phosphatidylinositol 4,5-biphosphate, PI(4,5)P2, in the plasma
membrane through phospholipase C activation.
Lee JH, Marcus DC (2003) Endolymphatic sodium homeostasis by Reissner’s membrane. Neuroscience 119:3-8
Leipziger J (2003) Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284:F419-432
Lehrmann H, Thomas J, Kim SJ, Jacobi C, Leipziger J (2002) Luminal
P2Y2 receptor-mediated inhibition of Na+ absorption in isolated perfused mouse CCD. J Am Soc Nephrol 13:10-18
requirements.
1Department of Otolaryngology, Seoul National University College
of Medicine, Seoul National University Hospital, Seoul, Republic
of Korea and 2Department of Otolaryngology-Head and Neck
Surgery, Sungkyunkwan University School of Medicine, Samsung
Medical Center, Seoul, Republic of Korea
The epithelial cells of Claudius’ cells form boundary separating endolymph from perilymph in the cochlea and are expected
to transport Na+ out of endolymph via epithelial Na+ channel
(ENaC). It has been reported in many other epithelia that extracellular ATP decreases ENaC activity through P2Y purinergic
signaling by depletion of phosphatidylinositol 4,5-biphosphate,
PI(4,5)P2, in the plasma membrane (Lehrmann et al., 2002;
Leipziger, 2003). Therefore, we investigated P2Y signaling as
a possible regulatory mechanism of ENaC in gerbil Claudius’
cells using voltage-sensitive vibrating probe technique. Gerbils (3-4 weeks old) were anesthetized with sodium pentobarbital (50 - 100 mg/kg; i.p.) and sacrificed to remove temporal bones. The methods for voltage-sensitive vibrating probe
have been previously described (Lee and Marcus, 2003). Results
showed that uridine triphosphate (UTP) induced partial inhibition of the amiloride-sensitive short-circuit current (Isc), but
not with pre-treatment of amiloride. We introduced the antagonists of purinergic receptors to characterize the subtype of
UTP-responsive P2Y receptor and compared the effects of UTP
on Na+ absorption in the absence and presence of each antagonist. The short circuit current (Isc) was reduced from 9.2±0.5μA/cm2 to -5.6±1.1μA/cm2 in the absence of 100μM
reactive blue 2 (n=5), and from -8.5±0.6μA/cm2 to 8.0±0.5μA/cm2 in the presence of reactive blue 2 (n=5). In contrast, neither 100 μM suramin nor 100 μM pyridoxalphosphate-
PCA111
Aldosterone and arginine vasopressin stimulate cultured
CCD18-Co myofibroblast proliferation and release EGF and
PDGFA, thereby inducing T84 colonic epithelial cell growth
and reducing transepithelial permeability
M. Moretó1, L. Miró1, M. Maijó1, R.J. Naftalin2 and A. PérezBosque1
1Fisiologia,
2Physiology,
Universitat de Barcelona, Barcelona, Spain and
King’s College London, London, UK
Aldosterone (Aldo) and Arginine-vasopressin (AVP) can regulate Na and water reabsorption at the crypt colon level (Cristià
et al, 2007). Raised Aldo, in response to low NaCl intake, or
raised AVP, in response to dehydration, induce pericryptal
myofibroblast proliferation in the pericryptal sheath surrounding rat distal colonic crypts with concomitant reduction
in colonic crypt wall permeability. We have reproduced these
phenomena in vitro, using co-cultures of human CCD-18Co
myofibroblasts and T84 colonic epithelial cell lines. Myofibroblast and epithelial cell proliferation was quantified from
5-Bromo-2’-deoxyuridine incorporation, the expression of
growth factors (EGF, PDGFA, TGFβ1 and VEGFa) by real-time
PCR, and the expression of epithelial junctional proteins
(claudin IV, β-catenin) by Western blot. Results (means ± SEM;
n=6) were analysed by ANOVA. 24 h incubation with 100 nM
Aldo or with 10 nM AVP stimulated myofibroblasts cell proliferation by 70% and 60%, respectively. Aldo stimulated the
myofibroblast expression of EGF by 30%, (p<0.05) and PDFGA
157P
(28%, p<0.05); while AVP increased myofibroblast PDGFA
expression by 32%, (p<0.05). The AVP induced effects were
prevented when Manning peptide, a V1 receptor antagonist,
and Tolvaptan, a V2 receptor antagonist, were present in the
incubation medium, and the effects of Aldo were prevented
by spironolactone. Conditioned medium from myofibroblasts
incubated with either Aldo or AVP, stimulated cell proliferation in T84 cells (42% and 54%, respectively; p<0.05), and both
hormones increased 24-34% the expression of claudin IV and
β-catenin (p<0.05). These results indicate that changes in
colonic permeability during dehydration in response to raised
AVP are mediated by PDGFA secreted by myofibroblasts;
whereas the similar changes in colonic epithelial cell permeability induced by increase in Aldo induced by a low salt diet
are mediated by myofibroblast secreted EGF and PDGFA.
Cristià E et al. (2007) J Physiol 578:413-424.
tification of CLOCK and BMAL1. Analysis indicates that CLOCK
and BMAL1 are present in the nucleus and the cytoplasm of
BeWo cells. Results also show CLOCK expression is significantly
down regulated (P<0.05) as glucose concentration increases
in both the nucleus and the cytoplasmic fraction. By contrast
BMAL-1 expression remains constant. These data suggest that
BeWo maintains some workings of the circadian clock that can
be explored to further our understanding about the placentas
robust circadian control mechanisms.
Hastings, M et al (2007) J Endocrinol 195: 187-198.
Zhang, E et al (2010) Nature Med 16: 1152-1156.
Asher, G. and U. Schibler (2011) Cell Metab 13: 125-137.
requirements.
Supported by BFU2006-08410, MEC, Spain.
requirements.
PCA112
PCA113
Characterization of the anion selectivity and mode of
transport of SLC26A8
L. Stouvenel and A. Toure
INSERM U1016, CNRS UMR 8104., Institut Cochin, Paris, France
The effect of glucose concentration on the expression of
key circadian regulators in the human choriocarcinoma cell
line BeWo
L. Sansby and A. Collett
School of Applied Science, University of Huddersfield, Huddersfield,
Yorkshire, UK
During gestation the developing fetus is dependent upon nutrient transfer from the mother which is mediated by the placenta. Fetal and placental growth responds to maternal influences such as circulating blood glucose levels and therefore
may be adversely affected by abnormal homeostasis. The
mammalian biological clock is based up on the rhythmic
expression of clock and clock-controlled genes. Thus transcripttranslational associations result in the synthesis of key regulatory and downstream proteins involved in various physiological and metabolic processes. It is thought that the master
clock, located in the suprachiasmatic nucleus, orchestrates
peripheral tissues in their own circadian rhythms and generates organism wide synchronicity [1]. Two essential components of the circadian clock which have been postulated to
regulate glucose homeostasis are CLOCK and BMAL1 genes
[2]. These two are considered ‘core clock genes’ and their activation leads to the subsequent synthesis of CLOCK and BMAL1
proteins that interact and inhibit protein production to create a negative feedback loop [3]. Both proteins have been identified in a range of peripheral tissues including the placenta,
and we have previously reported that intermittent fasting
causes an increase in CLOCK expression in the rat placenta. In
this study we set out to identify whether the key circadian regulators were present in the human choriocarcinoma cell line,
BeWo, and to establish whether their expression in this cell
line can be manipulated via mechanisms previously reported
to have this function in other models.
BeWo cells were cultured 30°C at 5% CO2 conditions in DMEM
media, 10% FBS, 2% penicillin-streptomycin at a range of glucose concentrations, 10, 20, 30, 45mmol for 3 passages. Nuclei
isolates (n=3) and cytoplasmic fractions (n=3) were collected
for further analysis and samples were subjected to separation via gel electrophoresis and transferred to PVDF membrane
though electro-blotting. Immunostaining allowed for quan-
158P
Members of the SLC26 family of anion exchangers display a
transport activity towards monovalent and/or divalent anions
including sulfate, chloride, bicarbonate, iodide and oxalate.
In humans, genetic abnormalities of SLC26A2, A3, A4, and A5,
have been causally associated with several diseases (1). Interestingly in various epithelia, SLC26 members (SLC26A3, A4,
A5, A6 and A9) form a complex with the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), the chloride/bicarbonate channel mutated in cystic fibrosis, and are able to regulate CFTR transport activity (2).
We previously described SLC26A8, also known as Testis Anion
Transporter 1 (TAT1), as a sperm-specific member of the SLC26
family (3). Ion fluxes play an essential role in the control of
sperm motility and capacitation (a maturation event occurring in the female genital tract and required for fertilization);
in particular, calcium, chloride and bicarbonate are essential
for both processes by activating cAMP-PKA-dependent phosphorylation pathways. By generating a knock out mouse model
of Slc26A8 we demonstrated that Slc26a8 is required for sperm
motility, sperm capacitation and male fertility (4). Recently
we confirmed the essential role of SLC26A8 in human too by
identifying mutations in SLC26A8, which impair the stability
of the SLC26-CFTR complex in men presenting with male infertility due to impaired sperm motility (i.e. asthenozoospermia)
(5).
Although the functional cooperation of SLC26A8 with CFTR is
now well established, the intrinsic activity of SLC26A8 remains
poorly defined. In particular the anion selectivity of SLC26A8
together with its mode of transport and the upstream regulatory signalling pathways are not defined. In order to better
characterize SLC26A8, we have set up a functional assay in Hela
cells transiently transfected with SLC26 proteins and tested
for their anion transport activity using N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide (MQAE), a chloride
sensitive probe. Fluorescence emission associated with chloride effluxes was recorded upon shifting the extra-cellular
medium composition from chloride/bicarbonate to gluconate/bicarbonate. The slopes of the curves associated with
the fluorescence emission of SLC26A3 and A8 transfected cells
were compared to that of cells transfected with an empty vector and not transfected cells. Our preliminary results indicate
that unexpectedly, in contrast to the SLC26A3 protein, SLC26A8
does not behave as a chloride-bicarbonate exchanger (n=8.
Student test: p=0,042).
Everett, L.A. and Green, E.D. (1999) A family of mammalian anion
transporters and their involvement in human genetic diseases. Hum
Mol Genet, 8, 1883-1891.
El Khouri, E., Toure, A. (2014) Functional Interaction of the Cystic Fibrosis Transmembrane conductance Regulator with members of the
SLC26 family of anion transporters (SLC26A8 and SLC26A9): physiological and pathophysiological relevance. (Review). Intern. J. Bioch.
Cell Biol. 2014 Feb 13
Toure, A., Morin, L., Pineau, C., Becq, F., Dorseuil, O. and Gacon, G.
(2001) Tat1, a novel sulfate transporter specifically expressed in human
male germ cells and potentially linked to rhogtpase signaling. J Biol
Chem, 276, 20309-20315.
Dirami, T., Rode, B., Jollivet, M., Da Silva, N., Escalier, D., Gaitsch, N.,
Norez, C., Tuffery, P., Wolf, JP., Becq, F., Ray, PF., Dulioust, E., Gacon,
G., Bienvenu, T. and Touré, A. Missense mutations in SLC26A8, encoding a sperm-specific activator of CFTR, are associated with human
asthenozoospermia. Am J. Hum. Genet. 2013 May 2;92(5):760-6
Rode B, Dirami T, Bakouh N, Rizk-Rabin M, Norez C, Lhuillier P, Lorès
P, Jollivet M, Melin P, Zvetkova I, Bienvenu T, Becq F, Planelles G, Edelman A, Gacon G and Touré A.
The Testis Anion Transporter TAT1 (SLC26A8) stimulates CFTR activity during sperm capacitation. Hum Mol Genet. 2012, 21(6):128798.
This work was supported by the Ministère de l’Education
Nationale et de la Recherche Scientifique, Institut National de
la Santé et de la Recherche Médicale, Centre National de la
Recherche Scientifique, Université Paris Descartes, Agence
Nationale de la Recherche (ANR-07-JCJC-0099; ANR-12-BSV10011-01 MUCOFERTIL) and Association Vaincre la
Mucoviscidose (RF20110600465).
requirements.
PCA114
hypoxia (1 mM dimethyloxalylglycine) increased extracellular
CA activity and CAIX expression in Colo357, AsPC-1 and BxPC3 cells. Inhibition of CA (100 μM acetazolamide) in cell monolayers did not affect the growth after 72h (BCA protein assay;
p>0.38[two tailed t-test], n=6), viability after 72h (CellTitre
Blue; p>0.65, n=6) or colony formation after 21 days (clonogenic assay; p>0.55, n=3). This lack of effect may relate to the
absence of large diffusion distances in thin monolayers, which
are otherwise necessary to drive CO2/HCO3- out-of-equilibrium
(the substrate for CA catalysis). To test whether extracellular
CA activity may exert a physiologically role in diffusivelyrestricted tissue, Colo357 cells (which had the highest extracellular CA activity) were cultured as 3D tissue-growths (spheroids). Imaging intracellular pH confocally (SNARF) revealed a
shallow surface-to-core gradient of intracellular pH (0.34±0.03
pH units, n=17) over a distance of 127±2.2μm (radius). Inhibition of CA activity with acetazolamide in size-matched spheroids (125±2.6μm) increased the degree of pH non-uniformity
(0.50±0.09 pH units, n=11), and resulted in a significantly
(p<0.01) more acidic spheroid-core. Thus, CA activity may only
exert a biological effect in a diffusively-restricted environment
of a spheroid, which resembles that in poorly-perfused solid
tumours. Extracellular CA catalysis may be important for facilitating the venting of CO2 and lactic acid, particularly from the
spheroid-core and thereby support higher metabolic and
growth rates, essential for disease progression.
Supported by the Marie Curie Initial Training Network
“IonTraC”, EU.
requirements.
PCA115
Inhibiting proteasomal degradation increases the activity
of the epithelial sodium channel (ENaC) via an SGK1dependent mechanism that mimics the response to
aldosterone
Carbonic anhydrase activity regulates intracellular pH in 3D
pancreatic cancer growths
M.K. Mansley, M. Bertog and C. Korbmacher
T.H. Dovmark, A. Hulikova, R.D. Vaughan-Jones and P. Swietach
Institut für Zelluläre und Molekulare Physiologie, FriedrichAlexander-Universität Erlangen-Nürnberg, Erlangen, Germany
Department of Physiology, Anatomy and Genetics, University of
Oxford, Oxford, UK
Pancreatic cancer severity has been associated with high
expression levels of extracellular carbonic anhydrases (CA),
such as CAIX, and reduced expression of intracellular isoforms,
such as CAII. The biochemical function of these enzymes is to
catalyse the otherwise slow conversion between CO2 and
HCO3- and H+ ions, but the role of this process in pancreatic
cancer biology is unclear. CA activity was measured in five
human pancreatic cancer cell lines: AsPC-1, PANC-1, BxPC3,
MIA PaCa-2 and Colo357 (ranked in order of decreasing reliance
on oxidative respiration, determined by effect of replacing glucose with galactose using CellTitre Blue viability assay). Intracellular CA activity (measured by cytoplasm-loaded SNARF pHreporter dye upon rapid CO2/HCO3- addition/removal) was
highest in AsPC-1 (9.98±0.54[SEM] above spontaneous, n>50)
and decreased in the order PANC-1, Colo357, BxPC3 to MIA
PaCa-2. In contrast, extracellular CA activity (measured by
membrane-loaded exofacial WGA-fluorescein pH-reporter dye
upon rapid NH3/NH4+ addition/removal) was highest in
Colo357 (2.63±0.04 above spontaneous, n>50) and decreased
in the order AsPC-1, BxPC3, MIA PaCa-2 to PANC-1. Chemical
The epithelial sodium channel (ENaC) is involved in the tightlyregulated absorption of sodium across the epithelia lining the
aldosterone-sensitive distal nephron (ASDN), critically important for maintaining total body sodium content and thus the
long term control of arterial blood pressure. ENaC activity
depends, in part, on the number of channels resident at the
apical membrane of the epithelia and this involves a balance
between insertion and retrieval processes [1]. Preliminary data
[2] revealed that the proteasomal inhibitors MG132 and bortezomib could stimulate ENaC-mediated Na+ transport by ~3
fold in mCCDcl1 cells, a mammalian model of principal cells
from the ASDN [3]. This stimulatory effect is likely mediated
by increased serum and glucocorticoid inducible kinase 1
(SGK1) activity [4], thought to stimulate ENaC by reducing
channel retrieval. The aim of this study was to clarify the molecular mechanisms underpinning ENaC stimulation by proteasomal inhibitors. ENaC-mediated Na+ transport was quantified
by measuring amiloride-sensitive short circuit current (ISC)
across monolayers of mCCDcl1 cells mounted in Ussing chambers. The surface abundance of β-ENaC was monitored by Western blot analysis of cell lysates following a biotinylation assay.
The activity of SGK1 was monitored by Western blot analysis
159P
Bertog M, Mansley MK and Korbmacher C (2012). Proc Physiol Soc 27,
C102.
by low temperature (27 °C) incubation for 24 h (n = 4 – 6). By
contrast, treatment of cells expressing A561E with CFFT-004
(5 μM) or corr-4a (10 μM) for 24 h at 37 °C failed to confer CFTRmediated iodide efflux on A561E-CFTR, whereas low temperature incubation restored some CFTR-mediated iodide efflux
(n = 5). Next, we tested the action of CFTR-004 as a CFTR potentiator. When low temperature-rescued F508del-CFTR was
treated with forskolin (10 μM) and CFFT-004 (5 μM), the magnitude of CFTR-mediated efflux was similar to that elicited by
forskolin (10 μM) and genistein (50 μM; n = 5). Interestingly,
treatment of low temperature-rescued A561E-CFTR with CFFT004 (5 μM) generated an efflux of iodide comparable to that
observed with low temperature-rescued F508del-CFTR treated
with CFFT-004 (5 μM; n = 5). Finally, we tested CFFT-004 for
dual-activity by incubating cells expressing F508del- and A561ECFTR with CFFT-004 (5 μM) for 24 h at 37 °C before stimulating iodide efflux with forskolin (10 μM) and CFFT-004 (5 μM).
CFFT-004 both corrected and potentiated F508del-CFTR,
whereas it was without effect on A561E-CFTR consistent with
the results of correction testing (n = 4 – 6). We conclude that
CFFT-004 acts as a dual-acting molecule to rescue, albeit partially, the expression and function of F508del-CFTR. Based on
our present and previous (2) data, we also conclude that the
processing defect of A561E-CFTR is distinct from that of
F508del-CFTR and different CFTR correctors will likely be
required to delivery A561E-CFTR to the cell surface.
Gaeggeler HP et al.(2005). J Am Soc Nephrol 16, 878-891.
Sheppard DN. Chem Biol. 2011; 18:145-7.
Mansley MK, Bertog M and Korbmacher C (2013). Acta Physiologica
207, Suppl. 694.
Wang Y, et al. Proc 37th IUPS. 2013; PCB192.
of a downstream target n-myc downstream-regulated gene 1,
NDRG1. Data are shown as mean ± SEM, statistical significance
was assessed using a Student’s unpaired t-test. The time-course
and magnitude of ENaC stimulation by proteasomal inhibitors
was similar to the 2.0 ± 0.2 fold (n = 5, p < 0.001) stimulation
seen with aldosterone (3nM, 3h). Following treatment with
MG132, SGK1 activity was increased as shown by a ~3 fold
increase in NDRG1 phosphorylation [4]. Inhibiting SGK1 activity, using GSK650394 (10μM), abolished the stimulation of
amiloride-sensitive ISC caused by MG132 (n = 6, p < 0.001) or
by aldosterone (n = 5, p < 0.001). Interestingly, the abundance
of the mature glycosylated form of β-ENaC at the apical surface of mCCDcl1 cells increased by 1.9 ± 0.2 fold with MG132
(n = 5, p < 0.01) and 4.1 ± 1.4 with aldosterone (n = 6, p <
0.001). This was associated with a decrease in the less glycosylated form of β-ENaC, indicating a shift in glycosylation status of β-ENaC rather than an overall increase in subunit abundance. Inhibiting SGK1 activity prevented this shift in
glycosylation. Together these data suggest that proteasomal
inhibitors stimulate ENaC activity by preventing the breakdown of SGK1 thus mimicking the stimulation of ENaC activity by aldosterone, which acts by upregulating transcription
of SGK1.
Rotin D and Staub O (2011). Pflug Arch Eur J Phy 461, 1-21.
Alexander von Humboldt Foundation
This work was supported by the CF Trust; we thank Cystic
Fibrosis Foundation Therapeutics for the kind gift of CFFT-004.
requirements.
requirements.
PCA116
PCA117
Rescue of F508del-CFTR by the dual-acting small-molecule
CFFT-004
PDZK1 expression is critical for the brush border membrane
localisation and membrane half life of Na+/H+ exchanger
isoform 3 in enterocytes
S. Adams, L.W. Lake, G.O. McFadden, D.A. Roberts, R. Tanner,
J. Liu, H. Li and D.N. Sheppard
G. Chodisetti1, M. Luo1, S. Yeruva1, U. Seidler1, P. He2 and
C. Yun2
Physiology and Pharmacology, University of Bristol, Bristol, UK
1Department
Cystic fibrosis (CF) is caused by mutations that disrupt the
expression and function of the cystic fibrosis transmembrane
conductance regulator (CFTR) Cl- channel. To develop transformational drug therapies for CF, small-molecules have been
identified that rescue the expression (CFTR correctors) and
function (CFTR potentiators) of mutant Cl- channels. Interestingly, some small-molecules have been identified with both
CFTR corrector and potentiator activity (CFTR corrector-potentiators or dual-acting compounds; 1). Here, we investigate the
action of the dual-acting small-molecule CFFT-004
(WO2010/068863) on F508del-CFTR using BHK cells stably
expressing F508del-CFTR and the iodide efflux technique. As
controls, we studied wild-type CFTR and A561E, a CF mutant
with a similar clinical phenotype and mechanism of dysfunction, but different location in CFTR (2) and used the CFTR corrector corr-4a, and the CFTR potentiator genistein. We began
by testing the action of CFFT-004 as a CFTR corrector. Incubation of BHK cells expressing F508del-CFTR with CFFT-004 (5
μM) or corr-4a (10 μM) for 24 h at 37 °C, but not the vehicle
(DMSO; 0.05% vv-1), restored CFTR-mediated iodide efflux to
F508del-CFTR that were similar in magnitude to those achieved
160P
of Gastroenterology, Hannover Medical School,
Hannover, Lower Saxony, Germany and 2Department of Medicine,
Division of Digestive Diseases, Atlanta, GA, USA
Genetic deletion or inflammation-associated downregulation
of the PDZ-adaptor PDZK1/NHERF3 results in a decreased transport activity of the Na+/H+ exchanger NHE3 in the murine
intestine, accompanied by increased enterocyte NHE3 mRNA
expression levels, but a borderline reduced NHE3 protein
expression in or near the apical plasmamembrane (Hillesheim
et al. Pflüger’s Arch 2007, Cinar et al. J. Physiol. 2007, Lenzen
et al. Plos One 2012). This suggested that PDZK1 may play a
role in targeting/stabilizing NHE3 in the enterocyte brush border membrane. To answer this question, we searched for an
intestinal cell line that endogenously expresses PDZK1 and
found the spontaneously differentiating cell line Caco-2BBe to
have robust endogenous PDZK1 expression. VSVG-tagged
NHE3 was stably expressed in Caco-2BBe cells (Caco2BBe/NHE3-VSVG), followed by stable knock donw of PDZK1
by using recombinant lentiviral sh-RNA transfer. We then studied the effect of PDZK1 knockdown on membrane NHE3 half
life, brush border membrane expression, and transport function of NHE3. Biotinylation of membrane proteins, followed
by Western analysis and immunocytochemistry revealed a
significantly decreased amount of apical membrane NHE3 in
PDZK1 knock down (KD) cells, with more cytoplasmic/endosomal and basolaterally localized NHE3, compared to control
cells. This correlated with a significantly decreased acid-activated NHE3 transport activity in the PDZK1 KD compared to
control cells. Membrane half life studies for NHE3 were performed by following the degradation of biotinylated NHE3 over
time. Our study revealed that that control cells have a biphasic time course for NHE3 membrane loss, with an early rapid
phase followed by a slower phase with a linear loss of the ensuing 48 hours. In PDZK1 KD cells, the initial percentage of NHE3
that was expressed in the membrane was significantly lower
than in control cells, and the early phase of rapid loss of membrane NHE3 was completely ablated while the second, slower
phase was identical to control cells. Conclusions: The PDZ-adaptor protein PDZK1 is important for the targeting and/or retention of the Na+/H+ exchanger isoform 3 to the brush border
membrane of intestinal epithelial cells. The data further suggest that human NHE3 is retrieved from the membrane and
degraded by different pathways with different time constants
and differentially requiring PDZ-adaptor proteins. Further work
is necessary to characterize the protein complex formation
that decides the pathway of NHE3 into recycling endosomes
versus lysosomal/proteasomal degradation.
Down regulation of small intestinal ion transport in PDZK1(CAP70/NHERF3) deficient mice.
Hillesheim J, Riederer B, Tuo B, Chen M, Manns M, Biber J, Yun C, Kocher
O, Seidler U.
Pflugers Arch. 2007 Jul;454(4):575-86. Epub 2007 Mar 9.
NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ-domain protein PDZK1-deficient murine enterocytes.
Cinar A, Chen M, Riederer B, Bachmann O, Wiemann M, Manns M,
Kocher O, Seidler U.
J Physiol. 2007 Jun 15;581(Pt 3):1235-46. Epub 2007 Mar 29.
Downregulation of the NHE3-binding PDZ-adaptor protein PDZK1
expression during cytokine-induced inflammation in interleukin-10deficient mice.
METHODS 1) Nuclear iASPP is a key ARP maintaining tumor
growth. We employed co-immunoprecipitation to search the
potential nuclear transport factors involved in iASPP nuclear
entry. Mutagenesis was applied to identify the essential
residues (barcode), in iASPP, that bind nuclear transport factors and trigger nuclear import. 2) The barcode in human proteomic ARPs was determined by protein sequence alignment,
aiming to predict their nuclear localization. 3) The barcode
explained the mechanism behind p16 (a key ARP suppressing
tumor) function in melanoma. Additionally, we screened for
compounds that mask the iASPP barcode to block iASPP
nuclear entry and kill cancer cells.
RESULTS 1) Co-immunoprecipitation identified NTF2/RanGDP
as an iASPP binding partner in the cytoplasm. In the nucleus,
RanGDP is converted to RanGTP, disassembling
NTF2/RanGDP/iASPP complex and releasing iASPP. Mutagenesis identified the hydrophobic 13th residues from two consecutive ARs as contributing to RanGDP binding and nuclear
import. 2) 17 ARPs were experimentally tested. The barcode
predicts the nuclear localization of >150 human proteomic
ARPs with high accuracy. Interestingly, > 70% of barcode containing ARPs participate in NF-kB signaling, implying a role of
the RaDAR (RanGDP-AR) guided nuclear import pathway in
human cancers, autoimmune and inflammatory diseases. 3)
The most commonly occurring p16 mutation, M13I, confers
the barcode to p16, resulting in aberrant p16 nuclear accumulation and uncontrolled melanoma growth. In the oncoprotein iASPP, masking the barcode by compounds
sequestered iASPP in cytoplasm, inhibiting tumor growth in
mice.
SIGNIFICANCE The finding shows a precise molecular barcode
that flags proteins for nuclear import. This discovery will help
illuminate the molecular dysfunction that underpins a broad
array of ailments. The RaDAR is being exploited to develop
entirely novel diagnostics and therapies (Lu et al, Cell, 2014).
We experimentally proved, in melanoma treatment (Lu et al,
Cancer Cell, 2013), it is a promising strategy (Nat. Rev. Cancer, 2013). The barcode masking compounds is scheduled for
further pre-clinical study (Lu et al, WIPO, 2013).
Lenzen H, Lünnemann M, Bleich A, Manns MP, Seidler U, Jörns A.
PLoS One. 2012;7(7):e40657. doi: 10.1371/journal.pone.0040657.
Epub 2012 Jul 27.
requirements.
PCA118
RaDAR guides nuclear membrane transport
M. Lu, J. Zak and X. Lu
NDM department, University of Oxford, Ludwig Institute for Cancer
Research, Oxford, UK
MOTIVATION Regulation of nuclear import is fundamental to
eukaryotic biology. The only general nuclear import pathway
identified in the past 30 years is the Nuclear Localization Signal (NLS) pathway, yet it cannot account for the entire nuclear
proteome. The ankyrin repeat (AR) is a common protein structural motif. ARs containing proteins (ARPs) play pivotal roles
in the development and maintenance of tissue homeostasis.
Mutation and mislocalization of ARPs are associated with various human diseases. It is mysterious how ARPs without a NLS
enter the nucleus and play their various physiological and
pathological roles.
Lu M, Zak J, Chen S, et al. A code for RanGDP binding in ankyrin repeats
defines a nuclear import pathway. Cell. 2014 (In press).
Lu M, Breyssens H, Salter V, et al. Restoring p53 function in human
melanoma cells by inhibiting mdm2 and cyclin B1/cdk1 phosphorylated nuclear iASPP. Cancer Cell. 2013. 23, 618-633.
Mccarthy N. Tumour suppressors: Restoring function. Nat Rev Cancer (2013 Research Highlight). 2013, 13: 379.
Lu X, Lu M, Breyssens H, Slee E. World Intellectual Property Organization (Patent number: WO 2013131019).
This work was funded by the Ludwig Institute for Cancer
Research Ltd. We thank Kimberley Bryon-Dodd for critical
reading of the abstract.
161P
requirements.
PCA120
A functional evaluation of SLC26A9-mediated chloride
secretion in epithelial cells
PCA119
Novel physiological roles of ezrin in the regulation of bile
fluidity
R. Hatano1, K. Akiyama1, A. Tamura2, S. Hosogi3, Y. Marunaka3,
M.J. Caplan4, S. Tsukita2 and S. Asano1
1Department of Molecular Physiology, College of Pharmaceutical
Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan,
2Laboratory of Biological Science, Graduate School of Frontier
Biosciences and Graduate School of Medicine, Osaka University,
Osaka, Osaka, Japan, 3Department of Molecular Cell Physiology
and Bio-Ionomics, Kyoto Prefectural University of Medicine, Kyoto,
Kyoto, Japan and 4Department of Cellular and Molecular
Physiology, Yale University School of Medicine, New Haven, CT,
USA
The secretin dependent biliary secretion of ions and water by
transporters and/or channels, which are located at apical membrane of cholangiocytes, is essential for the regulation of biliary flow. The cystic fibrosis transmembrane conductance regulator (CFTR) plays a key role in the chloride secretion into the
bile. In the cystic fibrosis (CF) patients, totally 5 to 10% of
patients develop the progressive biliary fibrosis resembling
primary sclerosing cholangitis. The loss of cystic fibrosis transmembrane conductance regulator (CFTR) leads to the onset
of liver disease in human. Ezrin, radixin and moesin (ERM) proteins are identified as cross-linkers between the plasma membrane proteins and actin cytoskeleton. Ezrin interacts with
Na+/H+ exchanger regulatory factor-1 (NHERF1) via its N-terminal binding domain and with actin cytoskeleton via its C-terminal actin-binding domain. CFTR is associated with NHERF1
via its c-terminal PDZ binding motif. In the liver, ezrin, but not
radixin or moesin, is only expressed in the cholangiocytes and
colocalizes with CFTR and NHERF1 at apical membrane of
cholangiocyte. In the present study, we used ezrin knockdown
(Vil2kd/kd) mice to investigate the physiological roles of ezrin
in the liver (1). All work with animals was performed with
approval from the Animal Ethics Committee of Ritsumeikan
University. We have found that Vil2kd/kd mice develop severe
hepatic failure c

1P PL1 PL2 PL3 PL4 Plenary Lectures

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