INVITED REVIEW Mechanisms of Rectal Gland Secretion Franklin H

Transcription

The Bulletin, MDI Biological Laboratory V. 44, 2005
INVITED REVIEW
Mechanisms of Rectal Gland Secretion
1
Franklin H. Epstein1, M.D. & Patricio Silva2, M.D.
Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA 02215
2
Department of Medicine, Temple University Hospital, Philadelphia, PA 19140
Introduction
Swimming in an ocean in which the
concentration of sodium chloride approximates
500 mEq/L, all marine vertebrates must
continually excrete salt to preserve the
constancy of their internal environment.
Teleosts, with a serum sodium and chloride that
varies from 150 to 180 mEq/L do this via the
specialized chloride-secreting cells located in
their gills. Elasmobranchs, with serum sodium
averaging 280 mEq/L, utilize the rectal gland, a
specialized chloride-secreting structure
emptying into the terminal large intestine 1.
This review summarizes information about
the mechanism of this process, developed at
MDIBL over the past 30 years through work on
the rectal gland of Squalus acanthias, using
intact sharks, whole isolated perfused rectal
glands 17, rectal gland slices, rectal gland
tubules, cultured cells and isolated membranes.
Many scientists have worked on the shark
rectal gland at MDIBL. We have not attempted
a complete or exhaustive review of their
contributions in this short paper, but have relied
heavily on our own work, to describe the
evolution of our own thoughts.
Hormonal Stimulation of Secretion
Chloride secretion by the rectal gland is
stimulated by two endogenous polypeptide
hormones: vasoactive intestinal peptide,(VIP) 29
and C-type cardiac natriuretic peptide (CNP)
14,28
. In the shark, VIP serves chiefly as a
neurotransmitter, rather than as a circulating
hormone 21,30. It is located in rectal gland nerves
and released in response to neurostimulation 30.
CNP is released from secretory granules within
cardiocytes in response to an increase in the
volume of circulating blood (sensed as cardiac
dilatation) 21,24-27. This humoral factor in the
blood perfusing the rectal gland causes the
gland to secrete chloride. If an isolated rectal
gland is perfused by blood from a shark whose
blood volume has been expanded, the perfused
gland begins to secrete chloride profusely in a
way that is blocked by an inhibitor of cellular
receptors for cardiac peptides 9.
Shark CNP has two actions on the rectal
gland. The first is to release vasoactive
intestinal peptide from rectal gland nerves 21.
The second is detected in isolated perfused
rectal glands when neurotransmission is
blocked by the local anesthetic procaine, or in
isolated rectal gland cells and tubules, where no
neuronal elements are present 18. It constitutes
a direct action of CNP on rectal gland cells
themselves to stimulate chloride secretion.
Secondary Active Transport of Chloride
Chloride secretion is accomplished in rectal
gland cells by the process known as “secondary
active transport”, diagrammed in Figure 1 5,19,20.
The motive power for active transport is
derived from the hydrolysis of ATP to ADP by
the enzyme Na-K-ATPase, located on the
basolateral border of rectal gland cells 4. The
furosemide-sensitive, Na-K-2Cl cotransporter,
also located on the basolateral border, permits
sodium to move down its electrochemical
gradient into the cell, accompanied by
potassium and chloride ions. The concentration
of chloride in the cell thus exceeds the
concentration predicted at electrochemical
equilibrium. Chloride exits the cell into the
duct through a chloride channel controlled by a
shark version of the cystic fibrosis
trasnmembrane regulator (CFTR). Activators
1
Figure 1. Model of secondary active chloride secretion
in shark rectal gland. The motive power for the
transcellular movement of C1− across the rectal gland
epithelium is supplied by the Na-K-ATPase pump, which
pumps Na+ out of the cell into the blood. Na+ moves into
the cell across the basolateral cell membrane down its
electrochemical gradient, through the Na+K+2C1− cotransporter, dragging C1− and K+ with it. Intracellular
C1− concentration therefore exceeds that predicted by the
Nernst equilibrium equation. When the gland is
stimulated to secrete, C1− channels open in the luminal
membrane (controlled by the cystic fibrosis
transmembrane regulator - CFTR protein) and chloride
exits the cell into the duct lumen. Na+ moves passively
down its electrochemical gradient through paracellular
pathways into the duct lumen.
of secretion open this channel, permitting
chloride to flow down its electrochemical
gradient into the duct. Stimulation of chloride
secretion also directly activates the ouobain
inhibitable, basolateral Na-K-ATPase 11,13.
Intracellular cascades
The intracellular signals by which VIP
and CNP stimulate chloride secretion in the
rectal gland appear to follow different
pathways. It is clear that, as in other vertebrate
tissues that secrete chloride, the chloride exit
channel of rectal gland cells is activated by
cAMP. Isolated rectal glands are stimulated to
secrete by activators of adenylate cyclase,
including VIP, forskolin and high
concentrations of adenosine. Infusion of cAMP
itself into the isolated perfused gland stimulates
secretion, as do inhibitors of phosphodiesterase
2
that slow the hydrolysis of cAMP. Inhibiting
adenylate cyclase (e.g., by infusing
somatostatin) blocks stimulation by VIP or by
forskolin 22. The action of cAMP to stimulate
chloride secretion is thought to occur via
stimulation of protein kinase A (PKA) which
then utilizes ATP to phosphorylate CFTR and
open its chloride exit channel.
Different cellular pathways are entrained
when the rectal gland is stimulated by CNP 18.
Unlike VIP, CNP does not stimulate the
adenylate cyclase of rectal gland membranes.
Instead, its action is exerted via protein kinase
C (PKC) and guanylate cyclase. Inhibitors of
PKC, such as staurosporine, chelerythrine, and
bisindolylmaleimide 2,18 block the direct
stimulatory action of C-type cardiac natriuretic
peptide in isolated perfused rectal glands.
These compounds do not inhibit stimulation by
VIP. Stimulation of isolated perfused glands
with CNP also increases the intracellular
accumulation of cyclic guanosine
monophosphate (cGMP). If the hydrolysis of
cyclic GMP is inhibited: stimulation of chloride
secretion by a small dose of CNP is enhanced16.
Simultaneous activation of PKC and guanylate
cyclase appears to be necessary to stimulate
rectal gland secretion, if cAMP levels are not
raised 18.
Contractile elements of the cytoskeleton
may play a role in chloride secretion stimulated
by CNP but not by VIP 15. Direct CNP
stimulation of the isolated perfused gland is
completely prevented by cytochalasin D, which
disrupts actin filaments, and by ML-7, an
inhibitor of myosin light chain kinase. These
agents have little effect on stimulation of the
rectal gland by VIP or by forskolin.
Interacting Pathways
An important question raised by the
foregoing studies is whether the intracellular
cascades initiated by VIP and by CNP are
entirely separate from beginning to end or
whether they interact. The patch-clamp studies
of rectal gland tubules carried out by Greger
and his associates at MDIBL suggested that
there is more than one chloride exit channel on
the luminal border of rectal gland cells 6,8. It
seemed possible that chloride secretion
stimulated by the direct action of CNP involved
a different chloride channel than that opened by
VIP or other stimulators of cAMP production.
The Forrest laboratory showed that when
Xenopus oocytes were transfected with shark
CFTR and shark receptor for CNP, exposure to
CNP could open the CFTR-associated chloride
channel 7. The discovery of a specific inhibitor
of CFTR-associated chloride channels by Dr.
Alan Verkman and his associates 12 permitted
this question to be investigated in isolated
perfused rectal glands. Preliminary exposure of
these glands to Verkman’s compound markedly
inhibited chloride secretion stimulated by CNP
as well as VIP 3.
It seems likely, therefore, that the final exit
step for chloride secretion, its downhill
movement through a CFTR- modulated
chloride channel, is the same for CNP and for
VIP. Another point of similarity between VIPinduced stimulation and CNP-induced
stimulation is that in isolated rectal gland
tubules, both are said to increase the
fluorescent signal generated by the reaction of
intracellular Ca++ with fura-2 7.
Additional interactions between the CNP
pathway and the VIP pathway have been
uncovered by recent studies of isolated
perfused rectal glands, summarized in this
edition of the Bulletin. Perfusion of glands
with small amounts of CNP greatly enhanced
their response to VIP and dibutyryl cAMP, as if
the CNP pathway, once activated, amplified the
secretory response to cAMP. This synergistic
action is consistent with studies by others in
mammalian cells, suggesting that CFTR may
be phosphorylated by other protein kinases than
PKA, and that such phosphorylation renders
CFTR more sensitive to activation by PKA 10.
Inhibition of PKA (by the compound H-89)
effectively blocks stimulation of intact perfused
glands or of rectal gland slices 23 by both VIP
and CNP. CNP stimulation thus appears to
require PKA activity. It seems possible that a
major component of CNP activated chloride
Figure 2. Schema of effect of CNP on secretion of
chloride in dogfish rectal gland. Volume expansion
causes release of CNP from the heart. CNP binds to a
guanylate cyclase-linked B-type receptor and activates
guanylyl cyclase producing cGMP. The increase in
cGMP is not itself sufficient to produce an increase in
chloride transport. A parallel stimulation of protein
kinase C (PKC) is probably mediated by phospholipase
C and the phosphoinositide pathway producing a
synergistic effect on chloride transport. CNP also has an
indirect effect to stimulate chloride secretion by eliciting
release of VIP from nerves within the rectal gland. VIP
stimulates adenylate cyclase, thus increasing cAMP,
which activates an apical chloride channel homologous
to the human cystic fibrosis transmembrane conductance
regulator (CFTR).
secretion involves the sensitization of shark
CFTR to the small amounts of cAMP that
might be produced within the cell
constitutively, that is, by the “idling” of the
adenylate cyclase of rectal gland cells at basal
levels.
In this perspective, the process of rectal
gland stimulation used by elasmobranchs for
salt homeostasis appears ingenious and
complex. Salt retention and volume expansion
lead to release of CNP from a dilated heart.
CNP not only stimulates the local release of
VIP from nerves within the rectal gland but
also greatly sensitizes the gland to stimulation
by the cAMP produced by VIP. The result is
an immediate burst of salt secretion, which rids
3
the body of salt and water, and returns the
volume of blood and extracellular fluid to
normal. These experiments strengthen the view
that several parallel and interacting intracellular
pathways are involved in the stimulation of
rectal gland secretion by endogenous
hormones. It seems possible that an analogous
pattern of interacting influences may control
active transport in other highly developed
secreting epithelial organs in man and other
vertebrates.
References
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Epstein FH. The sea within us. J. Exp. Zool. 1999;
284:50-54
Epstein FH, Sighinolfi C, Hessler K, Silva P.
Effects of chelerythrine and bisindolylmaleimide
on rectal gland function. Am J. Physiol. Submitted
for publication.
Epstein FH, Sighinolfi C, Hessler K, Verkman
AS, Riordan JR, Silva PS. A synthetic CFTR
inhibitor blocks stimulation of Squalus acanthias
rectal gland secretion by CNP and VIP. Bull
MDIBL, 2004, 43:13-14
Eveloff J, Karnaky KT Jr, Silva P, Epstein FH,
Kinter WB. Elasmobranch rectal gland cell:
Autoradiographic localization of (3H) ouabainsensitive Na,K –ATPase in rectal gland of dogfish,
Squalus acanthias. J Cell Biol 1979; 83:16-32
Eveloff J, Kinne R, Kinne-Saffran E, Murer H,
Silva P, Epstein FH, Stoff J, Kinter WB.
Coupled sodium and chloride transport into plasma
membrane vesicles prepared from dogfish rectal
gland. Pflufers Arch, 1978, 378:97-92
Gogelein H, Schlatter E, Greger R. The “small”
conductance chloride channel in the luminal
membrane of the rectal gland of the dogfish
(Squalus acanthias). I: Pflugers Arch, 1987, 409:
122-5
Greger R, Bleich M, Warth R, Thiole I, Forrest
J N . The cellular mechanisms of Cl secretion
induced by C-type natriuretic peptide (CNP).
Experiments on isolated in vitro perfused rectal
gland tubules of Squalus acanthias. Pflugers Arch,
1999, 438:15-22
Greger R, Schlatter E, Gogelein H, Chloride
channels in the luminal membrane of the rectal
gland of the dogfish (Squalus acanthias).
Properties of the “larger” conductance channel. I:
Pflugers Arch, 1987, 409:114-21
Gunning M, Solomon RJ, Epstein FH. Role of
guanylyl cyclase receptors for CNP in salt secretion
by shark rectal gland. Am J Physiol, 1997, 273:
R1400-R1406.
Jia Y, Mathews CJ, Hanrahan JW.
Phosphorylation by protein kinase C, is required for
acute activation of cystic fibrosis transmembrane
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regulator by protein kinase A. J Biol Chem,
1997,272: 49 78-94
Lear S, Cohen BJ, Silva P, Lechene C, Epstein
FH. cAMP activates the sodium pump in cultured
cells of the elasmobranch rectal gland. J Am Soc
Nephrol 1992; 2:1523-1528
Ma T Thiagarajah Jr, Yank H, Sonawane ND,
Folli C, Galietta LJ, Verkman AS.
Thiazolidionone CFTR inhibitor identified by highthroughput screening blocks cholera toxin-induced
intestinal fluid secretion. J Clin Invest, 2002, 110:
1651-8
Marver D, Lear S, Marver LT, Silva P, Epstein
FH. Cyclic AMP-dependent stimulation of Na,KATPase in shark rectal gland. J Membr Biol 1986;
94: 205-215
Schofield, J.P., D. S. C. Jones, and J. N. Forrest.
Identification of C-type natriuretic peptide in the
heart of spiny dogfish shark (Squalus acanthias).
Am. J. Physiol,1991, 261: F734-739
Silva P, Epstein FH. Role of the cytoskeleton in
secretion of chloride by shark rectal gland. J Comp
Physiol B 2002, 172:719-723
Silva P, Sighinolfi C, Hessler K, Spokes K, Hays
R, Epstein FH. Sildenafil citrate enhances the
stimulation of chloride by CNP in S q u a l u s
acanthias rectal gland. Bull MDIBL 2004, 43: 1516
Silva P, Solomon R, Epstein FH. Perfusion of
shark rectal gland. Methods in Enzymology,
volume 192, Academic Press 1991; pp 754-766
Silva P, Solomon RJ, Epstein FH. Mode of
activation of salt secretion by C-type natriuretic
peptide in the shark rectal gland. Am J. Physiol.
1999;277 : R1725-R1732
Silva P, Solomon RJ, Epstein FH. The rectal
gland of Squalus acanthias: a model for the
transport of chloride. Kidney Int 1996; 49:15521556
Silva P, Stoff J, Field M, Fine L, Forrest JN,
Epstein FH. Mechanism of active chloride
secretion by shark rectal gland: role of Na-KATPase in chloride transport. Am J Physiol, 1977,
233:F298-F306
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Silva, P., J. S. Stoff, R. J. Solomon, S. Lear, D.
Kniaz, R. Greger, and F. H. Epstein. Atrial
natriuretic peptide stimulates salt secretion by shark
rectal gland by releasing VIP. Am J Physiol, 1987,
252: F99-F103
Silva, P., J.S. Stoff, D.R. Leone, and F.H.
Epstein. Mode of action of somatostatin to inhibit
secretion by shark rectal glands. Am J Physiol,
1985, 249: Physiol. 18: R329-R334
Solomon R, Castelo L, Franco E, Taylor M,
Silva P, Epstein FH. Preliminary data on
intracellular signaling mechanisms in the rectal
gland of Squalus acanthias: a pharmacologic
approach. Bull MDIBL, 1995, 34:47-48
Solomon R, Taylor M, Dorsey D, Silva P, and
Epstein FH. Atriopeptin stimulation of rectal gland
function in Squalus acanthias: Am. J. Physiol,
1985, 249: R348- 354
Solomon R, Taylor M, Sheth S, Silva P, Epstein
FH. Primary role of volume expansion in
stimulation of rectal function. II. Hemodynamic
changes. Am J Physiol, 1984;246: R67-R71
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F H . Primary role of volume expansion in
stimulation of rectal gland function. Am J Physiol,
1985, 248:R638-R640
Solomon R, Taylor M, Stoff JS, Silva P, Epstein
FH. In vivo effect of volume expansion on rectal
gland function. I. Humoral factors. Am J Physiol,
1984, 246:R63-R66
Solomon, R., A. Protter, G. McEnroe, J.G.
Porter, F.H. Epstein, and P. Silva. C-type
natriuretic peptides stimulate chloride secretion in
the rectal gland of Squalus acanthias. Am J.
Physiol 1992, 262 : R707-711. 1992
Stoff JS, Rosa R, Hallac R, Silva P, Epstein FH.
Hormonal regulation of active chloride transport in
the dogfish rectal gland.
Am J Physiol
1979,237:F138-F144
Stoff, J. S., P. Silva, R. Lechan, R. Solomon, and
F. H. Epstein. Neural control of shark rectal
gland. Am J Physiol, 1988, 255 24: R212-R216
5
Potassium secretion in winter flounder (Pseudopleuronectes americanus) intestine:
Effects of K+ channel blockers on electrogenic NaCl absorption
Scott M. O’Grady and Peter J. Maniak,
Department of Physiology, University of Minnesota, St. Paul MN 55108.
Marine teleosts like the winter flounder, maintain plasma osmolarities and serum K+ concentrations
(2 to 5 mM) that are significantly lower than sea water. Water balance and regulation of plasma Na+
and Cl- concentrations are primarily achieved through the coordinate absorptive and secretory
functions of the small intestine and gill epithelium, respectively18. The ingestion of sea water,
containing significantly higher [K+] (≈ 10 mM) relative to plasma, poses the problem of increasing
extracellular fluid [K+] to levels that could seriously compromise function of excitable tissues. To
limit increases in plasma [K+], the ion is actively secreted by the small intestine and urinary
bladder4,5,6.
K+ secretion by the intestinal epithelium occurs in parallel with NaCl and fluid absorption. The
mechanism of NaCl transport is similar to that present in the thick ascending limb of Henle’s loop in
mammalian kidney19. Electroneutral uptake of Na+, K+ and Cl- across the apical membrane is
mediated by a Na-K-2Cl cotransporter that is thought to be homologous to the isoform previously
characterized in mammalian kidney22,. Na+ entering across the apical membrane is transported across
the basolateral membrane by the Na-K-ATPase. [Cl-]i, which is maintained above electrochemical
equilibrium, exits the cell across the basolateral membrane through both conductive and electroneutral
pathways. K+ entering the cell by both Na-K-2Cl cotransport and Na-K-ATPase activities exits across
the apical membrane through K+ channels that have yet to be identified. Apical K+ efflux is critical for
sustaining the electrical driving force for Cl- exit through Cl- channels present in the basolateral
membrane7,11. It also ensures that [K+] within the luminal fluid does not become rate limiting with
respect to the activity of the Na-K-2Cl cotransporter.
The apical K+ channel responsible for K+ recycling and secretion in thick ascending limb cells of
mammalian kidney was identified as the ROMK (Kir1.1) K+ channel12,21,23. Kir1.1 is an inwardly
rectifying K+ channel22 that is blocked by Ba2+ and Cs+. Previously, whole cell amphotericin B
perforated patch clamp studies using freshly dissociated winter flounder enterocytes failed to detect
inwardly rectifying K+ channels, but instead identified a Ba2+-sensitive voltage-gated (Kv) K+
channel17. This Kv channel was activated by membrane depolarization above -60 mV and exhibited
slow, C-type inactivation similar to Kv channels identified in mammalian alveolar epithelium15.
Analysis of activation and steady-state inactivation curves revealed that an overlap (window current)
exists between -60 and -20 mV, indicating constitutive Kv channel activity within this range of
voltages. The channel was blocked by charybdotoxin (CTX) and by treatment with 8-Br-cGMP17.
The major objective of the present study was to characterize the effects of known K+ channel
blockers and peptide toxins on K+ transport pathways in the apical membrane of the winter flounder
intestine to obtain pharmacologic data that would aid in the molecular identification of channels
involved in K+ recycling and secretion. Intestinal mucosa, stripped of submucosal muscle layers, was
mounted in Ussing chambers (area = 0.64 cm2) and bathed on both aspects with flounder saline
solution containing, (in mM) 150 NaCl, 5 KCl, 1CaCl2, 1MgSO4, 3Na2HPO4, 5 HEPES, pH 7.8.
Dextrose (10 mM) was added to the basolateral solution and 10 mM mannitol was added to the apical
solution. The tissues were gassed with air and kept at a constant temperature of 15oC. Under these
conditions the tissues exhibited a serosa negative transepithelial potential difference that ranged
between 2-5 mV and a mean tissue conductance of 22.7 ± 0.73 mS (n = 25). Short circuit current (Isc)
6
responses are presented as means ± SE and were normalized to an area of 1 cm2. Statistically
significant differences between mean current values were determined using an unpaired Student’s t test
with the level of significance set at p < 0.05.
A representative Isc tracing showing the effects of apical Ba2+ (1 mM at each addition) and
bumentanide (100 µM) is shown in Figure 1. Note that treatment with Ba2+ produced only a partial
inhibition of the Isc and that at a concentration of 2 mM, maximal inhibition was achieved.
Subsequent addition of the Na-K-2Cl cortansport inhibitor bumetanide produced a slow, but
continuous inhibition of the Isc and reduced the total current by approximately 95%.
BaCl2 (1 mM)
100
Basal Isc
120
Barium (2 mM)
100
Quinidine (10µM)
2
Isc (µA/cm )
Bumetanide (100 µM)
80
2
Isc (µA/cm )
BaCl2 (1 mM)
60
40
80
60
20
0
0
1000
2000
*
Bumetanide (100 µM)
40
20
0
Quinidine (200µM)
*
*
*
3000
Time (seconds)
Figure 1: Representative Isc trace showing effects of apical
Ba2+ and bumetanide.
Figure 2: Bar graph summarizing the effects of Ba2+,
quinidine and bumetanide on Isc. (*) significantly
different from the basal Isc.
A summary comparing the mean basal Isc with the effects of Ba2+, quinidine and bumetanide is
shown in Figure 2. Ba2+ at a concentration of 2 mM and quinidine at 10 µM produced approximately
50% inhibition of the Isc. Treatment with 200 µM quinidine or 100 µM bumetanide inhibited the basal
Isc by more than 80% and 95%, respectively.
Quinidine
Ba
80
60
40
20
0
-7
100
2+
% Inhibition of Isc
% Maximal Response
100
Barium (2 mM, n= 18)
Quinidine (0.2 mM, n = 17)
80
60
CTX (300 nM, n = 11)
DTX-K (100 nM, n = 5)
DTX-I (100 nM, n = 5)
40
Cesium (1 mM. n = 7)
TEA (5 mM, n = 6)
20
0
-6
-5
-4
-3
-2
Log Concentration (M)
Figure 3: Concentration-response curves for Ba2+ and quinidine. Figure 4: Effects of K+ channel blockers on basal Isc.
7
Concentration response relationships for Ba2+ and quinidine are presented in Figure 3. Data were
normalized by expressing the maximum change in Isc produced at the highest blocker concentration as
100%. The data were fit using a four parameter logistic function with Prism© software. The IC50
values for quinidine and Ba2+ were 8.2 ± 0.2 µM (n = 5) and 0.29 ± 0.03 mM (n = 5), respectively.
Correlation coefficients (R2) for both concentration-response relationships exceeded 0.99.
Figure 4 shows the effects of several K+ channel blockers and peptide toxins on Isc. Compared to
all blockers tested, quinidine (200 µM) was the most efficacious followed by Ba2+. CTX and Cs+,
previously shown to inhibit Kv channel currents in dissociated winter flounder enterocytes, produced a
modest 10% inhibition of Isc, similar in magnitude to the effects of dendrotoxin (DTX-K). In contrast,
the effects of dendrotoxin-I (DTX-I) and TEA (5 mM) were not significantly different from zero.
The results of these experiments suggested that at least two distinct pathways for K+ exit were
present in the apical membrane of winter flounder enterocytes. One of them was blocked by Ba2+, and
the other appeared to be blocked by either Ba2+ or quinidine. Previous in vitro studies of winter
flounder intestinal mucosa showed that Ba2+ significantly reduced apical membrane K+ conductance,
measured using conventional microelectrode techniques and decreased net K+ secretion, as determined
by isotopic flux measurements7,11. The results of the present study were consistent with these earlier
findings, but indicated that 50% or more of basal NaCl absorption is sustained in the presence of 2 mM
Ba2+. Thus the quinidine-sensitive pathway (presumably a K+ channel) appears to be necessary in
supporting a significant portion of NaCl absorption. CTX, DTX-K and Cs+ consistently inhibited 10%
of the Isc response, suggesting the possibility that previously identified Kv channels in flounder
enterocytes could be localized to the apical membrane, but if so, their role in sustaining NaCl
absorption appears to be relatively minor.
The findings of this study may have significance in understanding the effects of certain heavy
metals that accumulate within coastal marine sediments and benthic invertebrates adjacent to heavy
industrial areas24. The observation that NaCl absorption by the winter flounder intestine is inhibited by
cations such as Ba2+ suggests that certain metal cation contaminants may block intestinal salt and fluid
absorption by inhibiting the activity of apical K+ channels. Silver for example, when present as free
silver ion or as silver chloride complexes, is one of the most toxic heavy metals affecting the
physiology of freshwater and marine fishes1,3,16. Experiments with sculpin, trout, lemon sole European
flounder, starry flounder and midshipmen previously showed that chronic exposure to low levels of
silver resulted in significant accumulation within the intestine and that intestinal osmoregulatory
function was a sensitive target for waterborne silver exposure2,8-10,13,14. Further studies are necessary to
determine whether apical K+ channels involved in K+ recycling and secretion are specifically affected
by heavy metal contaminants.
This study was supported by funds from the Center for Marine Toxicity Studies. Dr. O’Grady was a
recipient of a New Investigator Award.
1.
2.
3.
8
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sodium uptake rate. Environ. Sci. Technol. 36(8): 176-136, 2002.
Brauner, C. J. and C.M. Wood. Effect of long-term silver exposure on survival and ionoregulatory development in
rainbow trout (Oncorhynchus mykiss) embryos and larvae, in the presence and absence of added dissolved organic
matter. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 3(12):161-173, 2002.
Brauner, C. J. and C.M. Wood. Ionoregulatory development and the effect of chronic silver exposure on growth,
survival, and sublethal indicators of toxicity in early life stages of rainbow trout (Oncorhynchus mykiss). J. Comp.
Physiol. [B].172(2):153-162, 2002.
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mucosa of the winter flounder, Pseudopleuronectes americanus. I. Functional and structural properties of cellular
and paracellular pathways for Na and Cl. J. Memb. Biol. 41(3):265-293, 1978.
Field, M., P/L. Smith, and J.E. Bolton. Ion transport across the isolated intestinal mucosa of the winter flounder,
Pseudopleuronectes americans: II. effects of cyclic AMP. J. Memb. Biol. 55(3):157-163, 1980.
Frizzell, R. A., D.R. Halm, M.W. Musch, C.P. Stewart, and M. Field. Potassium transport by flounder intestinal
mucosa. Am. J. Physiol. 46(6):F946-951, 1984 .
Grosell, M., G. De Boeck, O. Johannsson, and C.M. Wood. The effects of silver on intestinal ion and acid-base
regulation in the marine teleost fish, Parophrys vetulus. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol.
24(3):259-270, 1999.
Grosell, M. and C.M. Wood. Branchial versus intestinal silver toxicity and uptake in the marine teleost Parophrys
vetulus. J. Comp. Physiol. [B] 71(7):585-594, 2001.
Guadagnolo, C. M., C.J. Brauner and C.M. Wood. Chronic effects of silver exposure on ion levels, survival, and
silver distribution within developing rainbow trout (Oncorhynchus mykiss) embryos. Environ. Toxicol. Chem.
20(3):553-560. 2001.
Halm, D. R., E.J. Krasny, Jr, and R.A. Frizzell. Potassium transport across the intestine of the winter flounder:
active secretion and absorption. Prog. Clin. Biol. Res.126:245-255, 1983.
Hebert, S. C. Bartter syndrome. Curr Opin Nephrol Hypertens 2003 12(5):527-532 .
Hogstrand, C., E.A. Ferguson, F. Galvez, J.R. Shaw, N.A. Webb, and C.M. Wood. Physiology of acute silver
toxicity in the starry flounder (Platichthys stellatus) in seawater. J. Comp. Physiol. [B] 169(7):461-473, 1999.
Hogstrand, C., C.M. Wood, N.R. Bury, R.W. Wilson, J.C. Rankin and M. Grosell. Binding and movement of
silver in the intestinal epithelium of a marine teleost fish, the European flounder (Platichthys flesus). Comp Biochem.
Physiol. C Toxicol. Pharmacol. 133(12):125-135, 2002.
Lee, S. Y., P.J. Maniak, D.H. Ingbar and S.M. O'Grady. Adult alveolar epithelial cells express multiple subtypes of
voltage-gated K+ channels that are located in apical membrane. Am. J. Physiol. 284(6):C1614-1624, 2003.
Lima, A. R., C. Curtis, D.E. Hammermeister, D.J. Call and T.A. Felhaber. Acute toxicity of silver to selected fish
and invertebrates. Bull. Environ. Contam. Toxicol. 29(2):18-49, 1982.
O'Grady, S. M., K.E. Cooper and J.L. Rae, Cyclic GMP regulation of a voltage-activated K+ channel in dissociated
enterocytes. J. Memb. Biol. 124(2):159-167, 1991.
O'Grady, S. M., M. Field, N.T. Nash and M.C. Rao. Atrial natriuretic factor inhibits Na-K-Cl cotransport in teleost
intestine. Am. J. Physiol. 249: C531-534, 1985.
O'Grady, S. M., M.W. Musch, and M. Field. Stoichiometry and ion affinities of the Na-K-Cl cotransport system in
the intestine of the winter flounder (Pseudopleuronectes americanus). J. Memb. Biol. 91(1):33-41, 1986.
Rao, M. C., N.T. Nash and M. Field. Differing effects of cGMP and cAMP on ion transport across flounder intestine.
Am. J. Physiol. 246:C167-171, 1984.
Starremans, P. G., A.W. Van der Kemp, N.V. Knoers, L.P. Van den Heuvel and R.J. Bindels. Functional
implications of mutations in the human renal outer medullary potassium channel (ROMK-2) identified in Bartter
syndrome. Pflugers Arch. 443(3):466-472, 2002.
Wald, H. Regulation of the ROMK potassium channel in the kidney. Exp. Nephrol. 7(3):20-16, 1999.
Wang, W. Regulation of the ROMK channel: interaction of the ROMK with associate proteins. Am. J. Physiol.
277:F826-831, 1999.
Warila, J., S. Batterman and D.R. Passino-Reader, A probabilistic model for silver bioaccumulation in aquatic
systems and assessment of human health risks. Environ. Toxicol. Chem. 20(2):432-441, 2001.
9
Effect of H89, an inhibitor of protein kinase A, on chloride secretion by shark rectal gland
stimulated by VIP and by CNP
Franklin H. Epstein1, Chris Sighinolfi2, Katherine Hessler3, and Patricio Silva4
1
Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA 02215
2
University of Pennsylvania College of Arts and Sciences, Philadelphia, PA 19155
3
Bowdoin College, Brunswick, ME 04011
4
Department of Medicine, Temple University Hospital, Philadelphia, PA 19140
Secretion of chloride by the shark rectal gland is stimulated by vasoactive intestinal peptide
(VIP), a neurotransmitter released by rectal gland nerves, and by C-type natriuretic peptide (CNP) a
circulating hormone secreted by the heart in response to an increase in blood volume. Different
intracellular cascades are entrained by these two agonists. VIP activates adenylate cyclase, producing
cAMP, which activates protein kinase A (PKA), which is thought to phosphorylate the shark version of
the cystic fibrosis transmembrane regulator (CFTR) at the apical membrane of rectal gland cells,
thereby opening an apical chloride channel to permit chloride to pass into the duct. CNP, in addition to
stimulating the release of VIP from rectal gland nerves, has a direct action on rectal gland cells, which
is blocked by inhibitors of protein kinase C (PKC) and is mediated in part by the stimulation of
guanylate cyclase. This direct action of CNP, apparent even when neurotransmission and VIP release
are blocked (e.g., by perfusing with procaine), does not involve the stimulation of adenylate cyclase,2
but is prevented by a specific inhibitor of the CFTR chloride channel.1
The present experiments were designed to see if inhibition of PKA (a maneuver expected to
inhibit secretion stimulated by VIP) would affect as well chloride secretion stimulated by the direct
cellular action of CNP. The compound used to reduce PKA activity was H-89, an isoquinoline said to
exert a strong selective inhibitory action on PKA.3
Shark rectal glands were perfused as described by Silva et al.4 Duct fluid was collected at 10
minute intervals in small tared plastic centrifuge tubes and the volume was estimated by weighing.
The concentration of chloride was measured by amperometric titration using a Buchler-Cotlove
chloridometer. An initial 30-40 minutes of control perfusion allowed the gland to reach a stable basal
state. At the end of the control period, a bolus of 1 ml of shark Ringer’s solution was infused directly
into the rectal gland artery over one minute, containing an amount of VIP (Sigma) calculated to deliver
a final concentration of 10-7M to the gland, or of recombinant human CNP (Sigma) in an amount
calculated to deliver a final concentration of 5 x 10-7M. In the experiments with CNP, procaine 10-2M,
was added to the perfusate in order to present the release of VIP from rectal gland nerves.6 In the
experiments with H-89, this compound was added to the perfusate from the start of the experiment.
Preliminary perfusion with 25 micromolar H-89 inhibited the secretion of chloride induced
during the first 10 minutes after a one minute bolus injection of 10-7M VIP, from 1052 + 272 (n=10) to
366 + 104 mEq/g/hr (mean + s.e.). At the same concentration of H-89, CNP-induced secretion of
chloride (5 x 10-7M CNP given over one min. in the presence of 10-2M procaine) was almost
completely inhibited (780 + 152 to 60 + 8 mEq/g/hr.
These experiments in intact perfused rectal glands suggest that PKA activity is necessary for
direct stimulation of chloride secretion by CNP in the shark rectal gland. They support earlier findings
10
that H-89 (at a concentration of 10-6M) suppressed stimulation by CNP as well as by VIP in slices of
shark rectal gland.5 The results are compatible with the notion that a major action of CNP is to
sensitize the CFTR chloride channel to its activation by small amounts of VIP, cAMP, or activated
PKA.
1.
2.
3.
4.
5.
6.
Epstein, F.H., C. Sighinolfi, K. Hessler, A. Verkman, J. Riordan, P. Silva. A synthetic CFTR inhibitor blocks
stimulation of Squalus acanthias rectal gland secretion by CNP and VIP. Bull MDIBL 43, 13-14, 2004.
Gunning M., C. Cuero, R. Solomon, P. Silva. C-type natriuretic peptide receptors and signaling in rectal gland of
Squalus acanthias. Am J. Physiol 264:F300-F305, 1993.
Penn R.B., J.L. Parent, A.M. Pronin, R.A. Panetteri, J.L. Benovic. Pharmacologic inhibition of protein kinases in
intact cells. J Pharmacol Exp Ther 288:428-37, 1999.
Silva P., R.J. Solomon, F.H. Epstein. Shark rectal gland. In: Methods in Enzymology, vol 192. pp 754-766, 1990,
Academic Press.
Solomon R., L. Castelo, E. Franco, M. Taylor, P. Silva, F.H. Epstein. Preliminary data on intracellular signaling
mechanism in the rectal gland of Squalus acanthias: A pharmacologic approach. Bull MDIBL, 34: 47-48,1995.
Stoff J.S., P. Silva, R. Lechan, R. Solomon, F.H. Epstein. Neural control of shark rectal gland. Am J. Physiol
255:R212-R216, 1988.
1
11
Protein kinase C modulates the activation of the secretion of chloride mediated
by protein kinase A in the rectal gland of the shark, Squalus acanthias.
Patricio Silva,1 Katherine Hessler,2 Christopher Sighinolfi,3 Katherine Spokes,4 and Franklin H.
Epstein.4
1
Department of Medicine Temple University Hospital, Philadelphia, PA 19140.
2
Bowdoin College, Brunswick, ME 04011.
3
University of Pennsylvania, College of Arts and Sciences, Philadelphia, PA 19155.
4
Department of Medicine, Harvard Medical School and Beth Israel-Deaconess Medical Center, Boston,
MA 02215.
The secretion of chloride by the shark rectal gland is normally activated by the release of CNP
from the heart in response to volume stimuli.5 Circulating CNP causes the release of vasoactive
intestinal peptide (VIP) from vipaminergic nerves within the rectal gland and, jointly with it, activates
the secretion of chloride by the gland. These two peptides use different intracellular pathways to
activate the final common pathway, the shark version of cystic fibrosis transmembrane regulator
(CFTR).4 VIP activates adenylate cyclase with the production of cAMP and subsequent activation of
protein kinase A (PKA). The effect of CNP is mediated by protein kinase C but also requires the
activation of guanylate cyclase and the production of cGMP.4 The regulatory domain of CFTR has
binding sites for both PKA and PKC and it can be activated in vitro by either of these two kinases.2
Given that both these pathways share a common final pathway, CNP and VIP should be expected to
have an additive effect on chloride secretion by the gland. Indeed, Aller et al. showed that the secretion
of chloride induced by CNP can be potentiated by a small dose of VIP or forskolin.1 In the present
experiments we examined the nature of the relation between the VIP- and CNP-activated intracellular
pathways.
Shark rectal glands were perfused as previously described.3 Duct fluid was collected at 10-minute
intervals in tared plastic centrifuge tubes and the volume assessed by weight. The concentration of
chloride in the samples was measured by amperometric titration using a Buchler-Cotlove
chlorhidrometer. All glands were perfused with shark-Ringers containing in addition, glucose 5 mM,
and in the experiments with CNP, procaine 10-2M to prevent release of endogenous VIP from the rectal
gland nerves. Initial ten-minute control periods for the first 30-40 minutes of perfusion were collected
to allow the gland secretion to reach a steady state. At the end of the control period a continuous
infusion containing CNP, forskolin, dibutyryl cAMP, sildenafil, phorbol ester, or nitroprusside, alone
or in combination, were started, and the experiment continued for an additional sixty minutes. When
VIP was used it was either infused as a constant solution at a concentration of 10-9M or given as a
bolus in 1 ml of shark Ringer’s solution infused directly into the rectal gland artery over one minute
containing an amount calculated to deliver a final concentration of 10-7M or 10-9M to the gland.
Perfusion with CNP 5 x 10-9M enhanced the response to a bolus infusion of VIP 10-9M but had no
effect on that of VIP at 10-7M. Figure 1 shows the increment in the secretion of chloride above the
control level after the bolus infusion of VIP. A constant infusion of VIP 10-9M did not enhance the
effect of an additional bolus of VIP 10-9M. In fact, there was no additional response to the bolus
(baseline 179±55; increment after the infusion of VIP 599±148, p <0.01; increment after the additional
VIP bolus 108±86, not significant, n=6).
12
Figure 1. CNP potentiates the effect of VIP. In the glands
perfused with CNP, an infusion of CNP 5 x 10-9M was started
thirty minutes after the beginning of the perfusion. Thirty
minutes later a bolus of VIP, calculated to deliver a final
concentration of 10-9M, was injected directly into the artery
over the course of one minute. In the control experiments,
without CNP, the bolus of VIP was given thirty minutes after
the start of the perfusion. CNP increased the secretory
response of the gland seven-fold, control n=12, CNP n=7, p<
0.001. CNP had no enhancing effect when the concentration of
VIP was 10-7M, control n=20, CNP n=7, NS.
Increment chloride secretion (µEq/h/g)
2500.0
Control
CNP 5 x 10-9
2000.0
1500.0
1000.0
500.0
0.0
10-9
10-7
Concentration of VIP
An infusion of CNP 5 x 10-9M also enhanced the effect of dibutyryl cyclic AMP (dbcAMP), an
analog of the intracellular messenger for VIP. Figure 2 shows the effect on chloride secretion of
perfusion with CNP 5 x 10-9M, dibutyryl cAMP 5 x 10-4M, and the combination of both. The
combination of CNP and cAMP had a significantly greater effect than CNP alone and a much faster
effect than that of dbcAMP alone.
3000.0
Figure 2. CNP potentiates the effect of
dbCAMP. After a control period of thirty minutes,
glands were perfused with CNP 5 x 10-9M, with
dbCAMP 5 x 10-4M, or the combination of both.
CNP increased the secretion of chloride
approximately five times, n=12, p < 0.001. Dibutyryl
cAMP increased the secretion of chloride sixteen
times but the effect was delayed by about twenty
minutes, n=6, p < 0.001. The combination of CNP
and dbcAMP had an immediate effect that reached a
similar peak thirty minutes earlier than that of
dbcAMP alone, n=5, p < 0.001.
Chloride secretion (µEq/h/g)
CNP 5 x 10-9
dbcAMP 5 x 10-4
2500.0
CNP 5 x 10-5 and
dbcAMP 5 x 10-4
2000.0
1500.0
1000.0
500.0
0.0
1
2
3
4
5
6
7
8
9
Time (minutes)
CNP activates protein kinase G and C, therefore, we examined the effect of cGMP and phorbol
ester, activators of protein kinases G and C respectively on the stimulation induced by VIP. This
combination had no effect on the secretory response to VIP (baseline 100±29; increment after the
infusion of the combination of cGMP and phorbol ester, 399±91, p < 0.01; increment after the addition
of a bolus of VIP 214±118, not significant, n=5). To examine further the effect of activation of protein
kinase C and G on the effect of protein kinase A, the glands were perfused with a combination of
nitroprusside 2.5 x 10-6M, sildenafil 10-6M, to activate guanylate cyclase and inhibit
phosphosdiesterase V, with the purpose of activating protein kinase G; phorbol ester 10-6M, to activate
protein kinase C; and forskolin 10-7M to activate protein kinase A. Figure 3 shows that at a
concentration of 10-7M forskolin induced a modest but significant stimulation of the secretion of
chloride, n=7, p < 0.05. The combination of nitroprusside, sildenafil and phorbol ester enhanced the
secretion of chloride about as much as CNP alone, n=4, p < 0.025. The addition of forskolin to the
13
combination of nitroprusside, sildenafil and phorbol ester markedly increased the secretion of chloride,
n=4, p < 0.005.
2500.0
Figure 3. The combination of phorbol ester, nitroprusside
and sildenafil potentiates the effect of forskolin. After a
control period of thirty minutes, glands were perfused with
forskolin10-7M, with phorbol ester 10-6M, nitroprusside
2.5x10-6 M, and sildenafil 10-6M, or the combination of both.
Forskolin increased the secretion of chloride approximately 4
times, n=7, p < 0.05. The combination of phorbol ester,
nitroprusside, and sildenafil increased the secretion of chloride
ten-fold, n=4, p < 0.001. The combination of phorbol ester
plus nitroprusside and sildenafil and forskolin stimulated the
secretion of chloride nineteen times, n=4, p < 0.001.
Chloride secretion (µEq/h/g)
Forskolin 10-7M
Forskolin + PE/NP/S 10-6
2000.0
Phorbol ester + Nitroprusside
+ Sildenafil 10-6
1500.0
1000.0
500.0
0.0
10
20
30
40
50
60
70
80
90
Time (minutes)
In all of these experiments, activation of protein kinase A simultaneously with or after activation of
protein kinase C resulted in increases in the secretion of chloride beyond those expected of an additive
effect. CNP enhanced to response to VIP seven-fold. The combination of nitroprusside, sildenafil and
phorbol ester enhanced the effect of forskolin five times. The effect of CNP on that of dbCAMP was
more complicated; during the first thirty minutes of perfusion CNP increased the effect of dbcAMP
almost eight-fold, but after an hour of perfusion the effect of dbCAMP alone was the same as that with
CNP. These results suggest that protein kinase C modulates the activation of chloride transport
mediated by protein kinase A. This modulatory enhancement of the effect of protein kinase A by
activation of protein kinase C may explain the dual mechanism of activation of the secretion of
chloride by both CNP and VIP. These experiments also confirm previous experiments showing that
activation of protein kinase C requires the concurrent activation of guanylate cyclase and production of
cGMP.
1.
2.
3.
4.
5.
14
Aller, S., S. Wood, C. Aller, D. Opdyke, J.K. Forrest, M. Ratner, Kelley, and J.N. Forrest. C-type natriuretic
peptide acts synergistically with vasoactive intestinal peptide to increase both chloride transport and cyclic nucleotide
content in the rectal gland of Squalus acanthias. Bull. MDIBL 33:90-92, 1994.
Chen, Y., G.A. Altenberg, and L. Reuss. Mechanism of activation of xenopus CFTR by stimulation of PKC. Am. J.
Physiol. 287:C1256-63, 2004
Silva, P., R.J. Solomon, and F.H. Epstein. Shark rectal gland. Methods Enzymol 192: 754-766, 1990.
Silva, P., R.J. Solomon, F.H. Epstein. Mode of activation of salt secretion by C-type natriuretic peptide in the shark
rectal gland. Am J Physiol. 1999;277(6 Pt 2):R1725-32.
Solomon, R., A. Protter, G. McEnroe, J.G. Porter, and P. Silva. C-type natriuretic peptides stimulate chloride
secretion in the rectal gland of Squalus acanthias. Am J Physiol 262: R707-711, 1992.
Inhibitors of 4TM 2P potassium channels inhibit chloride secretion in the perfused rectal gland
of the spiny dogfish (Squalus acanthias)
Sarah Decker1, Catherine Kelley2 Eleanor Beltz3, Connor Telles1, Martha Ratner6, Kentrell Burks4,
Max Epstein6, Alexander Peters6, William Motley5, and John N. Forrest Jr1,6.
1
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510
2
Skidmore College, Saratoga Springs, NY 12866
3
Colby College, Waterville, ME 04901
4
Morehouse College, Atlanta, GA 30314
5
Middlebury College, Middlebury, VT 05753
6
Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
The shark rectal gland is a sodium chloride secretory organ comprised of polarized epithelial cells
that was first described by Wendell Burger2 over 40 years ago. The Na-K-ATPase pump and the NaK-2Cl cotransporter on the basolateral membrane are required to drive basolateral chloride entry and
basolateral recycling is required to maintain a favorable electrochemical gradient driving luminal
chloride exit through CFTR channels. In the perfused gland, when basolateral K+ exit is inhibited by
the non-specific K channel blocker barium, chloride secretion ceases5. By cloning studies we have
identified both a KIR 6.1 channel8 and a KVLQT channel6,7 in this tissue. However, in perfusion
studies inhibitors of these subtypes had no effect on chloride secretion suggesting that they are not the
dominant channels for basolateral K+ exit3. Only one K+ channel inhibitor, quinidine, had a significant
effect on chloride secretion3. Quinidine is an inhibitor of several families of K+ channels, including
rapid delayed rectifier K+ channels, the KA channel, cell-volume sensitive K+ channels, and the 4
transmembrane, 2 pore (4TM-2P) family of K+ channels.
+
We sought to further classify the basolateral K+ channel in
shark rectal gland epithelial cells by examining the effects of
other putative inhibitors of quinidine sensitive K+ channel
subtypes on maximally stimulated chloride secretion. These
effects were examined in in vitro perfused rectal glands and in
cultured monolayers of shark rectal gland epithelial cells.
Inhibitors tested were sotalol, a blocker of rapid delayed
rectifier K+ channels; TEA, and 3,4 DAP, blockers of the KA
channel; lidocaine, a blocker of cell volume sensitive K+
channels and 4TM, 4P K+ channels; and quinine, bupivicaine,
anandamide and acidic pH, also blockers of the 4TM, 2P
subtype. The effects of these inhibitors were compared to those
of BaCl2, a potent, non-specific K+ channel inhibitor.
Table 1. Effect of K channel inhibitors on
chloride secretion in in vitro perfusion studies
DRUG
Max
Dose
BaCl2
5 mM
Chromanol
200 !M
Tolbutamide
100 !M
Glybenclamide
1 !M
Clotrimazole
10!M
Charybdotoxin
50nM
Phrixotoxin
12 nM
Phentolamine
200!M
Quinidine
200 !M
Sotalol
100!M
TEA
10 !M
3,4 DAP
1 !M
Lidocaine
1 mM
Quinine
1 mM
Bupivicaine
2.5 mM
Anandamide
150 !M
Acidosis
PH 5
0
no inhibition
+
0-20 % inhibition
++
20-40% inhibition
+++ 40-80% inhibition
++++ 80-100% inhibition
N = Inhibition of
Cl- Secretion
6
++++
6
+
15
0
4
0
3
0
3
0
1
0
4
0
6
++++
2
0
8
0
9
0
6
++
6
+++
6
++++
9
+++
6
++++
Freshly excised rectal glands were perfused in vitro using methods previously described4. Glands
were perfused to basal levels with shark Ringer’s only, and then chloride secretion was activated by
continuous perfusion of forskolin (1µM) and IBMX (100µM) from t=30 min to the end of the
experiment. Individual K+ channel blockers were perfused continuously from t=50 to t=70 min.
Perfusate concentrations of these blockers were chosen based on known Ki values. Rectal gland
tubular cells were cultured and grown on collagen coated nylon membranes and Cl- secretion measured
as Isc in intact monolayers as described previously1.
15
Whereas Ba2+ completely blocked Cl- secretion in the perfused gland, inhibitors of calcium
sensitive, delayed rectifier, and ATP sensitive K+ channels (chromanol, tolbutamide, glybenclamide,
clotrimazole, charybdotoxin, phrixotoxin, phentolamine) had no effect on secretion. Likewise, the K+
channel inhibitors sotalol, TEA, and 3,4 DAP had no effect on stimulated chloride secretion (Table 1).
In contrast, the potassium channel blockers quinine and bupivicaine dramatically inhibited chloride
secretion. Acidic perfusate also resulted in dramatic inhibition of Cl- secretion. Addition of the K+
channel blockers anandamide and lidocaine inhibited chloride secretion to a lesser extent (Table 1).
The effects of quinine, bupivicaine, acidic pH, and lidocaine were reversible. Figure 1 depicts the time
course for inhibition and reversal by barium and acidic pH. Both barium and acidic pH (6.0) abruptly
decrease Cl- secretion in the perfused gland with a similar time course. With both, the half maximal
effect is seen within 1-2 min, maximal inhibition is 80-90% and the effects are promptly and
completely reversed within minutes.
Figure 1. A) Effects of BaCl2 (5mM) on forskolin + IBMX stimulated Cl- secretion (n=6 barium, n= 10 controls. B) Effects of
acidic pH on forskolin + IBMX stimulated Cl- secretion (n= 23 pH=7.6, n=5 pH=6.4, n=6 pH=6.0).
The effects of quinine were further examined in cultured SRG monolayers (Figure 2). The
inhibitory effect was more potent when added to the basolateral solution versus the apical solution.
Inhibition of Isc was reversible following washout of the inhibitor (Figure 2, Panels A and B).
Figure 2. Effects of increasing concentrations of quinine added apically (Panel A) on Isc of SRG epithelial cells, representative of 5
16
experiments compared to quinine added basolaterally (Panel B) on Isc of SRG epithelial cells, representative of 9
experiments.
Previous studies by this laboratory have suggested that Ca++ sensitive K+ channels, voltage
sensitive K+ channels, and ATP sensitive K+ channels are not the dominant subtypes regulating
chloride secretion3. The lack of inhibition by sotalol, TEA, and 3,4 DAP suggests that rapid delayed
rectifier K+ channels, and KA channels also do not play important roles in the regulation of chloride
secretion.
The inhibition of chloride secretion by the K+ channel blockers quinidine3, quinine, bupivicaine,
anandamide, and lidocaine, as well as by acidic perfusate, all of which are inhibitors of the 4TM, 2P
subtype of potassium channels, suggests that this family of K+ channels is dominant in the regulation
of chloride secretion. Specifically, each of these inhibitors has effects on the TASK subfamily of 4TM
2P channels. We propose that a TASK channel is present on the basolateral membrane of the shark
rectal gland cell, and that this channel plays a dominant role in the regulation of chloride secretion.
This work was supported by NIH grants DK 34208, NIEHS 5 P30 ES03828 (Center for Membrane
Toxicity Studies), and NSF grant DBI-0139190 (REU site at MDIBL).
1.
2.
3.
4.
5.
6.
7.
8.
Aller, S.G., I.D. Lombardo, S. Bhanot S. JN Forrest Jr. Cloning, characterization, and functional expression of a
CNP receptor regulating CFTR in the shark rectal gland. Am. J. Physiol. 276:C442-9, 1999.
Burger, W. Function of the rectal gland of the spiny dogfish. Science 131:670-671, 1960.
Decker, S, C. Klein, M. Ratner, C. Kelley, M. Epstein, K. Burks, W. Motley, A. Peters, and J.N. Forrest Jr.
Effects of quinidine and other K+ channel inhibitors on chloride secretion in the rectal gland of the spiny dogfish,
Squalus acanthias. Bull. Mt Desert Isl. Biol. Lab. 43: 30-32, 2004.
Kelley, G.G., E.M. Poeschla, H.V. Barron, J.N. Forrest Jr. A1 adenosine receptors inhibit chloride transport in the
shark rectal gland. Dissociation of inhibition and cyclic AMP. J. Clin. Invest. 85 (5): 1629-36,1990.
Silva P, Epstein JA, Myers MA, Stevens A, Silva P Jr, Epstein FH. Inhibition of chloride secretion by BaCl2 in the
rectal gland of the spiny dogfish, Squalus acanthias. Life Science, 38(6):547-52, 1986.
Waldegger, S., M. Bleich, P. Barth, J. N. Forrest Jr.,, F. Lang, R. Greger. Cloning and expression of the KvLQT
potassium channel from rectal gland of Squalus acanthias Bull. Mt Desert Isl. Biol. Lab. 38: 30-31, 1999.
Waldegger S., B. Fakler, M. Bleich, P. Barth, A. Hopf, U. Schulte, A.E, Busch, S.G. Aller, J.N. Forrest Jr. R.
Greger, F. Lang. Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of
Squalus acanthias. Pflugers Archiv – Eur. J. of Physiol. 437(2):298-304, 1999.
Weber, G., S. G. Aller, F. N. Plesch, and J.N. Forrest, Jr. Identification and partial seqeuencing of a KIR 6.1
potassium channel from the shark rectal gland and a ROM-K potassium channel from skate kidney. Bull. Mt Desert Isl.
Biol. Lab. 38: 112-113, 1999.
17
Differential Expression of CFTR and three G protein coupled receptors
in tissues of Squalus acanthias
1
Ali Poyan Mehr1, William Motley2, Diana Swett1, Sarah Decker1, and John N. Forrest, Jr.1,3
2
3
In the shark rectal gland, chloride secretion through CFTR is regulated by several G protein
coupled receptors, including the vasoactive intestinal peptide receptor (VIP-R), the A0 adenosine
receptor, and the growth hormone releasing hormone (GHRH) receptor. The shark CFTR chloride
channel was first sequenced by Marshall et al.4 and has been extensively studied by patch clamp2 and
oocyte expression studies1,3. Our laboratory has cloned and sequenced the A0 adenosine receptor, the
VIP-R and GHRH-R in this tissue6,7. Little is known about the expression of these proteins in other
shark tissues. In the present studies, we used shark specific primers in quantitative PCR to examine the
level of mRNA expression of these membrane proteins in 11 shark tissues.
To compare the tissue expression levels of these membrane proteins in tissues of Squalus acanthias
we applied the Stratagene Brilliant SYBR Green QPCR System using the Stratagene MX4000 RealTime PCR instrument at MDIBL. Total RNA was extracted from tissues using Trizol reagent
(Invitrogen). Prior to cDNA synthesis, RNA samples were DNase digested to avoid contamination
with genomic DNA (Ambion). RNA quality was analyzed using a Agilent 2100 Bioanalyzer. A total of
3µg RNA was reverse transcribed using SuperScript First-Strand cDNA Synthesis system (Invitrogen).
1µl of cDNA was amplified with primers designed to produce an amplification of approximately
300bp.
CFTR sense: TCTCTGCCTTGGACGAATAATAGC
CFTRantisense: CACTGCCACGCCCTCATCA
VIPR sense primer: GTCCTGAGGGCCATCGCTGTCTT
VIPR antisense primer: GGGCCCGAATGATCCACCAATAC
A0 adenosine receptor sense: AGCCGAGCGCCACATCAACATCAG
A0 adenosine receptor antisense: CGGGGTACGGGAGGCAGGAGAC
GHRHR sense primer: ACAGAGCAAGGGTGGAGTGAAT
GHRHR antisense primer: TGGCGCGGAGAATGAAGGAC
beta actin sense primer: CTGGCATTGTGCTAGATTCTGGTG
beta actin antisense primer: AAGAGCTAGCCGTCTGCATCTCAG
Primer specificity was tested by conventional PCR and all primer pairs yielded a single band.
Samples were prepared in triplicate and relative expression levels were calculated using the
comparative threshold cycle (Ct) method. By subtracting the average beta-actin Ct value from the
average target gene Ct-value, the expression levels of the target gene were normalized to the
expression level of the reference gene (beta-actin). We picked one tissue from one shark as the
standard and set its expression level as one. Expression levels in other tissues and sharks (n=6) were
set in relation to this standard. The expression level was calculated using the following equation:
2 - (target gene[sample] Ct – Actin[sample] Ct) - (target gene[standard])
Ct – Actin[standard] Ct)
18
A
1.40
1.20
Relative Expression
1.00
0.80
0.60
0.40
0.20
0.00
RG
KID
SPL
BRAIN
INT
LIV
STOM
MUSC
GILL
GON
HEART
Tissue
B
12.00
10.00
Relative Expression
8.00
6.00
4.00
2.00
0.00
RG
KID
SPL
BRAIN
INT
LIV
STOM
MUSC
GILL
GON
HEART
Tissue
Figure1. Relative expression of CFTR (Panel A) AND VIP-R (Panel B) in shark tissues. Error bars indicate SE, N=6.
Figure 1 (Panel A) shows the relative expression of CFTR in 11 shark tissues. The highest level of
expression was in rectal gland, followed by intestine and gonads. CFTR is well established as the
pathway for chloride exit in rectal gland cells. Modest levels were seen in brain and spleen. There was
19
minimal expression in kidney, liver, stomach and heart. Expression of VIP-R was also highest in
rectal gland, followed by intestine, stomach and brain. There were low levels of expression in kidney,
liver, gill and gonad.
2
1.8
1.6
relative expression
1.4
1.2
1
0.8
0.6
0.4
0.2
0
RG
KID
SPL
BRAIN
INT
LIV
STOM
MUSC
GILL
GON
HEART
tissue
Figure 2. Relative expression of A0 adenosine receptor in 11 shark tissues. Error bars indicate SE.
The A0 adenosine receptor is known to be highly expressed in shark rectal gland. Unexpectedly, we
also observed high levels of mRNA expression in shark stomach, with low levels of expression in
other tissues (Figure 2).
18.00
16.00
14.00
Relative Expression
12.00
10.00
8.00
6.00
4.00
2.00
0.00
RG
KID
SPL
BRAIN
INT
LIV
STOM
MUSC
GILL
GON
HEART
Tissue
Figure 3. Relative expression of GHRH-R in 11 shark tissues. Error bars indicate SE, N=6.
The results show very high expression of GHRHR in heart, which is 40 times higher than brain and
14 times higher than rectal gland. This result is consistent with human tissue, where myocardium
20
shows the largest number of ligand binding sites for growth hormone peptides5. The next highest level
of expression was seen in rectal gland (Figure 3).
These studies provide the first measurements of mRNA expression of CFTR, VIP-R, A0, and
GHRH-R in multiple shark tissues. High expression levels of these membrane proteins are consistent
with their known function in mediating NaCl secretion in the rectal gland. Their high expression
levels in other tissues, particularly epithelia, brain and heart, suggest likely physiological roles in these
tissues.
1. Aller S.G., I.D. Lombardo, S. Bhanot, and J.N. Forrest Jr. Cloning, characterization, and functional expression of a
CNP receptor regulating CFTR in the shark rectal gland. Am. J. Physiol. 276: C442-9, 1999.
2. Devor D.C., J.N. Forrest, Jr., W.K. Suggs, R.A. Frizzell. cAMP-activated Cl- channels in primary cultures of spiny
dogfish (Squalus acanthias) rectal gland. Am. J. Physiol. 268(1Pt1): C70-9, 1995.
3. Lehrich RW, S.G. Aller, P Webster, C.R. Marino, and J.N Forrest Jr. Vasoactive Intestinal Peptide Forskolin and
Genistein Increase Apical CFTR Trafficking in the Rectal Gland of the Spiny Dogfish, Squalus acanthias: Acute
Regulation of CFTR Trafficking in an Intact Epithelium. J Clin Invest, 101:737-745, 1998.
4. Marshall, J., K.A. Martin, M. Picciotto, S. Hockfield, A.C. Nairn, L.K. Kaczmarek. Identification and localization
of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 266(33): 2274954, 1991.
5. Papotti, M., C. Ghe, P. Cassoni, F. Catapano, R. Deghenghi, E. Ghigo, G. Muccioli. Growth hormone secretagogue
binding sites in peripheral human tissues. Journal of Clinical Endocrinology and Metabolism. 85(10):3803-7, 2000.
6. Pena, J.T., H. Seeger, S.G. Aller, J.N. Forrest Jr. Cloning and sequence analysis of a VIP receptor expressed in the
rectal gland of the spiny dogfish, Squalus acanthias. Bull. Mt Desert Isl. Biol. Lab. 38: 116-117, 1999.
7. Seeger, H, S.G. Aller, P. Burrage, and J.N. Forrest Jr. Cloning of a GHRH-like Receptor From the Shark Rectal
Gland and Functional Expression in Xenopus laevis oocytes. Bull. Mt Desert Isl. Biol. Lab, 40:119-121, 2001.
21
The Effect of Isoproterenol and Nifedipine on Electrically Stimulated Cai-transients in
Ventricular Myocytes of Squalus acanthias
Steve Belmonte, Jane Wang, and Martin Morad
Department of Pharmacology
Georgetown University, Washington, DC 20057
Mammalian cardiac myocytes contract via a process called Ca2+-induced Ca2+ release (CICR), in
which ICa through voltage gated Ca2+channels causes the opening of Ca2+ release channels (RyR) on
the sarcoplasmic reticulum 5. There is little functional or ultrastructural evidence, however, for such
Ca2+ release stores in shark heart 3. Instead, Ca2+ influx through L-type Ca2+ channels and Na+/Ca2+
exchanger (NCX) directly causes contraction, while the removal of Ca2+ from the cell on NCX results
in relaxation. It has been shown that β-adrenergic agonists augment the force of contraction by
increasing ICa (secondary to phosphorylation of Ca2+ channels) and enhance relaxation through
phosphorylation of phospholamban and decreased myofilament sensitivity 4. Here, we have
investigated the effects of isoproterenol and nifedipine on Cai-transients in single, intact ventricular
myocytes from dogfish sharks (Squalus acanthias).
1.0
2+
F340/F410
10
*
15
*
20
5µ M Isoproterenol Bath
1.0
0.5
*
5
*
5
*
*
10
400
350
300
250
200
*
15
Time(s)
% ∆ in Cai- transients
grey = Isoproterenol
black = Control
0.5
*
C
B
Control 5mM Ca Bath
white = control
grey = 5µM Isoproterenol
*
20
*
.75
F340/F410
F340/F410
A
.50
1
2
3
4
5
Time(s)
Figure 1. Comparison of electrically stimulated (*) Cai-transients in
control vs. 5μM isoproterenol (Panel A). Magnified trace from panel A
showing differential kinetics of control and isoproterenol Caitransients (Panel B). Quantification of enhancement of rate of rise
(ΔF/rise time), magnitude (ΔF), and rate of decay (ΔF/relax half time)
with isoproterenol treatment in paired cells (n =5).
150
Ventricular myocytes were isolated from hearts of
dogfish. Following complete spinal pithing, hearts were
50
excised
and placed in Ringer solution containing (in mM)
0
270 NaCl, 6 K+-glutamate, 5 CaCl2, 10 glucose, 10
Rate of Rise
Magnitude
Rate of Decay
MgCl2, 10 HEPES, 350 Urea, 0.5 KH2PO4, 0.5 Na2SO4, at
pH 7.4 and refrigerated at 2-8 °C until ready for use. For cell isolation, the heart was mounted on a
Langendorff apparatus and the aorta and two major coronary vessels were perfused with Ringer
solution containing zero CaCl2 for 15 min, with zero CaCl2 Ringer plus 1 mg/ml Collagenase A
(Roche) and 0.2 mg/ml Protease type XIV (Sigma) for 15 min, and finally with 0.2 mM CaCl2 Ringer
solution for 10 min to washout the enzyme. The ventricle was cut off and gently agitated to dissociate
ventricular myocytes. Cells were then plated on glass coverslips and loaded with ratiometric Ca2+
fluorescent dye Fura-2 AM (Molecular Probes). Cai-transients were measured using alternating 340
22
100
and 410 nm excitation waves at 1.2 kHz to monitor the fluorescence of Ca2+-bound and Ca2+-free Fura2 respectively 2.
To confirm the absence of intracellular Ca2+ release pools, an electronically controlled microbarrel
“puffer” system was used to apply a solution of 10 mM caffeine upon well-attached ventricular
myocytes. In agreement with the previously published data 3, we found no evidence for intracellular
Ca2+ release upon caffeine treatment (n=10). Interestingly, the puffing of bathing solution onto these
myocytes failed to elicit the shear stress-activated Cai-transients so prevalent in rat atrial myocytes 1.
To probe possible effects of isoproterenol on the shark NCX, we also investigated the effect of
isoproterenol on electrically stimulated Cai-transients. Figure 1 (upper trace, Panel A) clearly shows
that depolarization causes a rapid [Ca2+]i rise followed by a slower relaxation as Ca2+ is removed from
the cell. After 2 minutes of exposure to isoproterenol (5 μM), the magnitude as well as rate of rise and
relaxation of Cai-transients were significantly increased (Panel C). This finding is consistent with the
idea that Ca2+ channel and NCX phosphorylation may be critically involved in enhancement of Caitransients and acceleration of its relaxation respectively.
Figure 2. Nifedipine
A 1.5 1µΜ Nifedipine
1µΜ Nifedipine
reversibly reduces the
B
black = Control (5µΜ Iso.) magnitude of electrically
0.5
F340 /F 410
1.5
*
5
*
10
*
*
15
20
(continued from above)1µΜ Nifedipine
1.0
0.5
1.5
*
*
*
*
*
*
5
10
1µΜ Nifedipine Washout
15
*
20
1.0
0.5
5
120
C
% ∆ in Cai- transients
100
80
*
F340 /F 410
1.0
stimulated
(*)
Caitransients
compared
to
5
1.0
μM isoproterenol (Panel
A). Panel B is a magnified
trace from panel A
showing
reduced
0.5
4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2
magnitude and relaxation
rate in 1 μM nifedipine.
Time(s)
Panel
C
shows
quantification of nifedipine effect on rate of rise (ΔF/rise time), magnitude
(ΔF), and rate of decay (ΔF/relax half time) in paired cells (n =3).
To probe whether Ca2+ channel blocker nifedipine could
reverse the effects of isoproterenol, we exposed the
Time(s)
white = 5 µM Iso.
myocytes to 1 μM nifedipine. Figure 2, panels A and
grey = 5 µM Iso. + 1 µM Nifedipine
B show that nifedipine reversibly reduces the
magnitude of Cai-transients while also reducing the
rate of relaxation (Panel C). The rate of rise, however,
is not significantly altered by nifedipine (n=3).
10
15
20
60
These findings provide support for the lack of
functional
intracellular Ca2+ stores in shark myocytes
40
and secondarily suggest that the presence of
functional Ca2+ release stores is critical for activation
20
of shear-stress responses in rat atrium. Even though
isoproterenol enhanced the rates of rise and relaxation
0
Rate of Rise
Magnitude
Rate of Decay
of Cai-transients, the selective suppression of
isoproterenol-induced rate of decay by nifedipine, but not the rate of rise of Cai-transients was
unexpected and requires further experimentation. It has been suggested that isoproterenol enhances the
magnitude and the rate of relaxation of Cai-transients via phosphorylation of putative PKA site on the
NCX 6, although our data show that suppression of peak Cai-transients also plays a critical role in
regulating relaxation rate of Cai-transients.
23
This work was supported by NIH grant RO1 15652
24
1.
Belmonte, S and M. Morad. Probing the signaling cascade mediating puff-triggered Ca2+ transients in rat atrial
myocytes (Abstract). Biophysical Society. 2005.
2.
Cleemann, L. and M. Morad. Role of Ca2+ channel in cardiac excitation-contraction coupling in the rat: evidence
from Ca2+ transients and contraction. Journal of Physiology. 432: 283-312, 1991.
3.
Näbauer, M. and M. Morad. Modulation of contraction by intracellular Na+ via Na+-Ca2+ exchange in single shark
(Squalus acanthias) ventricular myocytes. Journal of Physiology. 457:627-37, 1992.
4.
Sham, J.S., Jones, L.R., and M. Morad. Phospholamban mediates the beta-adrenergic-enhanced Ca2+ uptake in
mammalian ventricular myocytes. American Journal of Physiology. 261: 1344-1349, 1991.
5.
Woo, S.H., Cleemann, L., and M. Morad. Ca2+ current-gated focal and local Ca2+ release in rat atrial myocytes:
evidence from rapid 2-D confocal imaging. Journal of Physiology. 543.2: 439-453, 2002.
6.
Woo, S.H. and M. Morad. Bimodal regulation of Na+-Ca2+ exchanger by β-adrenergic signaling pathway in shark
ventricular myocytes. PNAS. 98: 2023-2028, 2001.
Immunodetection of activation state of human and shark CFTR
April Mengos, J.D. Campbell, Tamas Hegedus, Tim Jensen and John R. Riordan
Mayo Clinic College of Medicine, Scottsdale, AZ 85268
CFTR plays a primary role in the regulation of chloride secretion in epithelial tissues of marine and
terrestrial vertebrates by responding to protein kinase A (PKA) mediated phosphorylation of its Rdomain. The PKA activation is due to cyclic AMP elevation on binding on extracellular secretory
agonists. In the absence of these secretory stimuli CFTR is maintained in a quiescent non-gating state
by phosphatases that very efficiently remove phosphoryl groups added by PKA. The mechanism by
which the phosphorylation of multiple (~ 10) R-domain sites permit channel gating on binding of the
ATP ligand to the nucleotide binding domains (NBDs) is not known but does involve conformation
changes at the level of the R-domain1 and the whole protein2. This conformation change can be readily
detected by a significant retardation of the mobility of the mature shark or human proteins in SDSPAGE. This mobility shift provides facile monitoring of the activation state of CFTR in isolated
membranes, whole cells or intact tissue such as shark rectal gland. In the case of the human protein we
have developed phosphorylation-sensitive monoclonal antibodies (mAbs) that can detect three of the
major PKA phosphorylation sites in the R-domain individually (Figure 1). The use of these reagents in
combination with the gel mobility shift enables detection of the reversible activation (phosphorylation)
and inactivation (dephosphorylation) of the CFTR channel. In addition to this simple diagnostic test of
activation state these methods begin to provide mechanistic insights. The kinetics of phosphorylation
of the three separate sites, that we can follow, reveal that they are occupied in an ordered fashion rather
than simultaneously and provide the basis for distinguishing between a processive and a distributive
mechanism. (Supported by the NIH).
1.
Dulhanty AM and Riordan JR. Phosphorylation by cAMP-dependent protein kinase causes a conformational change
in the R domain of the cystic fibrosis transmembrane conductance regulator. Biochemistry 33: 4072-4079, 1994.
2.
Grimard V, Li C, Ramjeesingh M, Bear CE, Goormaghtigh E, and Ruysschaert JM. Phosphorylation-induced
conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total
reflection-Fourier transform IR spectroscopy and fluorescence spectroscopy. J Biol Chem 279: 5528-5536, 2004.
25
Measurements of local pH-transients in K+-depolarized PC12 cells
Lars Cleemann, Kristian Errebo Krantz, Esben Vedel-Larsen, and Martin Morad
Georgetown University, 3900 Reservoir Road, NW, Washington, District of Columbia 20057, USA
Recently we reported that rapid (~20 ms) acidification of the cloned “slow” rat α3β4 nicotinic
receptors enhances the agonist-activated current and accelerates its gating kinetics by increasing the
affinity of the receptor to agonists 1. This is a novel and distinctly different effect than the
acidification-induced suppression of muscle nicotinic receptors and neuronal N-methyl-D-aspartate
receptor and raises the possibility that that brief transient acidification or alkalization of the synaptic
cleft may modulate postsynaptic receptors thereby providing plasticity to synaptic signaling. In support
of synaptic acidification it has been found that: 1) postsynaptic potentials may be accompanied by brief
(10 ms) acidic shifts 5,6; 2) protons, stored in the vesicles 7 and co-released with transmitters, can
modulate presynaptic Ca2+ channels 2,4; and 3) buffering capacity of synaptic cleft is limited during
brief synaptic events 10. On the other hand, it has also been found that the initial acidification is
followed by longer-lasting alkalization 6.
Here we describe experiments undertaken to directly measure local pH transients associated single
exocytotic events. It is our goal to measure such pH transients within synaptic clefs, but as a first step
in this direction, we decided to measure pH transients in the confined space between catecholaminesecreting PC12 cells (a line of rat pheochromacytoma cells) and the underlying glass substrate on
which they are cultured. In this configuration we compared confocal and total internal reflectance
fluorescence (TIRF) microscopy in conjunction with water-soluble (Snarf 4F, S23920 from Molecular
Probes) and lipophilic (Fluorescein DHPE, F362 from Molecular Probes) fluorescent pH-indicator
dyes. The dual objective was to improve the sensitivity of our pH-imaging techniques and to obtain
preliminary recordings of local pH
transients.
To improve signal-to-noise ratio (S/N),
we have purchased a new CCD camera
(iXon DV860 from Andor Technology,
Northern Ireland), and added a green diode
laser (λex = 534 nm) to our Argon ion laser
(λex = 364, 457, 488, 514 nm). The new
CCD camera (128 x 128 pixels) is
exquisitely sensitive due to a novel CCD
chip (E2V 60 from e2v Technologies, UK)
with high quantum efficiency (~95%, back
thinned design) and low readout noise (onchip electron multiplication, Peltier cooling
to –90 oC). With flexible binning, the frame
rate can be increased from 450 per second
for full frames (128 x 128 pixels), to well
over 1 kHz.
Figure 1. pH-sparks in a cultured PC-12 cell depolarized with K+-rich solution. Panel A shows the average TIRF signal
measured in 1000 frames over a period of 10.5 sec. The cell is ~20 µm in diameter. pH-sparks were observed within four
regions (Panel, C: blue, green, orange and red) of the upper right hand quadrant (Panel B). The fluorescence intensity in
these areas increased and declined again as indicated by the curves in panel H. Panels D to G show sample frames with
background subtraction where each of the four pH-sparks appear red on a green background (Arrows). The used color scale
is shown as an inset.
26
TIRF experiments with cultured catecholamine-secreting PC-12 cells stained with F-362,
demonstrate the typical patters of adhesion (Fig.1A). With optimal staining the camera gives excellent
S/N at frame/rates as high as 1200 Hz. Most intriguingly, when activated by depolarizing K+-rich
solution pulses, the PC12 cells produced focal pH-transients lasting ~200 ms (Fig. 1H). Surprisingly,
our findings suggest that the “spark-like” increases in fluorescence reflect transient alkalization. The
confocal measurements in Fig. 2A, demonstrate the F-362 dye responds as expected to imposed pHchanges both at the cell surface and in the “cleft-space” between the cells. In particular, they verify that
alkaline
solution
increases
fluorescence intensity as observed
for the pH-transients in Fig. 1.
Attempts to measure paracellular
pH with a water-soluble pHindicator (SNARF 4F) were not
encouraging (Fig. 2B) since
fluorescence from dye within the
narrow spaces between (and under)
cells was swamped by the
fluorescence from the bulk of the
solution.
Figure 2. Comparison of confocal measurements with lipophilic (Panel A, F-362) and water-soluble (Panel B, SNARF 4F)
dyes. Panel A: Measurement of interstitial pH measured between 2 PC12 cells stained with F362 and exposed to test
solutions with pH 7.4 (control), 8.5, 6, and 7.4 (sample frames at top corresponding to *s in graph). The curves and regions
of interest correspond the exposed membrane surface (green) and membranes at different depths (red and blue) within the
cleft between the cells. Notice, that on return to control solution (at time ~7s), the pH-response in the cleft lags ~1s after
that at the surface. Panel B: Fluorescence from SNARF 4F distributed around, and possibly between (?) a string of adhering
PC-12 cells .
Application of K+-rich depolarizing solution to PC-12 cells stained by F-362 produced clear TIRF
images where transient focal fluorescence signals (Fig. 1) were superimposed on the steady state image
(Fig. 1 AB). The focal signals were seen roughly along a line, were stronger in areas of adhesion than
in between (Fig. 1 trace/panel D vs. E) and lasted ~200 ms, so that they were clearly seen in multiple
frames when recording at 95 frames per second (Fig. 3). These focal fluorescence signals may
correspond to pH-transients produced by exocytosis of single secretory vesicles. Detailed analysis of
pH-sparks in consecutive frames, was performed with a computerized algorithm that determined a
Gaussian approximation and quantified the amplitude, center and full-width at half amplitude (FWHA)
as well as the “size” of the pH-spark, i.e. its overall fluorescence (proportional to amplitude times
FWHA2, 3). From the first appearance of the pH-spark, the size increased over a period of ~50 ms, and
then declined slowly in the following period of ~ 200 ms. FWHA was remarkably constant at ~ 2µm
compared the slowly spreading Ca-sparks observed e.g. in atrial cells 11. This may reflect that the
membrane-bound pH-dye, unlike the dyes generally used for measurements of Ca-sparks, are not
freely diffusible in paracellular spaces.
The main finding of these experiments is that TIRF microscopy used in conjunction with a lipohilic
fluorescent pH-indicator dye is capable of measuring highly localized fluorescence transients that may
represent alkalization associated with exocytosis of single secretory vesicles. The alkalization is
surprising considering the acidic contents of secretory vesicles. To substantiate the present findings, it
would be useful to perform more extensive experiments with a ratiometric lipophilic pH-dye in order
to ascertain that the action spectrum of the local pH-transients display the same wavelengthdependence as observed when the cell are exposed to test solutions with different pH. Similarly,
higher frame rates should be used to test if the observed alkalization is preceded by brief acidification
27
(Cf. 6). It should also be noticed that the local pH-transients produced by exocytosis may be expected
to depend not only on the excess of protons within the secretory vesicles, but also on their contents of
pH-buffers and proton transporters. In fact at pH 5, it may be calculated that a secretory vesiscle
contains only about one free proton, but has ~10,000 protons and transmitter molecules that are
possibly bound to proteoglycans, the ion-exchange properties of which, remain to be fully explored 8.
Thus it is possible that both the magnitude and polarity of local pH-transients may depend on proton
fluxes generated by a number of proton-binding and –transporting molecules. Furthermore, it has been
proposed that the exocytotic process does not always run rapidly to completion, but that the fusion
pore only briefly opens and then closes again (“kiss-and-run” model9). Considering the sensitivity of
the TIRF technique, as demonstrated in the present results, it is likely that this technique may help to
elucidate these processes and their role in controlling pH within synaptic clefts. Supported by NIH
RO1 HL 16152 and R21 EB003473.
Figure 3. Consecutive
frames
showing
the
development and decline
of one of the pH-sparks
(Spark D, blue in Fig. 1.).
The graph shows the time
course of the size (overall
fluorescence
intensity)
full-width
at
half
maximum
(FWHA)
determined with same
computer program used
routinely for analysis of
Ca2+-sparks 3.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
28
Abdrakhmanova G., Dorfman J., Xiao Y., and Morad M. Protons enhance the gating kinetics of the alpha3/beta4
neuronal nicotinic acetylcholine receptor by increasing its apparent affinity to agonists. Mol Pharmacol. 61, 369-378,
2002.
Callewaert G., Johnson R. G., and Morad M. Regulation of the secretory response in bovine chromaffin cells. Am J
Physiol. 260, C851-860, 1991.
Cleemann L., Wang W., and Morad M. Two-dimensional confocal images of organization, density, and gating of
focal Ca2+ release sites in rat cardiac myocytes. Proc Natl Acad Sci U S A. 95, 10984-10989, 1998.
DeVries S. H. Exocytosed protons feedback to suppress the Ca2+ current in mammalian cone photoreceptors.
Neuron. 32, 1107-1117, 2001.
Kosmachev A. B., Beliaev V. A., Khrabrova A. V., Libman N. M., and Dolgo-Saburov V. B. [Effect of M3choline receptor blockade on the ability of muscarinic antagonists to prevent catalepsy induced by haloperidol in rats].
Eksp Klin Farmakol. 64, 10-12, 2001.
Krishtal O. A., Osipchuk Y. V., Shelest T. N., and Smirnoff S. V. Rapid extracellular pH transients related to
synaptic transmission in rat hippocampal slices. Brain Res. 436, 352-356, 1987.
Miesenbock G., De Angelis D. A., and Rothman J. E. Visualizing secretion and synaptic transmission with pHsensitive green fluorescent proteins. Nature. 394, 192-195, 1998.
Reigada D., Diez-Perez I., Gorostiza P., Verdaguer A., Gomez de Aranda I., Pineda O., Vilarrasa J., Marsal J.,
Blasi J., Aleu J., and Solsona C. Control of neurotransmitter release by an internal gel matrix in synaptic vesicles.
Proc Natl Acad Sci U S A. 100, 3485-3490, 2003.
Schneider S. W. Kiss and run mechanism in exocytosis. J Membr Biol. 181, 67-76, 2001.
Tong C. K., Brion L. P., Suarez C., and Chesler M. Interstitial carbonic anhydrase (CA) activity in brain is
attributable to membrane-bound CA type IV. J Neurosci. 20, 8247-8253, 2000.
Woo S. H., Cleemann L., and Morad M. Spatiotemporal characteristics of junctional and nonjunctional focal Ca2+
release in rat atrial myocytes. Circ Res. 92, e1-11, 2003.
Endothelin-1, superoxide and adeninediphosphate ribose in vascular smooth muscle of Squalus
acanthias
1
Susan K. Fellner 1,2 & Laurel A. Parker 2
Department of Cell & Molecular Physiology
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599-7545
2
Mount Desert Island Biological laboratory
Salisbury Cove, ME 04672
In vascular smooth muscle (VSM) of both mammals and Squalus acanthias, endothelin-1 (ET-1)
causes an increase in cytosolic Ca2+ ([Ca2+]i) via activation of the inositol trisphosphate receptor (IP3R)
and release of Ca2+ from the sarcoplasmic reticulum (SR). We have previously presented evidence that
calcium-induced calcium release (CICR), operating via the ryanodine receptor (RyR) and stimulated
by cyclic adenine diphosphate ribose (cADPR), occur in VSM of the anterior mesenteric artery of S.
acanthias2. We asked two questions: does inhibition of the ADPR cyclase diminish the [Ca2+]i
response to ET-1 and what is the mechanism by which ET-1 may activate the ADPR cyclase?
The anterior mesenteric artery of the dogfish shark was dissected and placed in ice-cold Ca2+-free
shark Ringers (pH 7.7) as previously described2. Arterial tissue was loaded with the Ca2+ sensitive
fluorescent dye, fura-2AM and [Ca2+]i was measured in 5-6 VSM cells. We employed two inhibitors
of the ADPR cyclase, nicotinamide (3mM)5 and Zn2+ (3mM)1, an inhibitor of NADPH oxidase (NOX),
diphenyl iodonium (DPI) (1µM) and a superoxide
dismutase mimetic, TEMPOL (1 mM)6. None of
A
the inhibitors caused a
Ca2+
change in baseline [Ca2+]i
300
Endothelin-1
values in these experiments.
250
Endothelin-1
[Ca2+]i (nM)
To
assess
the
participation of ET-1 in
activating
the
ADPR
cyclase, we pretreated VSM
with nicotinamide or Zn2+
in nominally Ca2+-free
Ringers.
These agents
reduced
the
[Ca2+]i
response by 62 and 72 %
respectively (n = 15 and 11,
P <0.01, figure 1).
200
Endothelin-1 +
nicotinam ide
150
100
50
0
50
100
150
200
250
300
350
400
450
Tim e (sec)
To address the question of whether Figure 1. Representative tracing of cytosolic Ca2+
there is a connection between ET-1 response of shark VSM to ET-1 in the presence and
2+
induced generation of superoxide (via absence of nicotinamide followed by the addition of Ca .
NOX), superoxide mediated activation of
ADPR cyclase and the [Ca2+]i response to ET-1, we pretreated VSM with the NOX inhibitor DPI or
the superoxide dismutase mimetic, TEMPOL. These two agents reduced the response by 63 % (n = 19
for both, P < 0.01, Figure 2). These data suggest that when the production or duration of superoxide is
diminished, the ability of ET-1 to mobilize Ca2+ from the sarcoplasmic reticulum (SR) is markedly
reduced.
29
120
2+
Difference in [Ca ]i (nM)
from baseline
100
80
60
*
*
40
20
0
Endothelin-1
ET-1 + DPI
To substantiate the premise
that ET-1 signal via two
independent pathways, namely
the classic IP3 pathway and
perhaps a NOX, superoxide,
ADPR cyclase pathway, we
measured the [Ca2+]i response to
ET-1 in the presence of
TEMPOL or DPI plus the IP3R
blockers 2-APB or TMB-82.
The combination of inhibitors of
the IP3R with a reduction in
superoxide reduced the [Ca2+]i
response to ET-1 by 82 % (P
<0.01).
ET-1 + TEMPOL
Evidence for a linkage
between ET-1 and the generation of
Figure 2. Effect of an NADPH oxidase inhibitor, DPI and a
2+
superoxide has previously been shown
superoxide dismutase mimetic, TEMPOL, on the [Ca ]I
in cultured A-10 VSM cells4 and in
response to ET-1 in shark mesenteric artery VSM.
human gluteal arterial cells6. Our data
suggest for the first time that there is a
non-phagocytic NOX in fish and that superoxide is involved in ET-1 induced increases in [Ca2+]i.
Because superoxide is known to dimerize ADPR cyclase 3,7, the active form of the enzyme, we propose
the following sequence of events: ET-1 activation of a G-protein-coupled receptor results in the
formation of IP3 and mobilization of Ca2+ from the SR. ET-1 also results in activation of NOX,
generation of superoxide, activation of ADPR cyclase, formation of cADPR and augmentation of
CICR. Without amplification of the Ca2+ signal by CICR, the IP3R – mediated mobilization would
account for less than 1/3 of the total response.
Supported in part by Research Awards from the University of North Carolina and the Thomas H.
Maren Foundation. Laurel Parker was supported by an award from the Thomas H. Maren Foundation.
1.
deToledo, F.G., Cheng, J, Liang, M., Chini, E.N. and Dousa, T.P. ADP-Ribosyl cyclase in rat vascular smooth
muscle cells: properties and regulation. Cir. Res. 86: 1153-1159, 2000.
2.Fellner, S.K. and Parker, L.A. Endothelin B receptor Ca2+ signaling in shark vascular smooth muscle: participation of
inositol trisphosphate and ryanodine receptors. J. Exp. Biol. 207:3441-3417, 2004.
3.Okabe, E., Tsujimoto, Y. and Kobayashi, Y. Calmodulin and cyclic ADP-ribose interaction in Ca2+ signaling related
to cardiac sarcoplasmic reticulum: superoxide anion radical-triggered Ca2+ release. Antioxid. Redox. Signal. 2: 47-54,
2000.
4. Sedeek, M.H., Llinas, M.T., Drummond, H., Fortepiana, L., Abram, S.R., Alexander, B.T., Reckelhoff, J.F. and
Granger, J.P. Role of reactive oxygen species in endothelin-induced hypertension. Hypertension 42:806-810, 2005.
5. Sethi, J.K., Empson, R.M. and Galione, A. Nicotinamide inhibits cyclic ADP-ribose-mediated calcium signalling in
sea urchin eggs. Biochem. J. 319: 613-617, 1996.
6. Touyz, R.M., Yao, G., Viel, E., Amiri, F. and Schiffrin, E.L. Angiotensin II and endothelin-1 regulate MAP kinase
through different redox-dependent mechanisms in human vascular smooth muscle cells. J. Hypertens. 22: 1141-1149, 2004.
7. Xie, G.H., Rah, S.Y., Yi, K.S., Han, M.K., Chae, S.W., Im M.J. and Kim, U.H. Increase of intracellular Ca(2+0
during ischemia/reperfusion injury of heart is mediated by cyclic ADP-ribose. Biophys. Res. Commun. 307: 713-718, 2003.
30
Activation of 5'-AMP activated protein kinase during anaerobiosis in the rock crab, Cancer irroratus
Ilka Pinz 1, Dylan J. Perry2 & Markus Frederich3
1
College of Osteopathic Medicine and 3Department of Biological Sciences,
University of New England, Biddeford, ME 04005
2
Carrabassett Valley Academy, Carrabassett Valley ME 04947
5’-AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism in mammals.
Depleted cellular energy pools, for example during prolonged exercise or in hypoxic states result in the
activation of AMPK 4. Since AMPK has been highly conserved during evolution 3, its presence in
marine crustaceans is likely, although no reports are available yet. Marine crustaceans are often
exposed to temperature fluctuations that could lead to hypoxic conditions. At too high and too low
temperatures a mismatch of O2 demand and O2 supply in the tissues of animals occurs despite
sufficient O2 availability in the environment. These temperature thresholds have been defined as
critical temperatures, Tc, and are characterized by the onset of anaerobic metabolism with the
accumulation of anaerobic endproducts such as lactate 2, 5. In addition, the switch from aerobic to
anaerobic metabolism causes a change in the cell's energy sources and energy status. Extended
exposure to temperatures above high Tc or below low Tc finally leads to death due to energy depletion.
It is not known whether AMPK regulates energy metabolism in marine invertebrates under such
conditions. Therefore, we tested the hypothesis that AMPK is present in the rock crab Cancer irroratus
and can be activated during anaerobiosis due to O2 depletion and temperature stress.
To investigate AMPK activation during hypoxia, C. irroratus were incubated in nitrogen
equilibrated seawater for 40, 60 and 120 min at 16ºC. At the endpoints the crabs were killed by a cut
through the cerebral ganglion and the different tissues quickly excised and freeze clamped. During the
incubations heart rate was recorded non-invasively using photoplethysmographs glued to the carapace.
Lactate in tissues was measured enzymetrically. AMPK activity was measured using the "SAMS"
peptide, a synthetic peptide with the specific target sequence for AMPK, synthesized after the AMPK
recognition sequence of acetyl-CoA carboxylase from rat liver (amino acid sequence
HMRSAMSGLHLVKRR). In this assay AMPK phosphorylates the SAMS peptide using radiolabelled g32P-ATP. The resulting amount of radioactive SAMS peptide represents therefore AMPK
activity, for details see 1.
Fourty minutes of hypoxia resulted in a significant lactate accumulation in heart, hepatopancreas
and muscle tissues (Figure 1a). Concomitant with the lactate accumulation in the hepatopancreas
AMPK activity increased 5.3 fold. In the heart, 120 min of hypoxia increased lactate 9.1 fold with a
1.7!fold increase in AMPK activity (Figure 1b). The faster and higher activation in the hepatopancreas
compared to the heart is probably due to either the difference in metabolic activity or different
functions of AMPK in various tissues. The hepatopancreas has a high energy expenditure during
hypoxia due to glycogen catabolism supplying the body with glucose. The lesser increase of AMPK
activity in the heart despite higher lactate accumulation could reflect the diminished contractile
performance of the heart at hypoxic conditions: heart rate dropped from 88.2±10.9 bpm during
normoxia to 16.7±9.6 bpm after 40 min hypoxia and 8.5±2.1 bpm after 120 min hypoxia, therefore the
energy demand was greatly reduced and the large increase in anaerobic ATP production might have
improved the energetic state of the muscle cells. More detailed tissue specific effects of AMPK need to
be determined in the future.
31
a
c
[lactate]:
temperature incubations
AMPK activity:
hypoxia incubations
1.6
1.6
6
0.8
0.6
0.4
heart
4
3
2
1
0.2
0.0
20
40
120
Time of hypoxia (min)
1.2
25ºC
30ºC
1.0
0.8
0.6
0.4
0.2
0.0
0
0
Lactate mmol/g protein
1.0
Change in AMPK activity
heart
1.4
liver
5
liver
1.2
18ºC
muscle
muscle
1.4
[lactate] mmol/g protein
Fig. 1. Lactate accumulation (a) and
respective increase in AMPK activity
(b) in various tissues of C a n c e r
i r r o r a t u s . Exposure to high
temperatures leads to lactate
accumulation comparable to
prolonged exposure to hypoxia (c)
and indicates the upper critical
temperature between 25 and 30ºC.
b
[lactate]:
hypoxia incubations
0
20
40
120
Time of hypoxia (min)
muscle
liver
heart
To test whether AMPK also plays a role in thermal tolerance, the critical temperatures (Tc) of C.
irroratus had to be determined. Exposure of the animals to various temperatures and subsequent tissue
sampling and lactate analysis showed a significant increase in lactate between 25 and 30ºC (Figure 1c).
Tc is therefore reached in this temperature range. The animals died above 30ºC. The lactate
accumulation at 30ºC in the leg muscle is comparable with an exposure to 40 min of hypoxia. The
lactate accumulation at 30ºC in the heart is comparable with an exposure to 120 min of hypoxia. The
various degrees of anaerobiosis are again a function of the metabolic activity of the respective tissues.
Further experiments will test the activation of AMPK as well as the AMPK gene expression after the
onset of anaerobiosis due to temperature stress.
We conclude that the highly conserved metabolic master switch, AMPK, is present in C. irroratus
and is activated during hypoxia. Additional measurements are currently done to characterize the
described AMPK activation in more detail. The specific effect of AMPK activity on downstream
targets, such as acetyl-CoA carboxylase, malonyl-CoA carboxylase, glycogen synthase, and others still
needs to be determined for C. irroratus. An enhanced understanding of the integrative regulation of
energy metabolism during environmental stress could lead to a better understanding of the animal's
tolerance of various stressors.
This research was supported by an MDIBL New Investigator Award and a faculty development grant
from the University of New England to M.F. and a Maine High School Research Fellowship supported
by the Betterment Fund to D.P.
1.
2.
3.
4.
5.
32
Frederich, M., Balschi, J.A. The relationship between AMP-activated protein kinase activity and AMP concentration
in the isolated perfused rat heart. J Biol Chem 277/3: 1928-1932, 2002.
Frederich, M., Pörtner, H.O. Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance
in spider crab Maja squinado. Am J Physiol 279: R1531-R1538, 2000.
Gao, G., Widmer, J., Stapleton, D., The, T., Cox ,T., Kemp, B.E., Witters, L.A. Catalytic subunits of the porcine
and rat 5'-AMP-activated protein kinase are members of the SNF1 protein kinase family. Biochim Biophys Acta
1266(1): 73-82, 1995.
Kemp, B.E., Stapleton, D., Campbell, D.J., Chen, Z.P., Murthy, S., Walter, M., Gupta, A., Adams, J.J., Katsis,
F., van Denderen, B., Jennings, I.G., Iseli, T., Michell, B.J., Witters, L.A. AMP-activated protein kinase, super
metabolic regulator. Biochem Soc Proc 31/1: 162-168, 2003.
Pörtner, H.O. Physiological basis of temperature-dependent biogeography: trade-offs in muscle design and
performance in polar ectotherms. J Exp Biol 205: 2217-2230, 2002.
Expressed sequence tags in a normalized cDNA library prepared from multiple tissues
of the American lobster Homarus americanus
David W. Towle and Christine M. Smith
Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672
Changes in temperature and salinity are known stressors in American lobsters that induce
expression of chaperone proteins, including hsp70 and hsp90, as well as polyubiquitin, a component of
the protein degradation pathway2,3. Although induction of these genes is likely to be protective during
environmental fluctuations normally encountered, they may be insufficient to resist extremes of
temperature or salinity, particularly on a background of environmental contamination. To develop a
more complete view of gene transcriptional changes during stress in adult lobsters, we are developing a
library of expressed sequence tags (ESTs) that will be employed in future microarray analyses.
Total RNA was prepared from three tissues in the branchial chamber (gill, epipodite, and
branchiostegite) as well as examples of major organ groups (brain, heart, antennal gland,
hepatopancreas, and abdominal muscle) and reproductive tissues (ovary and testis). Analysis of total
RNA with an Agilent Bioanalyzer revealed two closely-spaced peaks of rRNA, the crustacean 28S
rRNA fragmenting to two smaller products, one or both of which may coincide with the 18S rRNA
peak. cDNA was reverse transcribed from pooled total RNA using oligo-dT as primer and was
directionally ligated into the pCMV Sport 6.1 vector. The resulting cDNA library was normalized by
subtraction at two different Cot values (Invitrogen). Normalization reduced the abundance of a highlyexpressed transcript (arginine kinase) from 2.47% in the un- normalized library to 0.0465% in the final
product, a 53- fold reduction. The average insert size was 2.41 kb.
Plasmids were purified from overnight cultures of randomly-picked clones using a Biomek 2000
liquid handling robot and 5,568 of these were 5’-end sequenced. Nucleotide sequences were processed
by automated editing of trace files and local blast analysis for direct submission to dbEST, using
trace2dbest on a dedicated Linux computer (Parkinson, Anthony and Blaxter, unpublished software,
University of Edinburgh). Resulting were 4,604 dbES T-submissable sequences with an average length
of 572 nucleotides, including an average high-quality length of 453 nucleotides. Clustering by
partigene software1 revealed 1,412 ESTs in 579 individual clusters, plus 3,192 singletons. Sequencing
5,568 clones thus produced a total yield of 3,771 unique sequences, representing a return of two unique
sequences for every three clones picked and sequenced. Following blastx analysis against the nonredundant database from NCBI, 41.6% of the unique sequences were tentatively identified via highscoring hits. ESTs of Homarus americanus and their blastx identifications are presently available at
www.ncbi.nlm.nih.gov.
The project described was supported by NIH Grant Number P20 RR-016463 from the INBRE
Program of the National Center for Research Resources.
1. Parkinson, J., A. Anthony, J. Wasmuth, R. Schmid, A. Hedley, and M. Blaxter. PartiGene—constructing partial
genomes. Bioinformatics 20: 1398-1404, 2004.
2. Spees, J.L., S.A. Chang, M.J. Snyder, and E.S. Chang. Osmotic induction of stress-responsive gene expression in the
lobster Homarus americanus. Biol. Bull. 203: 331-337, 2002.
3. Spees, J.L., S.A. Chang, M.J. Snyder, and E.S. Chang. Thermal acclimation and stress in the American lobster,
Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones
and polyubiquitin. Cell Stress & Chaperones 7: 97-106, 2002.
33
Splice variants in hsp70 cDNAs from the marine copepod Calanus finmarchicus
1
Kelly Baehre1 , Petra Lenz2 , Céline Spanings-Pierrot3 and David W. Towle 4
Bates College, Lewiston, ME 04240; 2 University of Hawaii at Manoa, Honolulu, HI 96822;
3
Université Montpellier II, UMR 5171 GPIA-AEO, 34095 Montpellier cedex 05, France;
4
The copepod Calanus finmarchicus is an abundant crustacean in the North Atlantic, providing a
major food source for many teleosts and some elasmobranchs. Its oceanic distribution depends at least
in part on water temperatures, which are increasing measurably as a result of global warming. In a
2003 study, we found that the expression of heat shock protein 70 (hsp70) increases following either
an acute (30- minute) temperature shock or an extended (48-hour) exposure to a warmer environment
(transfer from 8 to 18o C)2 . In a follow- up study, we have analyzed in greater detail the hsp70 cDNAs
prepared from C. finmarchicus and have found evidence for at least two hsp70-encoding genes in the
Gulf of Maine population, with one of them showing apparent splice variants.
Total RNA was prepared from whole copepods preserved in RNAlater (Ambion). Analysis of
total RNA with an Agilent Bioanalyzer revealed a single sharp peak of rRNA, the crustacean 28S
rRNA fragmenting to two smaller products, both of which coincide with the 18S rRNA peak. cDNA
was reverse transcribed from total RNA using oligo-dT as primer and a partial hsp70 sequence was
amplified using Calanus-specific primers and Redtaq DNA polymerase (Sigma) at an annealing
temperature of 55o C. Following electrophoresis on agarose gels, single bands were excised and the
DNA prepared for sequencing (Qiagen). Raw sequence traces, analyzed by Chromas software,
revealed numerous instances of multiple overlapping peaks, particularly in the third codon position.
To isolate individual cDNAs, the PCR product was ligated into the TA cloning vector (Invitrogen)
and seven individual clones were isolated for plasmid extraction. Following a subsequent round of
sequencing, we obtained evidence for the existence at least two hsp70 genes (or alleles) encoding
nearly the same amino acid sequence (Fig. 1). mRNAs transcribed from one of these ge nes, however,
were clearly the result of alternative splicing, producing proteins that differed in length by 26 amino
acids (Fig. 1, clones 5R and 6R).
Numerous instances of third-position sequence variations were noted. Of 23 variations, 20 were in
the third codon position, and only 3 of these represented amino acid substitutions. Some of these
variants may have resulted from the lack of proofreading of the taq polymerase used to generate the
amplification products, notable in clone 8R that contained several unique variants, including one not in
the third position. Most of the variations, however, were exhibited by two or more of the clones,
suggesting that they existed in the organism and were not artifacts of amplification. Our data suggest
that a family of hsp70 genes exists in Gulf of Maine C. finmarchicus, similar to the phenomenon
observed in other species1 . Variant-specific probes and primers will be required to sort out the
expression pattern of these heat shock proteins in response to environmental perturbation.
Supported by NSF Grant Number IBN-0340622 to DWT and OCE-9906223 to PHL, and by NIH Grant Number P20
RR-016463 from the INBRE Program of the National Center for Research Resources.
1. Feder, M.E., and G.E. Hofmann. Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary
and ecological physiology. Ann. Rev. Physiol. 61:243-282, 1999.
34
2. Voznesensky, M., P.H. Lenz, C. Spanings-Pierrot, and D.W. Towle. Genomic approaches to detecting thermal stress
in Calanus finmarchicus (Copepoda: Calanoida). J. Exp. Mar. Biol. Ecol. 311: 37-46, 2004.
G02_KB-9R_
E01_KB-3R_
C01_KB-1R_
C02_KB-7R_
E02_KB-8R_
G01_KB-5R_
A02_KB-6R_
:
:
:
:
:
:
:
*
100
*
120
*
140
*
160
TTGGTACAGCTTGGTAATAACGGGACTGCAGACCGCCTCGACCTCTTTCTGTTTCTCATTAAACTCTTCAACTTCTGCAA
TTGGTACAGCTTGGTAATAACGGGACTGCAGACCGCCTCGACCTCTTTCTGTTTCTCATTGAACTCATCAACTTCTGCAA
TTGGTACAGCTTGGTAATAACGGGACTGCAGACTGCCTCTACCTCTTTCTGTTTCTCATTAAACTCTTCAACTTCTGCAA
TTGGTACAGCTTGGTAATAACGGGACTGCAGACTGCCTCTACCTCTTTCTGTTTCTCATTAAACTCTTCAACTTCTGCAA
TTGGTACAGCTTGGTAATAACGGGACTGCAGACCGCCTCAACCTCTTTCTGTTTCTCATTAAACTCATCAACTTCTGCAA
TTGGTACAGCTTGGTAATAACGGGACTGCAAACAGCCTCGACTTCTTTCTGTTTCTCATTGAACTCATCAAATTCTGCAA
TTGGTACAGCTTGGTAATAACGGGACTGCAAACAGCCTCGACTTCTTTCTGTTTCTCATTGAACTCATCAAATTCTGCAA
:
:
:
:
:
:
:
160
158
160
158
157
160
158
G02_KB-9R_
E01_KB-3R_
C01_KB-1R_
C02_KB-7R_
E02_KB-8R_
G01_KB-5R_
A02_KB-6R_
:
:
:
:
:
:
:
*
180
*
200
*
220
*
240
GTTGGTTAGCATCTAGCCATTTAATGGCTTCTTCACATTTTTCCGATATCTTTTTCTTGTCATCATCTGATATCTTATCC
GTTGGTTAGCATCCAGCCATTTTATGGCTTCTTCACATTTTTCTGATATCTTTTTCTTGTCATCATCTGATATCTTATCC
GTTGGTTAGCATCTAGCCATTTAATGGCTTCTTCGCATTTTTCCGATATCTTTTTCTTGTCATCATCTGATATCTTATCC
GTTGGTTAGCATCTAGCCATTTAATGGCTTCTTCGCATTTTTCCGATATCTTTTTCTTGTCATCATCTGATATCTTATCC
GTTGGTTAGCATCAAGCCATTTTATGGCTTCTTCACATTTTTCCGATATCTTTTTCTTATCATCATCGGATATCTTACCC
GTTGGTTAGCATCAAGCCATTTTATGGCTTCCTCACATTTCTCATATATCTTTTTCTTGTCATCATCTGATATCTTATCC
GTTGGTTAGCATCAAGCCATTTTATGGCTTCCTCACATTTCTCATATATCTTTTTCTTGTCATCATCTGATATCTTATCC
:
:
:
:
:
:
:
240
238
240
238
237
240
238
G02_KB-9R_
E01_KB-3R_
C01_KB-1R_
C02_KB-7R_
E02_KB-8R_
G01_KB-5R_
A02_KB-6R_
:
:
:
:
:
:
:
*
260
*
280
*
300
*
320
TTGACCTTTTCATCTTCAATGGTAGTTTTCATGTTGAAACAATAAGACTCAAGGCCATTTTTTGCAGAAATTCTGTCCTT
TTGACCTTTTCATCTTCAATGGTAGTTTTCATGTTAAAGCAATATGACTCAAGGCCATTCTTTGCAGAAATTCTGTCCTT
TTGACCTTTTCATCTTCAATGGTAGTTTTCATGTTGAAACAATAAGACTCAAGGCCATTCTTTGCAGAAATTCTGTCCTT
TTGACCTTTTCATC-----------------------------------------------------------------TTGACCTTTTCATC------------------------------------------------------------------
:
:
:
:
:
:
:
320
318
320
318
317
254
252
G02_KB-9R_
E01_KB-3R_
C01_KB-1R_
C02_KB-7R_
E02_KB-8R_
G01_KB-5R_
A02_KB-6R_
:
:
:
:
:
:
:
*
340
*
360
*
380
*
400
CTGCTTTTCATCATCAGCCTTGAACTTTTCTGCATCATTGACCATCCTTTCAATGTCCTCCTTAGACAACCTTCCTTTGT
CTGCTTTTCATCATCAGCCTTGAACTTTTCTGCATCATTGACCATCCTTTCAATATCCTCCTTAGACAACCTTCCTTTGT
CTGCTTTTCATCATCAGCCTTGAACTTTTCTGCATCATTGACCATTCTTTCAATATCCTCCTTAGACAACCTTCCTTTGT
------------ATCAGCCTTGAACTTTTCTGCATCATTGACCATTCTTTCAATATCCTCCTTAGACAACCTTCCTTTGT
------------ATCAGCCTTGAACTTTTCTGCATCATTGACCATTCTTTCAATATCCTCCTTAGACAACCTTCCTTTGT
:
:
:
:
:
:
:
400
398
400
398
397
322
320
G02_KB-9R_
E01_KB-3R_
C01_KB-1R_
C02_KB-7R_
E02_KB-8R_
G01_KB-5R_
A02_KB-6R_
:
:
:
:
:
:
:
*
420
*
440
*
460
*
480
AAGGGCGAATTCGCGGCCGCTAAATTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACG
:
:
:
:
:
:
:
480
478
480
478
477
402
400
G02_KB-9R_
E01_KB-3R_
C01_KB-1R_
C02_KB-7R_
E02_KB-8R_
G01_KB-5R_
A02_KB-6R_
:
:
:
:
:
:
:
*
500
*
520
*
540
*
560
TCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG
:
:
:
:
:
:
:
560
558
560
558
557
482
480
Fig. 1. Multiple nucleotide alignment of partial hsp70 cDNA sequences of individual recombinant plasmids obtained by
PCR with Calanus-specific hsp70 primers. Clones 5R and 6R are clearly splice variants lacking a 78-nucleotide region that
is present in the remaining 5 clones. Third-position variations, indicated by thin arrows, are particularly notable in the
region upstream of the splice site. Several variations occur in other codon positions as well (heavy arrows). An in-frame
stop codon appears in the insert within the extended sequence of clones 1R, 7R, 8R, and 9R (double arrow), indicating that
these mRNAs may yield an incomplete protein product.
35
Quantitative analysis of hsp70 mRNA expression under salinity stress
in the euryhaline shore crab Pachygrapsus marmoratus
Nishad Jayasundara1, Céline Spanings-Pierrot2, and David W. Towle3
1
College of the Atlantic, Bar Harbor, ME 04609 ; 2Université Montpellier II, 34095 Montpellier cedex
05, France ; 3Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672
Marbled rock crabs (Pachygrapsus marmoratus) inhabit coastal regions of the Mediterranean Sea
where they may be subjected to osmotic stress due to large and rapid fluctuations in seawater salinity.
We have previously shown that the expression of genes encoding ion transport proteins in the gills of
this species is responsive to salinity change2,3. Based on work in other laboratories on the heat shock
response to osmotic stress4, we hypothesized that extremes of salinity might also induce changes in the
expression of heat shock proteins (hsps) in the gills of P. marmoratus. We were particularly interested
in the possible differentiation of gill function as noted with ion transporter expression.
Using degenerate oligonucleotide primers based on hsp70 sequences in several aquatic species, we
successfully identified and sequenced hsp70 cDNA from P. marmoratus. Following amplification and
sequencing of an initial hsp70 fragment, species-specific primers were used with 5’-RACE (Clontech)
and 3’-RACE (Invitrogen) protocols, yielding a 2,189-bp cDNA encoding a 650-amino-acid protein
that showed high homology to proteins identified as hsp70 in other arthropods (Fig. 1).
Pac hyg rap sus
Lit ope nae us
Pen aeu s
Mac rob rac hiu m
Api s
:
:
:
:
:
*
20
*
40
*
60
*
80
*
1 00
*
12 0
MS KGA AVG IDL GTT YSC VGV FQH GKV EII AND QGN RTT PSY VAF TDT ERL IGD AAK NQV AMN PNN TVF DAK RLI GRK FND HNV QSD MKH WPF DVI DDN TKP KIK VEY KGE AKS FYP EEI S
MA KAP AVG IDL GTT YSC VGV FQH GKV EII AND QGN RTT PSY VAF TDT ERL IGD AAK NQV AMN PNN TVF DAK RLI GRK FED HTV QSD MKH WPF TII NES TKP KIQ VEY KGD KKT FYP EEI S
MA KAP AVG IDL GTT YSC VGV FQH GKV EII AND QGN RTT PSY VAF TDT ERL IGD AAK NQV AMN PSN TVF DAK RLI GRK FED HTV QSD MKH WPF TII NES TKP KIQ VEY KGD KKT FYP EEI S
MA KSA AVG IDL GTT YSC VGV FQH GKV EII AND QGN RTT PSY VAF TDT ERL IGD AAK NQV AMN PNN TVF DAK RLI GRK FDD GVV QSD MKH WPF TVI NDN TKP KIQ VDY KGE TKT FFP EEI S
MA KAP AVG IDL GTT YSC VGV FQH GKV EII AND QGN RTT PSY VAF TET ERL IGD AAK NQV AMN PNN TIF DAK RLI GRR FED PTV QAD MKH WPF TVV NDG GKP KIQ VYY KGE AKT FFP EEV S
Pac hyg rap sus
Lit ope nae us
Pen aeu s
Mac rob rac hiu m
Api s
:
:
:
:
:
*
140
*
1 60
*
18 0
*
200
*
2 20
*
24 0
SM VLI KMK ETA EAY LGS VVK DAV VTV PAY FND SQR QAT KDA GTI SGL NVL RII NEP TAA AIA YGL DKK VGG ERN VLI FDL GGG TFD VSI LTI EDG IFE VKS TAG DTH LGG EDF DNR MVN H
SM VLI KMK ETA EAY LGS TVK DAV VTV PAY FND SQR QAT KDA GTI SGL NVL RII NEP TAA AIA YGL DKK VGG ERN VLI FDL GGG TFD VSI LTI EDG IFE VKS TAG DTH LGG EDF DNR MVN H
SM VLI KMK ETA EAY LGS TVK DAV VTV PAY FND SQR QAT KDA GTI SGL NVL RII NEP TAA GIA YGL DKK VGG ERN VLI FDL GGG TFD VSI LTI EDG IFE VKS TAG DTH LGG EDF DNR MVN H
SM VLI KMK ETA EAF LGS TVK DAV ITV PAY FND SQR QAT KDA GTI SGL NAL RII NEP TAA AIA YGL DKK VGG ERN VLI FDL GGG TFD VSI LTI EDG IFE VKS TAG DTH LGG EDF DNR MVN H
SM VLV KMK ETA EAY LGK TVS NAV ITV PAY FND SQR QAT KDA GTI SGL NVL RII NEP TAA AIA YGL DKK TTS ERN VLI FDL GGG TFD VSI LTI EDG IFE VKS TAG DTH LGG EDF DNR MVN H
Pac hyg rap sus
Lit ope nae us
Pen aeu s
Mac rob rac hiu m
Api s
:
:
:
:
:
*
260
*
2 80
*
30 0
*
320
*
3 40
*
36 0
FL QEF KRK YKK DPS ESK RAL RRL RTA CER AKR TLS SSA QAS VEI DSL FEG IDF YTS ITR ARF EEL CAD LFR GTL EPV EKA LRD AKM DKA QIH DIV LVG GST RIP KIQ KLL QDF FNG KEL N
FI QEF KRK YKK DPS ENK RSL RRL RTA CER AKR TLS SST QAS VEI DSL FEG IDF YTS ITR ARF EEL CAD LFR GTL EPV EKS LRD AKM DKA QIH DIV LVG GST RIP KIQ KLL QDF FNG KEL N
FI QEF KRK YKK DPS ENK RSL RRL RTA CER AKR TLS SST QAS VEI DSL FEG IDF YTS ITR ARF EEL CAD LFR GTL EPV EKS LRD AKM DKA QIH DIV LVG GST RIP KIQ KLL QDF FNG KEL N
FI QEF KRK YKK DPS ENK RAL RRL RTA CER AKR TLS ASA QAS IEI DSL YEG TDF YTS VTR ARF EEL CGD LFR GTL EPV EKS LRD AKM DKA QIH DIV LVG GST RIP KIQ KLL QDF FNG KEL N
FV QEF KRK YKK DLT ANK RAL RRL RTA CER AKR TLS SST QAS IEI DSL YEG IDF YTS ITR ARF EEL CAD LFR GTL EPV EKS LRD AKM DKA QIH DIV LVG GST RIP KIQ KLL QDF FNG KEL N
Pac hyg rap sus
Lit ope nae us
Pen aeu s
Mac rob rac hiu m
Api s
:
:
:
:
:
*
380
*
4 00
*
42 0
*
440
*
4 60
*
48 0
KS INP DEA VAY GAA VQA AIL CGD KSE AVQ DLL LLD VTP LSL GIE TAG GVM TAL IKR NTT IPT KQT QTF TTY SDN QPG VLI QVY EGE RAM TKD NNL LGK FEL TGI PPA PRG VPQ IEV TFD I
KS INP DEA VAY GAA VQA AIL CGD KSE AVQ DLL LLD VTP LSL GIE TAG GVM TAL IKR NTT IPT KQT QTF TTY SDN QPG VLI QVY EGE RAM TKD NNL LGK FEL SGI PPA PRG VPQ IEV TFD I
KS INP DEA VAY GAA VQA AIL CGD KSE AVQ DLL LLD VTP LSL GIE TAG GVM TAL IKR NTT IPT KQT QTF TTY SDN QPG VLI QVY EGE RAM TKD NNL LGK FEL SGI PPA PRG VPQ IEV TFD I
KS INP DEA VAC GAA VQA AIL CGD KSE AVQ DLL LLD VTP LSL GIE TAG GVM TAL IKR NTT IPT KQT QTF TTY SDN QPG VLI QVY EGE RAM TKD NNL LGK FEL SGI PPA PRG VPQ IEV TFD I
KS INP DEA VAY GAA VQA AIL HGD KSE EVQ DLL LLD VTP LSL GIE TAG GVM TAL IKR NTT IPT KQT QTF TTY ADN QPG VLI QVY EGE RAM TKD NNL LGK FEL SGI PPA PRG VPQ IEV TFD I
Pac hyg rap sus
Lit ope nae us
Pen aeu s
Mac rob rac hiu m
Api s
:
:
:
:
:
*
500
*
5 20
*
54 0
*
560
*
5 80
*
60 0
DA NGI LNV SAV DKS TGK ENK ITI TND KGR LSK EEI ERM VQD AEK YKV EDE KQR DRI GAK NAL ESY CFN MKS TVE EDK FKD KVS EED RNK IME ACN ETI KWL DAN QLG EKE EYE HKQ KDI E
DA NGI LNV SAV DKS TGK ENK ITI TND KGR LSK EEI ERM VQD AEK YKA DDE KQR DRI SAK NSL ESY CFN MKS TVE DEK FKE KIS EED RNK ILE TCN ETI KWL DMN QLG EKE EYE HKQ KEI E
DA NGI LNV SAV DKS TGK ENK ITI TND KGR LSK EEI ERM VQD AEK YKA DDE KQR DRI SAK NSL ESY CFN MKS TVE DEK FKE KIS EED RNK ILE TCN ETI KWL DMN QLG EKE EYE HKQ KEI E
DA NGI LNV SAA DKS TGK ENK ITI TND KGR LSK EEI ERM VQE AEK YKA DDE KQR DRI AAK NSL ESY CFN MKS TVE DDK FKD KVP EED RNK IME ACN DAI KWL DSN QLG EKE EYE HKL KEI E
DA NGI LNV SAV DKS TGK ENK ITI TND KGR LSK EDI ERM VNE AEK YRS EDE KQK ETI AAK NGL ESY CFN MKS TVE DEK LKD KIS ASD KQV VLD KCN DII KWL DAN QLA DKE EYE HKQ KEL E
Pac hyg rap sus
Lit ope nae us
Pen aeu s
Mac rob rac hiu m
Api s
:
:
:
:
:
*
620
*
6 40
*
QI CNP IIT KMY QAA GGA PPG GMP GGF PG- AGG APG AAP G-G GSS GPT IEE VD
QV CNP IIT KMY AAA GGA PPG GMP GGF PGG APG AGG AAP GAG GSS GPT IEE VD
QV CNP IIT KMY AAA GGA PPG GMP GGF PGG APG AGG AAP GAG GSS GPT IEE VD
QI CNP IIT KMY QAA GGA PPG GMP GGF PGA PGG --G AAP G-G GSS GPT IEE VD
AI CNP IVT KLY QGT GGM P-G GMP GGM PGG FPG AGG GAP G-G GAS GPT IEE VD
Fig. 1. Multiple alignment of hsp70 amino acid sequences from four crustacean species and honeybee. Pachygrapsus
marmoratus (present study), Litopenaeus vanname (Acc. No. AAT46566), Penaeus monodon (Acc. No. AAQ05768),
Macrobrachium rosenbergii (Acc. No. AAS45710), and Apis mellifera (Acc. No. XP_392933).
Species-specific primers and real-time quantitative PCR with SYBR green were used to measure
the relative expression of hsp70 mRNA in triplicate samples of the equivalent of 0.1 µg of total RNA
prepared from gills of P. marmoratus sampled over a time course following transfer from 32 ppt
36
seawater to either 10 ppt or 45 ppt seawater. In the lower salinity, P. marmoratus effectively hyperosmoregulates its hemolymph via increased ion uptake across the gills, mediated at least in part by
marmoratu s
mRNA expression of Hsp70 in individual gills from
marmoratu smRNA expression of Hsp70 in individual gills from
induction
of ion transporter genePachygrapsus
transcription.
In the higher
salinity,
the crab
isPachygrapsus
a (45ppt)
hypo-osmoregulator,
during short-term
acclimation
to high salinity
during short-term acclimation to low salinity (10ppt)
1
most likely by
enhanced
salt
excretion
across the gills .
0
2
4
6
24
48
0
2
4
6
24
48
B
1.8
1.8
1.6
1.6
1.4
1.4
1.2
1
0.8
0.6
0.4
0.2
0
48
24
6
4
G5
2
G6
G7
Gill number
Hours in 10 ppt
0
G8
Relative mRNA
expression
Relative mRNA
expression
A
G9
1.2
1
0.8
0.6
0.4
0.2
0
48
24
6
4
2
G5
G6
G7
Gill number
Hours in 45 ppt
0
G8
G9
Fig. 2. Relative mRNA expression of hsp70, determined by quantitative PCR, in gills of Pachygrapus marmoratus pooled
from at least three animals per sampling interval, in relation to gill 9 after 48 hours of exposure of crabs to 10 ppt seawater.
Our analysis showed that the expression of arginine kinase (AK), a putative housekeeping gene,
was at nearly equal levels at all times (data not shown). Moreover, hsp70 mRNA was expressed
equally under control conditions (zero time) in anterior (G5, G6) and posterior (G7, G8, G9) gills.
Following transfer to low salinity, hsp70 mRNA expression increased in all gills (Fig. 2A). The
degree of increase was about 2-fold in G5, G6 and G9 at 24 and 48 h. However, in G7 and G8 hsp70
mRNA started to increase by 3 to 4-fold within the first 6 h and then slightly decreased by 48 h. These
data suggest that response to osmotic stress experienced by the gill tissue may lead to enhanced hsp70
expression in all gills, with a more rapid response in the two posterior gills that are believed to be most
involved in ion uptake2,3.
Following transfer to high salinity, G5, G6, and G9 did not show any significant change in hsp70
mRNA throughout the study period. However, G8 showed a 2-fold increase within 4 h and by 48 h
decreased gradually to the level observed under control conditions. In G7, on the other hand, hsp70
mRNA expression increased about 3-fold within 4 h and about 4-fold in 6 h, then slightly decreased at
24 and 48 h to a level that was still twice the amount expressed in controls. These data, coupled with
our observation of a dramatic induction of ion transporter gene expression in G7 under similar
conditions2,3, suggests that G7 plays an important role in the response to hypersaline conditions and
may indeed be primarily responsible for salt excretion during hyperosmotic stress.
Supported by NSF Grant Number IBN-0340622 to DWT.
1
Pierrot, C., A. Péqueux, and P. Thuet. Perfusion of gills isolated from the hyper-hyporegulating crab Pachygrapsus
marmoratus (Crustacea, decapoda): adaptation of a method. Arch. Physiol. Biochem. 103: 401-409, 1995.
2
Spanings-Pierrot, C., and D.W. Towle. Expression of Na+,K+-ATPase mRNA in gills of the euryhaline crab Pachygrapsus
marmoratus adapted to low and high salinity. Bulletin MDIBL 42: 44-46, 2003.
3
Spanings-Pierrot, C., and D.W. Towle. Salinity-related expression of the Na+/K +/2Cl- cotransporter and V-type H+ATPase in gills of the euryhaline crab Pachygrapsus marmoratus. Bulletin MDIBL 43: 6-8, 2004.
4
Spees, J.L., S.A. Chang, M.J. Snyder, and E.S. Chang. Osmotic induction of stress-responsive gene expression in the
lobster Homarus americanus. Biol. Bull. 203: 331-337, 2002.
37
Cloning and Expression of Pax8 from Adult Kidney of the Little Skate, Leucoraja erinacea
Jennifer Litteral1, Michaela Beese2, Torsten Kirsch2, Holly Fletcher3 and Hermann Haller2
1
2
Hannover Medical School, Nephrology Department, Hannover, Germany
3
College of the Atlantic, Bar Harbor, Maine
Previously, we have shown that the adult kidney of the little skate Leucoraja erinacea
possesses a nephrogenic zone that resembles the embryonic mammalian metanephric kidney1. The cell
types of the elasmobranch and the mammalian nephrons are highly similar2 and this nephrogenic zone
contains the essential structures characteristically found in the embryonic mammalian metanephros.
Therefore, we hypothesized that the same genes critical in mammalian embryonic kidney development
also play a role in the ongoing nephrogensis of the adult skate kidney. As nephrons form in the kidney
they express critical transcription factors such as WT1, Pax2 and Hoxa11 which condense and secrete
Wnt4. Wnt4 is required for nephrogenesis to continue. Wnt4 acts as an autocrine loop to stimulate its
own synthesis and is required for cells to differentiate into epithelia3. The homeobox gene Pax8, is
also expressed in the condensed mesenchyme and S-shaped bodies of the forming ureteric bud and
may be responsible for glomeruli maturation4. Pax8 has also been linked to co-transcription factors to
induce pronephric tubule differentiation and growth5. The aforementioned genes Pax2 and Wnt4 have
been fully characterized and our previous research has shown their expression in the developing niche
of adult skate kidneys, demonstrating that the roles of these genes were conserved through evolution.
However, there has been no molecular characterization of Pax8 expression in adult skate kidneys.
The original 827 base pair Pax8 sequence originated from a pooled organ library of Leucoraja
erinacea and was found in the marine genomics database (http://www.marinegenomics.org). Primer
walking was utilized to retrieve additional length of the initial sequence. Running in the 3’ to 5’
direction an 18bp primer GCTGAGTACAAGCGGCAG was designed to sit at the 564bp site. An
additional 410bp were retrieved after sequencing at the MDIBL DNA Sequencing Facility. Primer
walking
was
repeated
at
the
new
1,237bp
site
with
a
27bp
primer
GCCAAGCCCAGTTACACATCATCTGCC. After sequencing, an additional 870bp were retrieved,
resulting in a final sequence length of the 2,175 bp.
Using BLAST search algorithms (www.ncbi.nlm.nih.gov/BLAST), the Pax8 2,175bp sequence
was analyzed and compared with database entries for homology. Pax8 homologues of Homo sapiens,
Mus musculus, and Xenopus laevis were found and aligned with the Pax8 homologue from Leucoraja
erinacea using MultAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html).
Because the Pax8 gene originated from a normalized library of combined Leucoraja erinacea
organs, the organ of origin was uncertain. Relative expression levels of Pax8 in various skate organs
were determined using Real-Time Quantitative PCR. Skate kidney, brain, muscle, spleen, rectal gland,
and heart tissue were obtained. RNA from each was extracted using Trizol (Invitrogen) as per
manufacturer’s specifications. After a DNase-digest 1 µg of total RNA was reversed transcribed using
random hexamers, poly (dT)-oligonucleotides and M-MLV reverse transcriptase (Invitrogen).
Real-time qPCR oligonucleotide primers were designed using Primer Express software. The
forward primer had a sequence of TGCGGCCTTGTGACATCTC, the reverse primer sequence was
TGCCAAGGATTTTGCTGACA. Expression levels of Pax8 in the various organs were determined
using SYBR- green chemistry. For normalization, b-actin expression was measured. The relative
mRNA expression was analyzed using qgene software6.
38
321
Leucoraja TGGAATCAAT
Homo AGGGCTGAAC
Mus AGGGCTGAAT
Xenopus AGGTTTGAAT
CAACTCGGAG
CAGCTGGGAG
CAACTAGGAG
CAGTTAGGTG
GCATGTTTGT
GGGCCTTTGT
GGGCCTTTGT
GGGCTTTTGT
GAATGGGAGA
GAATGGCAGA
GAATGGCAGG
GAATGGACGA
CCGCTTCCCG
CCTCTGCCGG
CCTCTGCCAG
CCTCTACCAG
ATGTGGTGAG
AAGTGGTCCG
AAGTTGTACG
AGGTGGTGAG
GCAGAGGATT
CCAGCGCATC
TCAACGCATT
GCAGCGGATT
400
GTCGACCTTG
GTAGACCTGG
GTGGACTTGG
GTTGACCTGG
401
CACACCAAGG
CCCACCAGGG
CCCACCAGGG
CGCACCAGGG
GGTGCGGCCT
TGTAAGGCCC
CGTGAGGCCC
GGTACGTCCC
TGTGACATCT
TGCGACATCT
TGTGATATTT
TGTGACATCT
CACGCCAGCT
CTCGCCAGCT
CTCGCCAGCT
CACGACAGCT
CAGGGTCAGT
CCGCGTCAGC
CCGTGTCAGC
CAGAGTCAGT
CACGGGTGTG
CATGGTTGCG
CATGGCTGTG
CATGGCTGTG
TCAGCAAAAT
TCAGCAAGAT
TAAGCAAGAT
TCAGCAAAAT
480
CCTTGGCAGG
CCTTGGCAGG
CCTTGGCAGG
TTTGGGCAGG
481
Leucoraja TACTATGAAA
Homo TACTACGAGA
Mus TACTACGAGA
Xenopus TACTATGAGA
CGGGCAGCAT
CTGGCAGCAT
CTGGCAGCAT
CAGGCAGTAT
CATGCCAGGA
CCGGCCTGGA
CCGGCCTGGA
CCGGCCAGGT
GTCATCGGGG
GTGATAGGGG
GTGATAGGGG
GTCATTGGAG
GCTCAAAGCC
GCTCCAAGCC
GCTCCAAGCC
GTTCCAAGCC
AAAGGTTGCC
CAAGGTGGCC
CAAGGTGGCC
CAAGGTTGCC
ACGCCAACAG
ACCCCCAAGG
ACCCCCAAGG
ACCCCCAAAG
560
TGGTGGAGAA
TGGTGGAGAA
TGGTGGAGAA
TGGTGGAGAA
561
GATAGCTGAG
GATTGGGGAC
GATAGGAGAC
AATCGGAGAT
TACAAGCGGC
TACAAACGCC
TACAAGCGGC
TACAAACGCC
AGAACCCGAC
AGAACCCTAC
AGAACCCTAC
AGAACCCAAC
AATGTTTGCG
CATGTTTGCC
CATGTTTGCT
AATGTTTGCC
TGGGAGATCA
TGGGAGATCC
TGGGAGATCC
TGGGAAATCA
GAGACAGGCT
GAGACCGGCT
GGGACCGGCT
GGGACCGGCT
GCTGGCAAAA
CCTGGCTGAG
CCTGGCAGAA
GCTGACAGAC
640
GGAGTGTGTA
GGCGTCTGTG
GGCGTTTGTG
GGGGTGTGCG
641
ATAATGATAC
ACAATGACAC
ACAATGACAC
ACAATGACAC
TGTGCCCAGC
TGTGCCCAGT
TGTCCCCAGT
AGTTCCCAGT
GTCAGCTCCA
GTCAGCTCCA
GTCAGCTCCA
GTCAGCTCTA
TAAACAGAAT
TTAATAGAAT
TCAACAGAAT
TCAACAGAAT
TATAAGAACC
CATCCGGACC
CATCCGGACC
CATACGCACT
AAAGTTCACA
AAAGTGCAGC
AAAGTGCAGC
AAAGTACAGC
GCCATTNCAT
AACCATTCAA
AGCCATTCAA
AACTTTTTAA
720
CTACCTCTTG
CCTCCCTATG
CCTCCCCATG
CCTGCCCATG
Leucoraja
Homo
Mus
Xenopus
Leucoraja
Homo
Mus
Xenopus
Leucoraja
Homo
Mus
Xenopus
Fig 1. Amino acid sequence of Pax8 from Leucoraja erinacea, Homo sapiens, Mus musculus, and Xenopus laevis were
aligned using MultAlin. Areas of high consensus are seen in gray, areas of low consensus are seen in black.
Pax8 mRNA Expression in Different Skate Organs
0.7
Realtive Expression
0.6
0.5
0.4
0.3
0.2
0.1
0
Kidney
Brain
Spleen
Muscle
Rectal Gland
Heart
Fig 2. Distribution of Pax8 transcript in different organs from adult skate. Total RNA from kidney, brain, spleen, muscle,
rectal gland and heart was extracted, reverse transcribed and applied to qPCR using SYBR-green chemistry. For
normalization expression of skate β-actin expression was measured.
Pax8 showed high expression in kidney and relatively low expression in other organs. Most
likely the original Pax8 sequence was derived from the skate kidney. These data also showed that the
developmentally regulated gene Pax8 displayed high levels of consensus between skate and other
species. This supports our hypothesis that the same genes critical in mammalian embryonic kidney
development also play a role in the ongoing nephrogensis of the adult skate kidney.
1. Elger M, Hentschel H, Litteral J, Wellner M, Kirsch T, Luft F and Haller H. Nephrogenesis is induced by partial
nephrectomy in the elasmobranch Leucoraja erinacea. J Am Soc Nephrol, 14:1506-1518, 2003,
2. Lacy ER, Castellucci M and Reale E. The elasmobranch renal corpuscle: fine structure of Bowman’s capsule and the
glomerular capillary wall. Anat Rec, 218(3)294-305, 1987.
3. Davies J and Fisher C. Genes and proteins in renal development. Exp Nephrol 10:102-113, 2002.
4. Poleev A, Fickenscher H, Mundlos S, Winterpacht A, Zabel B, Fidler A, Gruss P and Plachov D. PAX8 a human
paired box gene: Isolation and expression in developing thyroid, kidney and Wilms' tumors. Development. Nov116:611-23,
1992.
5. Eccles MR, Yun K, Reeve AE and Fidler AE. Comparative in situ hybridization analysis of PAX2, PAX8, and WT1
gene transcription in human fetal kidney and Wilms' tumors. Am J Pathol. Jan;146(1):40-5, 1995.
6. Muller PY, Janovjak H, Miserez AR and Dobbie Z. Processing of gene expression data generated by quantitative realtime RT-PCR. Biotechniques. Jun;32(6):1372-4, 1376, 1378-9, 2002.
Partial funding provided through BRIN Maine Biomedical Research Infrastructure Network (NCRR 1 P20 RR16463-01).
39
Partial nucleotide sequence and expression of plasma membrane Ca-ATPase in the
hypodermis of the blue crab, Callinectes sapidus
Robert Roer1 and David Towle2
1
Department of Biological Sciences
University of North Carolina at Wilmington
Wilmington, NC 28403
2
Mount Desert Island Biological Laboratory
Being bound by an exoskeleton, crustaceans must molt in order to grow. The exoskeleton of
the brachyuran crabs is hardened, in many areas, by both sclerotization and impregnation with
calcium carbonate6,7. During the preparation for an ensuing molt, mineral is resorbed from the
old, calcified exoskeleton by the underlying hypodermis. Following the molt, mineral is deposited
by the hypodermis in the new exoskeleton4. In addition to the temporal changes in mineralization,
there are spatial differences as well. The exoskeleton of the branchial chamber, gills and
arthrodial membrane of the joints does not calcify.
Physiological5,10,11, biochemical3, molecular12,13, histological3,7 and immunocytochemical12
data indicate that a Ca-ATPase is involved in the hypodermal transport of calcium out from the
exoskeleton during premolt resorption and into the exoskeleton during postmolt deposition. The
precise timing of the induction of Ca-ATPase expression in the hypodermis during the pre- and
postmolt stages is not known. Moroever, the differences in expression pattern between those
tissues that mineralize (e.g. the dorsal carapace) compared to those that do not (e.g. the arthrodial
membrane) has not been studied. The latter question is particularly important in understanding
the control of the mineralization.
In order to address these questions, blue crabs were collected prior to the molt (stages D2 and
D3), immediately upon molting (0 h), and at 1, 2, 3, 4, 6, 8, 12, 24 and 48 h postmolt. Pieces of
dorsal carapace and arthrodial membrane were excised, the hypodermis was separated from the
exoskeleton with forceps, and the hypodermis stored in RNA Later (Ambion) and stored at -20°C.
Total RNA was extracted using the spin-column RNeasy Protect Mini Kit (Qiagen), with the
following modifications to increase yield and quality of RNA. Tissue was homogenized in 1ml
TRIzol (Invitrogen). RNA was eluted from the column in 30 µl nuclease-free water (Ambion),
and the eluate was passed through the column a second time to increase the yield. mRNA was
reverse-transcribed to make first-stand cDNA (SuperScript II kit, Invitrogen).
The following specific forward and reverse primers were constructed for the plasma membrane
Ca-ATPase (PMCA) and for arginine kinase (AK, as a constitutively expressed control4):
40
PMCA F3:
PMCA R4:
TTG AAC CGA TGG CGT GTA AT
TGA TGT CTG AGG CTT CTT TTG
AK F51:
AK R31:
CGC TGA GTC TAA GAA GGG ATT
GAT ACC GTC CTG CAT CTC CTT
The PCR product was run on an agarose gel, stained with ethidium bromide, photographed, and
the appropriate band was cut from the gel and extracted using the QiaQuick kit and protocol
(Qiagen). The gel-purified cDNA was quantified by agarose gel electrophoresis and sequenced on
an ABI Prism 3100 sequencer at the Marine DNA Sequence Center at MDIBL.
The mRNA was contaminated with
genomic DNA. The segment of the genomic
DNA that amplified along with the cDNA
bore one intron, resulting in two PCR
products. It was, therefore, not possible to run
quantitative PCR. However, the results of a
semi-quantitative comparison showed low
levels of expression of Ca-ATPase, relative to
arginine kinase, until 4 h postmolt in both
dorsal carapace and arthrodial membrane (Fig.
1). Between 4 h and 48 h postmolt, there is a
marked upregulation in the expression of the
Ca-ATPase mRNA.
The translation of the portion of the
Callinectes Ca-ATPase sequence that was
amplified revealed a high degree of amino acid
identity with that of the enzyme from the
crayfish, Procambarus clarkii2 (Fig. 2).
Fig. 1. Semi-quantitative analysis of the
expression of Ca-ATPase mRNA (PMCA)
compared to arginine kinase (AK) in the
hypodermis of dorsal carapace (C) and
arthrodial membrane (A), before the molt
(D2, D3) and from 0 to 48 h postmolt.
The upregulation of Ca-ATPase during early postmolt supports previous data implicating this
enzyme in the postecdysial mineralization of the pre-exuvially deposited epi- and exocuticle and
the post-exuvially deposited endocuticle4. Interestingly, there was at least as much Ca-ATPase
expressed in the non-calcifying arthrodial hypodermis as there was in the calcifying dorsal tissue.
This suggests that the lack of calcification in the arthrodial membrane is not due to the inability of
the hypodermis to transport calcium, but to differences in the nature of the organic matrix
comprising the two cuticle types1,8,9.
This work was supported by a MDIBL New Investigator Award (RDR) and by grant (IBN 0114597) from the National Science Foundation (RDR).
41
callinectes_
procambarus_
-----------------------------------------------------------MGDDANSSIEFHPRPNQRRDGNQAGGFGCSLMELRSLMELRGLEAVVKIQEDYGDVEGLC
callinectes_
procambarus_
-----------------------------------------------------------RRLKTSPTEGLADNTNDLEKRRQIYGQNFIPPKKPKTFLQLVWEALQDVTLIILEIAAIV
callinectes_
procambarus_
-----------------------------------------------------------SLGLSFYRPPGETGGGAAAGGAEDEGEAEAGWIEGAAILLSVVCVVLVTAFNDWSKEKQF
callinectes_
procambarus_
-----------------------------------------------------------RGLQSRIEQEQKFTVVRNGQVLQIPVAELVVGDIAQVKYGDLLPADGVLIQGNDLKIDER
callinectes_
procambarus_
-----------------------------------------------------------SLTGESDHVRKSADKDPMLLSGTHVMEGSGRMVVTAVGVNSQTGIIFTLLGAGAEEEEVE
callinectes_
procambarus_
-----------------------------------------------------------AKKRKKEAKKQRKKQKKGDSGEELIDANPKKQDGEMESNQIKAKKQDGAAAMEMQPLKSA
callinectes_
procambarus_
-----------------------------------------------------------EGGEADEEEEKKVNTPKKEKSVLQGKLTKLAVQIGKAGLVMSAITVIILVLYFGIETFVV
callinectes_
procambarus_
-----------------------------------------------------------EGRPWTPVYIQYFVKFFIIGVTVLVVAVPEGLPLAVTISLAYSVKKMMKDNNLVRHLDAC
callinectes_
procambarus_
-----------------------------------------------------------ETMGNATAICSDKTGTLTTNRMTVVQSYIGDEHYKEIPDPGSLPPKILDLLVNAISINSA
callinectes_
procambarus_
-----------------------------------------------------------YTTKILPPDKEGDLPRQVGNKTECALLGFVLDLKRDYQPIRDQIPEEKLYKVYTFNSVRK
callinectes_
procambarus_
----------------------------SFIHGKDGKLESFSKSMQDRLVREVIEPMACN
SMSTVVPMRDGGFRIYSKGASEIVLKKCSQILNRDGELRSFRPRDKDDMVRKVIEPMACD
* * .:**:*.**
:* :**:*******:
callinectes_
procambarus_
GLRTISIAYRDFVRGKAEINQVHFENEPHWDDEDHIINNLTCLCVLGIEDPVRPEVPDAI
GLRTICIAYRDFVRGCAEINQVHFENEPNWDNENNIMSDLTCLAVVGIEDPVRPEVPDAI
*****.********* ************:**:*::*:.:****.*:**************
callinectes_
procambarus_
HKCQRAGITVRMVTGDNINTARSIASKCGILKPGDNSLILEGKEFNRRVRDSTGKIQQHL
QKCQRAGITVRMVTGANINTARAIASKCGIIQPGEDFLCLEGKEFNRRIRDESGCIEQER
:************** ******:*******::**:: * *********:**.:* *:*.
callinectes_
procambarus_
VDKVWVNLRVLARSSPTDKYTLVKGIIESKVSANREVVAVTGDGTNDGPALKMADVGFAM
IDKVWPKLRVLARSSPTDKHTLVKGIIDSTTNDQRQVVAVTGDGTNDGPALKKADVGFAM
:**** :************:*******:*... :*:**************** *******
callinectes_
procambarus_
GIAGTDVAKEASDIILLDDNFNSIVKAVMWGR---------------------------GIAGTDVAKEASDIILTDDNFTSIVKAVMWGRNVYDSISKFLQFQLTVNVVAVIVAFTGA
**************** ****.**********
callinectes_
procambarus_
-----------------------------------------------------------CITQDSPLKAVQMLWVNLIMDTFASLALATEPPTESLLLRKPYGRTKPLISRTMMKNILG
callinectes_
procambarus_
-----------------------------------------------------------HAVYQLLIIFTLLFVGEGFFDIDSGRNAPLHSPPSEHYTIIFNTFVMMQLFNEINARKIH
callinectes_
procambarus_
-----------------------------------------------------------GERNVFDGIFSNPIFCTIVLGTFGIQIVIVQFGGKPFSCTPLPAEQWLWCLFVGAGELVW
callinectes_
procambarus_
-----------------------------------------------------------GQVMATIPTSQLKSLKGAGHEHRKDEMNAEDLNEGQEEIDHAERELRRGQILWFRGLNRI
callinectes_
procambarus_
-------------------------------------------------QTQIEVVNAFKSGSSVQGAVRRPSSILSQNQDVTNVSTPSHASSGMPLAL
Fig. 2. Comparison of the deduced amino acid sequence of the partial transcript of Callinectes sapidus Ca-ATPase
with that from Procambarus clarkii.
42
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Coblentz, F.E., T.H. Shafer and R.D. Roer. Cuticular proteins from the blue crab alter in vitro calcium
carbonate mineralization. Comp. Biochem. Physiol. 121B: 349-360, 1998.
Gao, Y. and M.G. Wheatly. Characterization and expression of plasma membrane Ca+2 ATPase (PMCA3) in
the crayfish Procambarus clarkii antennal gland during molting. J. Exp. Biol. 207: 2992-3002, 2004.
Greenaway, P., R.M. Dillaman and R.D. Roer. Quercitin-dependent ATPase activity in the hypodermal tissue
of Callinectes sapidus during the moult cycle. Comp. Biochem. Physiol. 111A: 303-312, 1995.
Kotlyar, S., D. Weihrauch, R.S. Paulsen and D.W. Towle. Expression of arginine kinase enzymatic activity
and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus. J. Exp. Biol. 203: 23952404, 2000.
Roer, R.D. Mechanisms of resorption and deposition of calcium in the carapace of the crab Carcinus maenas. J.
Exp. Biol. 88: 205-218, 1980.
Roer, R.D. and R.M. Dillaman. The structure and calcification of the crustacean cuticle. Am. Zoologist 24:
893-909, 1984.
Roer, R.D. and R.M. Dillaman. Molt-related change in integumental structure and function. In: The Crustacean
Integument - Morphology and Biochemistry, edited by M. Horst and J.A. Freeman. Boca Raton, FL: CRC Press,
1993, p. 1-37.
Shafer, T.H., R.D. Roer, C.G. Miller and R.M. Dillaman. Postecdysial changes in the protein and
glycoprotein composition of the cuticle of the blue crab, Callinectes sapidus. J. Crust. Biol. 14: 210-219,
1994.
Shafer, T.H., R.D. Roer, C. Midgett-Luther and T.A. Brookins. Postecdysial cuticle alteration in the
blue crab, Callinectes sapidus: Synchronous changes in glycoproteins and mineral nucleation. J. Exp. Zool.
271: 171-182, 1995.
Wheatly, M.G. An overview of calcium balance in crustaceans. Physiol. Zool. 69: 351-382, 1996.
Wheatly, M. Calcium homeostasis in Crustacea: The evolving role of branchial, renal, digestive and hypodermal
epithelia. J. Exp. Zool. 283: 620-640, 1999.
Wheatly, M., Z. Zhang, J.R. Weil, J.V. Rogers and L.M. Stiner. Novel subcellular and molecular tools to
study Ca2+ transport mechanisms during the elusive moulting stages of crustaceans: Flow cytometry and
polyclonal antibodies. J. Exp. Biol. 204: 959-966, 2001.
Ziegler, A., D. Weihrauch, D.W. Towle and M. Hagedorn. Expression of Ca2+-ATPase and Na+/Ca2+exchanger is upregulated during epithelial Ca2+ transport in the hypodermal cells of the isopod Porcellio scaber.
Cell Calcium 32: 131-141, 2002.
43
Long-term cultures of cells from Strongylocentrotus purpuratus and Schistosoma mansoni.
Christopher J. Bayne1,2 and Angela Parton2
1
Department of Zoology, and Marine & Freshwater Biomedical Science Center,
Oregon State University, Corvallis, OR 97331
2
Selection of the echinoderm Strongylocentrotus purpuratus as a model species for genomic
sequencing has enhanced the need for cell lines from this species. Previously2, we determined that
several tissues from the closely related, locally available urchin S. droebachiensis could be placed in
culture, and the cells would multiply. Interrogation of the public databases for putative growth factors
(GF) and GF receptors expressed in this genus allowed us to identify several GFs that were
commercially available and might be evaluated for their ability to provoke cell proliferation. Therefore,
in September 2004, six specimens of S. purpuratus were brought from their native Oregon to MDIBL
as sources of cells for in vitro culture.
Earlier we had also determined protocols and conditions that are well suited for the long term
culture of cells of the human blood fluke, Schistosoma mansoni1, but we had been unable to surmount
difficulties inherent in provoking these cells to proliferate in vitro. As S. mansoni continues to take a
severe toll on human health in over 70 countries, and as knowledge of genetic sequences are
accumulating rapidly for this species, derivation of cell lines from this species remains a highly desired
goal. Consequently, several thousand sporocyst larvae of S. mansoni were also transported from
Oregon to MDIBL in September, 2004. As with the urchin cells, we added putative GFs and some
additional receptor ligands to the media used to maintain the cells. These molecules included insulin,
transferrin, basic fibroblast GF, epidermal GF, acetyl choline, activin-A, ciliary neurotrophic factor,
glial-derived GF, heregulin, insulin-like GFI and II, interleukin-6, interleukin-7, interferon-γ,
neuropeptide Y, hepatocyte-derived GF, osteopontin, progesterone, bone morphogenetic protein-4,
bone morphogenetic protein-5, thyroid hormone (T3), thyroglobulin and γ-amino butyric acid.
Drawing on the extensive cell culture resources that are uniquely available in the Center for Marine
Functional Genomics Studies at MDIBL, we established primary tissue cultures from both the urchin
and the blood fluke, and cells have been sustained in a healthy, germ-free state for 4 months. Major
departures from conditions used previously included the use of nitrogen gas to create an hypoxic
atmosphere for the schistosomes, the use of media that have lowered levels of organic components,
and the addition to media (for both species) of growth factors and other biologically active molecules
which, from knowledge of cell biology and of the transcriptomes of these 2 species, could be expected
to have growth-promoting activities. Mining of DNA sequence databases for Strongylocentrotus and
Schistosoma was the method used to select the molecules for these tests.
44
Fig. 1. Sporocysts of Schistosoma
mansoni after 3 weeks in culture.
Many mother* sporocysts and one
of their smaller daughters** are
present. The tegument has
produced extensions*** into the
growth space around some
sporocysts. A cluster of bright
cells****, thought to be germinal
cells that have migrated from the
internal cavity of a sporocyst, is
seen towards the center of the
photomicrograph.
*
**
*
*
***
****
Fig. 2. Cells derived from tissues
located between the lateral faces
of the main teeth of Aristotle’s
lantern of Strongylocentrotus
purpuratus. These cells have the
appearance of cells that will
proliferate in vitro once they have
access to required growth factors
that are either provided by the
investigator or synthesized by the
cells themselves.
As illustrated in Figures 1 and 2, both projects were fruitful. It is the nature of cell line
development to require several weeks to several months in order to obtain indefinitely propagating cell
lines. Consequently, it is not to be expected that a cell line will be obtained in the course of a month.
After up to one month of intense husbandry (on the departure of the first author from MDIBL), the
cultures were placed on a maintenance regime. At the time of writing, both urchin and schistosome
cells remain viable. No explicit data on DNA synthesis or cell proliferation has been sought. However,
cells of the type shown in Figure 2 have been passaged successfully. The ability to maintain healthy
cell cultures over such a long term is fundamental to the development of permanent lines. It is
anticipated that further experimental manipulations of growth conditions including media additives and
manipulation of the physical environment will be evaluated as means to provoke cell multiplication in,
and derivation of cell lines from, these cultures.
45
Funds were kindly provided through a New Investigator Award from MDIBL to Christopher
Bayne and a grant from the NIH: P30 ES-03828. We thank David Barnes for helpful advice and
encouragement. He and Angela Parton are members of the MDIBL Membrane Toxicity Center
(MFBSC).
1. Bayne, C.J. and Barnes, D.W. Culture of cells from two life stages of Schistosoma mansoni. Cytotechnology 23:205210. 1997.
2. Bayne, C.J. and Parton, A. Derivation of cell lines from Strongylocentrotus droebachiensis, the Northern sea urchin.
Bull. Mount Desert Biological Laboratory. 43: 62-64, 2004.
46
Cloning the cDNA for serum- and glucocorticoid-regulated kinase (SGK) from killifish,
Fundulus heteroclitus
J. Denry Sato1, Ciara C. Clarke1,2, Joe Shaw1,3, and Bruce A. Stanton1,3
1
2
High School for Environmental Studies, New York, NY 10019
3
Dept. of Physiology, Dartmouth School of Medicine, Hanover, NH 03775
The euryhaline teleost Fundulus heteroclitus (killifish) adapts to rapid changes in environmental
salinity and thus is an excellent model for studies on the regulation of salt transport. In killifish
adaptation to seawater is accompanied by a transient increase in plasma cortisol levels, a sustained
increase in CFTR chloride ion transporter expression, and increased Cl- secretion by opercular
epithelia1. Stanton and colleagues2 (and unpublished results) found that non-toxic doses of arsenic (5
umol/kg) for 24h significantly reduced both CFTR-mediated opercular Cl- secretion and CFTR gene
expression, and killifish exposed to arsenic were unable to adapt to seawater. Since arsenic inhibits the
transcription factor activity of the glucocorticoid receptor3, the Stanton lab is investigating whether
arsenic blocks adaptation to seawater by inhibiting a cortisol-induced glucocorticoid receptor-mediated
increase in CFTR expression. Serum and glucocorticoid-regulated serine/threonine kinase (SGK),
which was originally discovered in rat mammary tumor cells4, has been shown to stimulate the Cltransport activity of co-expressed CFTR molecules in Xenopus oocytes5, and we have found that a 24h
treatment with arsenic at low concentrations inhibits the activation of SGK in killifish gill tissue (data
not shown). These results suggest that SGK is involved as a signaling intermediate in regulating
CFTR activity and in adaptation to seawater. In order to be able to assess the effects of salinity and
arsenic on SGK gene expression by quantitative PCR we decided to use RT-PCR to clone and
sequence killifish SGK cDNA.
SGK cDNAs encoding highly conserved protein sequences have been cloned and sequenced from a
number of vertebrate species. Figure 1 illustrates the phylogenetic relationships between SGK protein
sequences from six species, including the zebrafish and the pufferfish Tetraodon nigroviridis, that
were obtained from GenBank. The complete deduced amino acid sequences were aligned using the
Clustal program6. Because of the high degree of similarity between the teleost SGK cDNAs, we
mSGK1.aas
rSGK1.aas
hSGK1.aas
ZfSGK.aas
Tn SGK.aas
xSGK.aas
9.0
8
6
4
Sequence divergence (%)
2
0
Figure 1. Phylogenetic relationships
between SGK protein sequences
deduced from mouse (m), rat (r), human
(h), frog (x), zebrafish (zf) and
pufferfish (Tn) cDNAs.
used unique rather than degenerate oligonucleotide PCR primers to amplify fragments of killifish SGK
cDNA. The primer sequences chosen corresponded to regions of zebrafish SGK cDNA that were
conserved in the pufferfish Tetraodon nigroviridis and Takifugu rubripes. Total killifish liver cDNA
was subjected to RT-PCR, and identities of the resulting specific cDNA fragments were verified by
sequencing and BLAST analysis. A composite killifish SGK cDNA sequence was generated from
47
overlapping cDNA fragments and 5’- and 3’-RACE products. The final 1,575 nucleotide sequence
included a short 5’-untranslated region, the entire coding region and part of the 3’-untranslated region
of mRNA. The coding region of killifish SGK cDNA was deposited in GenBank and has been
Figure 2. Alignment of the zebrafish (Zf)
and killifish (Kf) SGK amino acid
sequences that were deduced from cDNA
sequences. The kinase domains are
underlined.
assigned the accession number
AY800243. The deduced 439
amino acid sequence of killifish
SGK is 87% identical to human
SGK1 (data not shown) and 92%
identical to zebrafish SGK (Fig.
2). Killifish SGK cDNA has an in
frame initiation codon upstream of
the codon corresponding to the
initiation codon in zebrafish SGK
cDNA, which predicts an eight
amino acid N-terminal extension.
The functional significance of this
peptide sequence is unknown.
We thank Christine Smith for sequencing RT-PCR products in the Marine DNA Sequencing Center.
JDS is supported by grant P20-RR016463 from the National Center for Research Resources, and JS
and BAS are supported by grant P42-ES07373 from the National Institute of Environmental Health
Sciences. JDS and BAS are investigators of the Center for Membrane Toxicity Studies, which is
supported by grant P30-ES03828 from the NIEHS. CCC was supported by the Mt. Sinai School of
Medicine Secondary Education through Health Program (SETH).
1.
2.
3.
4.
5.
6.
48
Marshall, W.S., T.R. Wimberley, T.D. Singer, S.E. Bryson, and S.D. McCormick. Time course of salinity
adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach. J. Exp. Biol. 202:
1535-1544, 1999.
Stanton, C.R., D. Prescott, A. Lankowski, K. Karlson, J.E. Mickle, J. Shaw, J. Hamilton, and B.A. Stanton.
Arsenic and adaptation to seawater in killifish (Fundulus heteroclitus) Bull. MDIBL 42: 117-119, 2003.
Kaltreider, R.C., A.M. Davis, J.P. Lariviere, and J.W. Hamilton. Arsenic alters the function of the glucocorticoid
receptor as a transcription factor. Environ. Health Persp. 109: 245-251, 2001.
Webster, M.K., L. Goya, Y. Ge, A.C. Maiyar, and G.L. Firestone. Characterization of sgk, a novel member of the
serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol. Cell
Biol. 13: 2031-2040, 1993.
Wagner, C.A., M. Ott, K. Klingel, S. Beck, J. Melzig, B. Friedrich, K.N. Wild, S. Broer, I. Moschen, A. Albers, S.
Waldegger, B. Tummler, M.E. Egan, J.P. Geibel, R. Kandolf, and F. Lang. Effects of the serine/threonine kinase
SGK1 on the epithelial Na(+) channel (ENaC) and CFTR: implications for cystic fibrosis. Cell. Physiol. Biochem. 11:
209-218, 2001.
Higgins, D.G., and P.M. Sharp. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer.
Gene 73: 237-244, 1988.
Determinants of zebrafish (Danio rerio) ES cell germ-line competency
J. Denry Sato1, Peter Alestrom2 and Paul Collodi3
1
2
Dept. of Biochemistry, Norwegian School of Veterinary Science, N-0033 Oslo, Norway
3
Dept. of Animal Sciences, Purdue University, West Lafayette IN 47907
The many favorable characteristics of the zebrafish make it an outstanding model for studies of
vertebrate development, toxicology and human disease. Despite these advantages, one deficiency of
the zebrafish model is the absence of a gene targeting approach by homologous recombination using
embryonic stem (ES) cells. The ability to combine the nearly complete zebrafish genomic sequence
with an ES-cell based strategy for introducing targeted mutations would greatly facilitate studies of
gene function in this model system. Towards the development of a gene-targeting approach, we have
established zebrafish ES cell lines that remain germ-line competent for multiple passages in culture1,4.
The ES cells can be transplanted into host embryos to generate germ-line chimeras and methods have
been developed to introduce targeted mutations into the ES cell cultures by homologous
recombination.
To improve the efficiency of germ-line chimera formation we are working to identify the factors
that are necessary to maintain the ES cells in a
Table 1. Receptor kinase expression in
germ-line competent condition in vitro. Once
germline incompetent zebrafish ES cells.
identified, the factors could be incorporated into
the culture system to improve the frequency of
Sequence ID
Identity (%)
germ-line chimera production and increase the
ActRIB
126/132 (95%)
length of time that germ-line competent ES cells
ActRIIB
100/101 (99%)
can be maintained in vitro for genetic
Alk8
118/121 (97%)
manipulation. The goal of this project is to
BMPR1a
74/76 (97%)
determine the receptor kinase expression profiles
BMPR1b
93/95 (97%)
of
germ-line
competent
and
germ-line
EGFR
116/116 (100%)
incompetent zebrafish ES cells. These studies will
FGFR1
455/462 (98%)
define the growth factors and cytokines to which
FGFR2
95/95 (100%)
pluripotent ES cells are likely to respond, and they
FGFR3
126/129 (97%)
will provide information on which receptor
FGFR4
109/110 (99%)
kinases are required for the maintenance of germIGF1R alpha
124/127 (97%)
line competency.
KDR
126/127 (99%)
c-Kit
PDGFR alpha
TGFbRII
Tie2
116/120 (96%)
119/121 (98%)
125/130 (95%)
105/107 (96%)
Receptor kinase expression by germ-line
incompetent zebrafish ES cells was determined in
a PCR assay using zebrafish-specific primers
corresponding to conserved nucleotide sequences
2,3
in kinase subdomains VIB and VIII . Receptor cDNA fragments were amplified from total RNA
isolated from ES cells that were rendered germ-line incompetent by propagating them in the absence
of RTS34st feeder cells for multiple passages. The identities of expressed receptor kinase cDNA
49
fragments were confirmed by the direct sequencing of PCR products in the Marine DNA Sequencing
Center at MDIBL. As shown in Table 1, cDNA fragments of sixteen receptor kinases were amplified
from total ES cell RNA, and the fragments were at least 95% identical to zebrafish receptor kinase
sequences in GenBank. Only two receptor kinase assays, those for colony stimulating factor 1 (CSF1)
receptor and c-Ret, were negative. At least 11 of the receptor kinases in Table 1 were also expressed by
germ-line competent zebrafish ES cells while EGFR and c-Kit, the receptors for epidermal growth
factor (EGF) and stem cell factor, respectively, were poorly expressed by those cells (data not shown).
These results indicate that differentiating non-germline competent zebrafish ES cells express many
receptor kinases in common with pluripotent and germ-line competent cells, but a subset of these
receptors may prove to be markers for pluipotency or germ-line competency.
We thank Christine Smith for sequencing RT-PCR products in the Marine DNA Sequencing Center.
JDS is supported by grant P20-RR016463 from the National Center for Research Resources, and he is
an investigator of the Center for Membrane Toxicity Studies (NIEHS grant P30-ES03828). PC is
supported by grant R01-GM69384 from the National Institute of General Medicine, and he is the
recipient of an MDIBL New Investigator Award. PA is supported by grant 159329/S10 from the
Norwegian FUGE program.
1.
2.
3.
4.
50
Fan, L., Crodian, J., Liu, X., Alestrom, A., Alestrom, P., and Collodi, P. Zebrafish embryo cells remain pluripotent
and germ-line competent for multiple passages in culture. Zebrafish Vol:1, 21-26, 2004.
Hanks, S.K., Quinn, A.M., and Hunter, T. The protein kinase family: conserved features and deduced phylogeny of
the catalytic domains. Science Vol: 241, 42-52, 1988.
Hanks, S.K., and Hunter, T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain
structure and classification. FASEB J. Vol: 9, 576-596, 1995.
Ma, C., Fan, L., Ganassin, R., Bols, N. and Collodi, P. Production of zebrafish germ-line chimeras from embryo cell
cultures, Proc. Natl. Acad. Sci. USA Vol: 98, 2461-2466, 2001.
Cloning and molecular identification of a TASK-1 channel cDNA and protein in the rectal gland
of the spiny dogfish shark, Squalus acanthias
Connor Telles1, William Motley2, Sarah Decker1, Eleanor Beltz3, Christine Smith4,
and J. N. Forrest, Jr.1
1
2
3
4
The dominant conductive basolateral K+ channel in the shark rectal gland (SRG) is unknown. The
SRG, composed of homogenous tubules of a single cell type, is an important model for secondary
active chloride transport1. Apical Cl- conductance in this tissue is tightly linked to a basolateral K+
conductance. Since the secreted fluid of the gland is essentially potassium free, K+ entry through the
basolateral Na-K-ATPase pump and Na-K-2Cl cotransporter must be accompanied by basolateral K+
exit to maintain the driving force needed for Cl- secretion5. An inwardly rectifying K+ channel in the
SRG was identified in electrophysiological studies6 and subsequently cloned (KIR 6.1)10 by our lab as
was the shark homolog of the human KvLQT19. However, based on inhibitor studies these channels
account for only for a minor part of the total K+ conductance in the SRG2.
We carried out further experiments to identify the K+ channel that plays a dominant role in chloride
secretion in this model tissue. We first performed perfusion studies2,3 with numerous K+ channel
inhibitors and perfusate solutions of varying pH which narrowed the search to the Two-Pore-Domain
(4TM 2P/KCNK/K2P) family7 of potassium channels. In this present study, we have used molecular
biological techniques to clone this 4TM 2P channel.
Eighteen pairs of degenerate primers were designed to target regions of high amino acid homology
in available mammalian and teleost 2P family potassium channel subtypes: TWIK, THIK, TASK,
TREK, and TRAAK. Using degenerate PCR and shark cDNA template reverse transcribed from RNA
extracted from shark tissues, one primer pair amplified a putative TASK-1 fragment (394 bp) in the
shark rectal gland, brain, gill, and kidney. The forward and reverse primers that amplified TASK-1 in
the shark tissues were: 5’- ATCCCCCTGACCCTGGTNATGTTYCA -3’ and 5’
GCACCACCAGGTTCAG GAANGCNCCDAT -3’ respectively and the PCR conditions for the
Touch Down PCR protocol were 94º for three minutes, 40 cycles of (95º 0:45, 65º 1:00 – 0.5º/cycle,
72º 1:30) and 72 ºC for 10 min. 5’ and 3’ RACE PCR was used to obtain the entire 3’ sequence and a
partial 5’ sequence of the shark gene. Genome walking (BD Biosciences GenomeWalker Universal
Kit) with shark genomic DNA was necessary to obtain the remainder of the 5’ sequence, including 335
bp of untranslated region sequence upstream of the methionine start codon.
Figure 1. Cloning of shark TASK-1 channel. A: Degenerate PCR fragments (394 bp) of shark TASK-1 with cDNA
template from SRG (lane 7), brain (lane 8), kidney (lane 9) and gill (lane 10). Lanes 2-5 represent a degenerate primer pair
that did not amplify template cDNA. cDNA was omitted in the PCR reaction in lane 6 and 11). 1 Kb plus ladder (lane 1).
51
B: full length clone, confirmed with nested PCR. 1 Kb plus ladder (lane 1), 1386 bp primary product (lane 2), 1282 bp
nested product (lane 3). C: Cloning strategy for obtaining full-length sequence of shark TASK-1. 394 bp fragment from
degenerate PCR identified as TASK-1. Using this sequence, primers were constructed for 5’ and 3’ RACE which yielded a
694 bp 3’ product that included the stop codon, and a partial 5’ product of 592 bp. To obtain the remainder of the 5’
sequence Genome Walking (GW) was used to obtain a 428 bp product which included the start codon and 334 bp of
upstream untranslated region. Using this complete sequence, primers were designed to amplify a 1282 bp full length
clone. * Position of the start and stop sequence of shark TASK-1
With the complete sequence information we designed primers to amplify full-length shark TASK-1
cDNA (Figure 1). This full length product was then cloned in TOPO TA cloning vectors (Invitrogen)
and transformed into One Shot TOP10 Chemically Competent E. Coli cells (Invitrogen). The full
length clone (1282bp) had an 1128 bp open reading frame encoding a protein of 375 amino acids
compared to the 1188 bp human TASK-1 of 394 amino acids. The sequence was then confirmed with
bidirectional sequencing at the MDIBL sequencing center using vector specific primers M13 F and
M13R yielding a nucleotide sequence that was 71% conserved between shark and human isoforms.
Figure 2.
Amino acid
alignment of shark and
human TASK-1 proteins.
Transmembrane
domains
(TM 1-4) and pore domains
(P 1-2) are identified with
boxes. Cytoplasmic domains
are underlined. The GYG/
GFG K+ selectivity motif
conserved in each pore region
is shaded with a gray box.
Human amino acid residues
that are identical to shark
TASK-1 are represented as
dots. Dashes indicate gaps
introduced when necessary
for proper alignment between
the two sequences.
PROTEIN DOMAIN
AA CONSERVATION
Whole Protein
316/394
80%
Cytoplasmic 1
8/8
100%
TM1
21/21
100%
P1
23/24
95.8%
TM2
19/21
90.5%
Cytoplasmic 2
21/30
70%
TM3
19/20
95%
P2
23/24
95.8%
TM 4
21/21
100%
Cytoplasmic 3
101/151
66.9%
TABLE 1. Percent conservation of amino acid sequence
comparing shark TASK-1 sequence to the human TASK-1
isoform10.
Figure 3. Western blot demonstrating that a
TASK-1 antibody recognizes a ~43kD protein
in crude SRG lysate. Protein concentration was
incrementally increased in lanes 1-5 (~200µg
maximum).
Shark TASK-1 was 80% identical at the amino acid level to the human TASK-1 protein (Table 1).
52
Major structural features of the human protein were conserved in the shark homolog, including the four
transmembrane segments (M1-M4), the 2P domains (P1 and P2), short NH2-terminal and long COOHterminal cytoplasmic parts, and an extended extracellular loop between M1 and P17 when analyzed via
Clustal W alignment (Figure 2 and Table 1). The presence of a TASK-1 protein in the rectal gland was
confirmed with Western Blot analysis using commercially available (Sigma) antibody raised against
mammalian TASK-1 revealing a 43kD protein (Figure 3). The target epitope of the Sigma antibody
differed in 3 amino acid residues from shark sequence.
The mammalian epitope was
EDEKRDAEHRALLTRNGQ (TASK-1252-269) and shark sequence was EDEKRDAEQKALLIRNGQ
(differences underlined).
With more than 70 types, K+ channels are the most diverse group of ion channels. These channels
are classified by their number of transmembrane and pore forming domains. The two-pore, four
transmembrane domain family of potassium channels includes the TWIK, THIK, TASK, TREK, and
TRESK subfamilies7. The first of these cloned from human tissue was TWIK-1 (Tandem of Pdomains in a Weakly Inward rectifying K+ channel). The TASK (TWIK-related acid-sensitive K+
channel) gene family encompasses 5 members, which encode background K+ channels that help set the
resting membrane potential. These channels are characterized by their sensitivity to changes in
extracellular pH. The TASK-1 subtype has been identified in human pancreas, placenta, brain, heart,
lung, and kidney6. TASK-1 has also been shown to have segment specific expression in the human
nephron, being present in the glomerulus and distal nephron segments8.
Shark rectal gland TASK-1 is the oldest family member identified to date, and the first TASK
orthologue found in lower marine vertebrates. These studies suggest that TASK-1 channels play a
major role in basolateral K+ conductance in the shark rectal gland model and provide the first evidence
that TASK-1 channels are coupled to chloride secretion.
1.
2.
Burger W. Function of the rectal gland of the spiny dogfish. Science 131:670-671, 1960.
Decker S., C. Klein, M. Ratner, C. Kelley, M. Epstein, K. Burks, W. Motley, A. Peters, and J.N. Forrest Jr.
Effects of quinidine and other K+ channel inhibitors on chloride secretion in the rectal gland of the spiny dogfish,
Squalus acanthias. Bull. Mt Desert Isl. Biol. Lab. 43: 30-32, 2004.
3. Decker, S., C. Kelley, E. Beltz, C. Telles, M. Ratner, K. Burks, M. Epstein, A. Peters, W. Motley, and J. N.
Forrest Jr. Inhibitors of 4TM 2P potassium channels inhibit chloride secretion in the perfused rectal gland of the spiny
dogfish (Squalus acanthias). Bull. Mt Desert Isl. Biol. 44: 2005.
4. Duprat F., F. Lesage, M. Fink, R. Reyes, C. Heurteaux and M. Lazdunski. TASK, a human background K+
channel to sense external pH variations. EMBO J. 16(17): 5464-5471, 1997.
5. Forrest JN Jr. Cellular and molecular biology of chloride secretion in the shark rectal gland: regulation by adenosine
receptors. Kidney Int. 49(6):1557-62, 1996.
6. Gogelein H, R. Greger, E. Schlatter. Potassium channels in the basolateral membrane of the rectal gland of Squalus
acanthias. Regulation and inhibitors Pflugers Arch. 409(1-2):107-13, 1987.
7. Lesage F and M. Lazdunski. Molecular and functional properties of two-pore-domain potassium channels. Am J
Physiol Renal Physiol. 279(5): F793-F801, 200.
8. Levy, D.I., H. Velazquez, S. Goldstein, and D. Brockenhauer. Segment specific expression of 2P domain potassium
channel genes in human nephron. Kidney Int. 65: 918-926, 2004.
9. Waldegger S, B. Fakler, M. Bleich, P. Barth, A. Hopf, U. Schulte, A.E. Busch, S.G. Aller, J.N. Forrest Jr, R.
Greger, and F. Lang. Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of
Squalus acanthias. Pflugers Arch. 437(2):298-304, 1999.
10. Weber G, S. Aller, F.N. Plesch, and J.N. Forrest Jr. Identification and Partial Sequencing of a KIR 6.1 potassium
channel from the shark rectal gland and a ROM-K potassium channel from skate kidney. Bull. Mt Desert Isl. Biol. Lab.
38:112-113, 1999.
53
Ribosomal binding protein L8 homologue detected in the gills of longhorn sculpin,
Myoxocephalus octodecimspinosus
1
Curtis E. Lanier1, Julia R. Curtis-Burnes, & James B. Claiborne1
Department of Biology, Georgia Southern, University, Statesboro, GA 30460
2
Wellesley College, 106 Central Street Wellesley, MA 02481
Analysis of gene expression using differential detection methods is a very valuable tool in
molecular biology. Once the gene of interest is characterized, the varying expression levels of mRNAs
must be confirmed. In order to confirm the identified transcript expression levels, a standard gene that
does not change in expression must be utilized to accurately determine differential expression patterns.
Choe et al. 2 showed that the highly conserved ribosomal binding protein L8 mRNA expression
remains constant during salinity and acid-base changes in the Atlantic stingray (Dasyatis Sabina). The
L8 protein complex consisting of L7/L12 and L10 in ribosomes is assembled on the conserved region
of 23 S rRNA and is termed the GTPase-associated domain 1 and is highly conserved. Here we show
the partial cDNA sequence detection of the ribosomal binding protein L8 gene in longhorn sculpin
(Myoxocephalus octodecimspinosus) for future use in differential gene expression analysis.
Sculpin gill total RNA isolation was performed using Tri Reagent (MRC, Inc.) according to the
company protocol. Reverse transcription using oligo dT primers was used to synthesis sculpin cDNA
(Invitrogen). Specific primers for ribosomal binding sequences in mammals were generated (L8F1
sense and L8R2 antisense) 2: 5′-AAGAAGGCTGAGTTGAACATTGGA-3′ in combination with
5′-TGTACTTGTGATAAGCCGAGCAG-3′ . Actin and NHE3 primers3 were used as controls for the
PCR reaction. The PCR reaction cycle parameters were performed in 50 µl volumes: initial
denaturation at 95 οC for 5 min., 25 cycle denaturation at 95 οC for 1 min., cycle annealing at 55 οC for
1 min, cycle extension at 72 οC for 1 min., and a final extension at 72 οC for 10 min. A PCR product of
approximately 400 bp was detected by gel electrophoresis and cloned into a Topo plasmid vector
system (Invitrogen) according to manufacturers protocol for sequencing at the
DNA Sequence facility Mount Desert Island Biological Laboratory.
Fig. 1: PCR product analysis by 1% agarose gel electrophoresis. DNA marker shown at left.
Lane #1 shows band of interest of 400 bp. NHE3 and actin positive controls shown in lanes 2
& 3 respectively.
Genebank sequence analysis of the 400 bp PCR product shown in Figure
1 shows a high homology to other known ribosomal binding L8 cDNAs. Future work will use the
longhorn sculpin L8 mRNA expression as a control for standardizing differential expression of
longhorn sculpin NHE3 previously characterized by Lanier and Claiborne 3.
Funding was provided by NSF IBN-0111073 to J.B.C. and REU Site award at MDIBL (NSF DBI0139190).
1.
2.
3.
54
Beauclerk, Alan A. D., Eric Cundliffe, and Jan Dijk. The Binding Site for Ribosomal Protein Complex L8 within
23 S Ribosomal RNA of Escherichia coli. J Bio. Chem. 259: 6559-6563, 1984.
Choe, Keith P., Jill W. Verlander, Charles S. Wingo, and David H. Evans. A putative H+-K+ -ATPase in the
Atlantic Stingray, Dasyatis sabina primary sequence and expression in gills. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 287: R981–R991, 2004.
Lanier, Curtis E. and James B. Claiborne. Analysis of branchial longhorn sculpin (Myoxocephalus
octodecimspinosus) Na+/H+ exchanger isoform 3 using Northern hybridization. Bull. Mt. Desert Isl. Biol. Lab. 43: 107,
2004.
Evolutionary and comparative analysis of aquaporin water channel genes in fish
1
Christopher P. Cutler1, Lara Meischke2 and Gordon Cramb2
Department of Biology, Georgia Southern University, Georgia GA 30460, and 2School of Biology,
University of St Andrews, St Andrews, Fife, Scotland, UK KY16 9TS
The role of aquaporin water channels in some teleost fish has been described for some (orthologues
of mammalian) aquaporins, especially aquaporins1 (AQP) 0, 1 and 3, however to date the role of
aquaporins in other more ancient fish lineages such as the Elasmobranches and Agnathans remains
almost completely uninvestigated. The goal of this project is to identify homologous aquaporin genes
from Elasmobranches (dogfish shark; Squalus acanthias and bullshark; Carcharhinus leucas) and
Agnathan (hagfish; Myxine glutinosa) species, and from ancient teleost fish species (eels; Anguilla
anguilla and Anguilla rostrata) in order to answer a number of questions regarding the kind of
aquaporin homologues that are present in these species and their relationship to aquaporins found in
higher vertebrates. A longer term objective is begin to discern the physiological role, that any
aquaporins found in Agnatha, Elasmobranches or Teleosts play.
The initial approach to identifying aquaporin genes in the dogfish shark and in hagfish, using
degenerate PCR cloning was unsuccessful. However, a partial cDNA fragment of an aquaporin gene
had previously been isolated using this approach in the bullshark. Cloning and sequencing of the
remainder of this cDNA was completed using 5’ and 3’ RACE PCR and bullshark kidney total RNA.
The bullshark aquaporin (AQP1e) cDNA sequence isolated was ~1.1kb in size and contained an open
reading frame of 256 amino acids. Comparison of the central conserved region of the bullshark amino
acid sequence with the sequences of the various aquaporins found in mammals (humans), revealed that
bullshark AQP1e had equally similar levels of homology to human AQP1 (47.9%), AQP2 (47.8%) and
AQP5 (48.9%) with slightly lower levels of homology to other water-selective aquaporins, AQP0, 4
and 6 (39-43%). Still lower levels of homology were seen between bullshark AQP1e and human AQP8
(27.9%), aquaglyceroporins (AQP3, 7, 9 and 10; 20-26%) and more distantly related aquaporin
homologues (AQP11 and 12; 14-23%). The similar level of homology between bullshark AQP1e and
human AQP’s 1, 2 and 5, suggests that bullshark AQP1e may represent an Elasmobranch descendent of
a common ancestral gene, which was subsequently duplicated to be become AQP1, 2 and 5 during the
further evolution that led to higher vertebrates. Further information that lends some weight to this idea
comes from the analysis of teleost sequence data. Analysis of the current fugu (Fugu rubripes) and
zebrafish (Danio rerio) databases as well as cDNA sequence data for eel (Anguilla anguilla)
aquaporins (unpublished), reveals that while there are homologues of AQP1 genes present in these fish,
no orthologues of either AQP2 or AQP5 appear to exist. This further suggests that the ancestral gene
may have been an orthologue of AQP1 from which the AQP2 and AQP5 genes developed presumably
by gene duplications and accumulation of subsequent mutations. Clearly more information from other
Elasmobranches, Agnathans or Teleosts (or even from Actinistia or Dipnoi species) is required to
confirm these hypotheses. With the increase in sequence data from bullshark AQP1e, as well as in
other gene databases, a further attempt has been made to amplify aquaporin fragments from the dogfish
shark and hagfish cDNA using redesigned degenerate PCR primers, this has resulted in the production
of three potential aquaporin cDNA fragments. Subsequent work will focus on cloning and sequencing
these fragments to determine if they encode aquaporin homologues.
Supported by a New Investigator Award Fellowship from MDIBL and by a grant from the Natural
Environment Research Council, UK, with assistance from Eda Kapinova (MDIBL Silk Fellow).
1. Cutler C. P. and G. Cramb. Molecular Physiology of Osmoregulation in Eels and Other Teleosts: The Role of
Transporter Isoforms and Gene Duplication. Comp. Physiol. Biochem. 130: 551-564, 2001.
55
Persistent neurogenesis in semi-isolated brains of the crab Carcinus maenas
David Sandeman
Wellesley College
Wellesley, MA 02481
Neurogenesis in proliferation zones in certain clusters of neurons in crustacean brains persists
throughout their lives1. Neuron precursors in these animals label with bromodeoxyuridine (BrdU) if this
is present when the cells pass through the S phase of their cell cycle. A single injection of BrdU with an
incubation time of 3-6 hours is sufficient to routinely label cells in the proliferation zones. Crabs that
are too small to inject can be labeled by immersing them in a sea water/BrdU solution. Neurogenesis in
the shore crab, Carcinus maenas was studied at the MDIBL to determine the following: 1, whether or
not a different number of new neurons is produced at different times of the day in the intact animals as
is the case for lobsters 2; 2, whether or not perfused, semi-isolated crab brains would, at room
temperature, remain physiologically active over the 3-6 hour incubation period, and 3, whether or not
neurogenesis would be detectable in such preparations when perfused with a solution of BrdU and
saline.
The first series of experiments were carried out in conjunction with the Beltz group (at the MDIBL
and in light-controlled rooms in Wellesley) on small crabs that were immersed in a sea water/BrdU
solution of 2mg/ml. It was found that, unlike lobsters and crayfish, Carcinus does not have a light
sensitive control on the rate of neurogenesis although there do appear to be tidal effects (see details in
Abstract of Beltz et al., in this issue).
The present study on Carcinus established that semi-isolated crab brain preparations, perfused at
rates as low as 0.45ml/min and at room temperature, remain physiologically active for 6 hours or more.
This was confirmed by stimulating the nerve root carrying afferent information from tactile receptors
around the eyes to the brain, which results in a reflexive withdrawal of the eyes 3. Neurons in the
proliferation zones of these preparations, perfused with 1mg/ml BrdU in saline for 6 hours, label in the
same way as they do in the intact animals. Such labeling is robust and persists in preparations perfused
with low Ca2+ or sea water (high Mg2+), treatments that reversibly block the eye withdrawal reflex.
Preparations that were first flushed with saline and then placed in a dish containing BrdU and saline but
without continued perfusion, were compared with preparations that were placed in the BrdU saline
mixture immediately after dissection, also without continued perfusion. In both cases, reflexive
responses were abolished, but cells labeled in the proliferation zones of both brains. A tentative
conclusion to be tested is that neurogenesis in the brains of Carcinus is not modulated in the short term
by afferent input to the brain, nor is it dependent on the maintenance of sensory-motor reflexive
synaptic pathways. In addition, the insensitivity of the Carcinus brain preparation to operational
trauma, and the ability to deliver agents directly to active cell proliferation zones, could provide us with
a new approach to the study of environmental toxins on neurogenesis in central nervous systems.
1.
Beltz, B. S. and D. C. Sandeman. Regulation of life-long neurogenesis in the decapod crustacean brain.
Arthropod
Structure and Development 32: 39-60 (2003).
2. Goergen, E., L. A. Bagay, K. Rehm, J. L. Benton, and B. S. Beltz. Circadian control of neurogenesis. Journal of
Neurobiology 53: 90-95 (2002).
3. Sandeman D.C. (1969) The synaptic link between the sensory and motor axons in the crab eye withdrawal reflex. J
Exp Biol 50:87-98
Sponsored by National Science Foundation Grant numbers IBN-0344448 (BSB) and DBI-0139190 (MDIBL), Fiske and
Staley Awards from Wellesley College, and the Maren Foundation (MDIBL).
56
Branchial expression of COX-2, nNOS, and transporters following rapid transfer of Fundulus
heteroclitus from fresh water to seawater
Keith P. Choe1, Justin C. Havird1, James B. Claiborne2, & David H. Evans1
1
2
Department of Zoology, University of Florida, Gainesville, FL 32611
Department of Biology, Georgia Southern University, Statesboro, GA 30460
Killifish can tolerate acute transfer from fresh water to seawater with only minor, transient
elevations in blood plasma osmolarity and Na+ concentration5 by rapidly stimulating NaCl secretion6,
and presumably inhibiting ion absorption. Although the key ion transporters involved in NaCl
secretion have been characterized and candidate transporters for ion absorption have been identified3,
little is known about transcriptional regulation of these transporters immediately following acute
salinity transfers. Transcriptional and post-transcriptional regulation of these transporters is thought to
be at least partially mediated by paracrine signaling pathways2. For example, we previously used
pharmacological inhibitors to demonstrate that prostanoids and nitric oxide (NO) inhibit short circuit
currents in the killifish opercular membrane (a model for salt secretion in teleost gills), suggesting that
cyclooxygenase (COX) and nitric oxide synthase (NOS) regulate NaCl transport in teleost gills4. In
this study, we measured a time course of mRNA expression changes for COX2, nNOS, and several
transporters for 24 hours following acute transfer from fresh water to seawater to determine if, and
how, transcription of these paracrine agents and transporters is altered to stimulate NaCl secretion and
inhibit NaCl absorption in the gills of teleosts.
Twenty killifish were shipped from the Mount Desert Island Biological Laboratory (MDIBL) to the
University of Florida and held in a 380 l tank containing buffered Gainesville tap water (fresh water:
conductivity ~1400 µs, pH ~8.2, 20˚C) for 37 days. Fifteen killifish were then transferred directly to
another 380 l tank containing buffered seawater (conductivity ~47 ms, pH ~8.2, 20˚C). After 3, 8, and
24 hours of exposure to seawater, killifish were removed and their gills were frozen; the five killifish
that remained in fresh water were used as controls. Poly A RNA was reverse transcribed from total
RNA as described previously1 and the resulting cDNA was subjected to PCR in the presence of
SYBR® Green (Molecular Probes, Inc., Eugene Oregon) binding dye at MDIBL. All PCR reactions
were run in triplicate and included 0.2 ul of cDNA (2.0 ul of a 1/10 dilution of original cDNA), 7.4
pmoles of each primer, and SYBR® Green Master Mix (Applied Biosystems, Foster City, CA) in a
total volume of 25 ul. Primers were designed from Fundulus specific cDNA sequences that we either
cloned using degenerate primers (L8, COX2:AY532639, nNOS:AY533030, NHE3:AY818825, NHE2,
and V-ATPase) or derived from GenBank (CFTR:AF000271, NKCC:AY533706, and
NKA1:AY057072). All real-time PCR reactions were run in a Stratagene MX4000 Real-Time
Quantitative PCR system (Stratagene, La Jolla, CA) with standard cycling parameters, and melting
curve analysis was used to verify the amplification of a single product in each well. Relative gene
expression was calculated from a relative standard curve that used serial dilutions of a pooled gill
cDNA sample as the template, and all results were normalized to ribosomal protein L8, a highly
conserved gene for which expression in the gills remains constant during salinity changes (unpublished
observation). ANOVAs with Dunnett’s post hoc tests were used to compare seawater to freshwater
control expression levels.
Interestingly, expression of the three NaCl secretion transporters increased with variable kinetics
and magnitudes. For example, NKCC and NKA1 expression increased only moderately after 24 hours,
but CFTR expression was elevated as early as three hours, reaching a maximum of 2.8 fold at eight
57
hours (Table 1). Our results confirm a previous study that observed rapid increases in CFTR
expression following transfer from fresh water to seawater7, and suggest that the apical Cl- channel
may be a rate-limiting step in the NaCl secretory mechanism. Expression of the basolateral NKCC and
NKA1 transporters may be constitutively high enough so that rapid transcription is not required. Of
the three Na+ and/or acid transporters that could potentially have a role in active Na+ absorption, only
NHE3 had a significant decrease in expression following transfer to seawater (Table 1). This is one of
the first demonstrations that salinity influences NHE expression in the gills of teleosts, and suggests
that NHE3 may be responsible for Na+ absorption. COX2 expression had the largest change in
expression for any mRNA with a transient, 3.4-fold increase three hours after transfer to seawater
(Table 1). Alternatively, nNOS expression did not change and had the latest threshold cycle numbers,
suggesting that its expression levels are low (unpublished observation). These data agree with our
previous pharmacological evidence that prostanoids have a larger quantitative role in controlling NaCl
transport in killifish opercular membranes than NO4. Further work is being done to localize COX2 and
NHE3 protein in the gills and to determine the effects of transfer from seawater the fresh water on the
same transcripts.
Table 1. Relative expression of putative paracrine and ion transport enzymes in the gills of killifish following acute transfer
from fresh water (FW) to seawater. Each cDNA was quantified by real-time RT-PCR and normalized to ribosomal protein
L8. N=5, * p < 0.05.
mRNA
FW
3h
8h
24 h
COX2
1.00 ±0.14
3.38 ±0.61*
1.39 ±0.28
1.04 ±0.20
nNOS
1.00 ±0.60
1.04 ±0.93
0.65 ±0.54
0.23 ±0.18
NKCC
1.00 ±0.13
1.20 ±0.16
1.07 ±0.16
1.87 ±0.27*
CFTR
1.00 ±0.13
1.99 ±0.28
2.77 ±0.41*
1.98 ±0.43
NKA1
1.00 ±0.09
1.07 ±0.10
1.04 ±0.12
1.38 ±0.05*
NHE3
1.00 ±0.08
0.98 ±0.05
0.49 ±0.10*
0.49 ±0.09*
NHE2
1.00 ±0.32
0.74 ±0.15
1.35 ±0.51
3.26 ±1.93
V-ATPase
1.00 ±0.09
0.98 ±0.07
0.78 ±0.08
0.89 ±0.08
This study was funded by National Science Foundation grant IBN-0089943 to D.H.E. Transporter
abbreviations are: NKCC = Na+/K+/2Cl- cotransporter, NKA1 = Na+/K+-ATPase α1, NHE = Na+/H+
exchanger, and V-ATPase = Vacuolar H+-ATPase.
1.
2.
3.
4.
5.
6.
7.
58
Choe, K.P., J.W. Verlander, C.S. Wingo, and D.H. Evans, A putative H+/K+-ATPase in the Atlantic stingray,
Dasyatis sabina: primary sequence and expression in gills. Am. J. Physiol., 287: R981-R991, 2004.
Evans, D.H., Cell signaling and ion transport across the fish gill epithelium. J. Exp. Zool., 293: 336-347, 2002.
Evans, D.H., P.M. Piermarini, and K.P. Choe, The multifunctional fish gill: dominant site of gas exchange,
osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev., 85: 97-177, 2005.
Evans, D.H., R.E. Rose, J.M. Roeser, and J.D. Stidham, NaCl transport across the opercular epithelium of Fundulus
heteroclitus is inhibited by an endothelin to NO, superoxide, and prostanoid signaling axis. Am. J. Physiol., 286: R560568, 2004.
Jacob, W.F. and M.H. Taylor, The time course of seawater acclimation in Fundulus heteroclitus L. J. Exp. Zool., 228:
33-39, 1983.
Marshall, W.S., T.R. Emberley, T.D. Singer, S.E. Bryson, and S.D. McCormick, Time course of salinity
adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: A multivariable approach. J. Exp. Biol.,
202: 1535-1544, 1999.
Singer, T.D., T. Stephen J, W.S. Marshall, and C.F. Higgins, A divergent CFTR homologue: highly regulated salt
transport in the euryhaline teleost F. heteroclitus. Am. J. Physiol., 274: C715-C723, 1998.
Quantitative expression of branchial carbonic anhydrase in the euryhaline green crab, Carcinus
maenas, in response to stepwise dilutions in salinity
Raymond P. Henry1,2, Kim L. Thomason1,2, & David W. Towle2
1
Auburn University, Auburn, AL 36849
2
The enzyme carbonic anhydrase (CA) has been shown to be a central component in the molecular
suite of adaptations to low salinity in euryhaline crustaceans. The cytoplasmic isoform is believed to
support general cation and anion transport by providing counterions in the form of H+ and HCO3through the catalyzed hydration of respiratory CO2.1 Cytoplasmic CA activity is highly salinitysensitive, being induced by six fold when green crabs are transferred from high (32 ppt) to low (10 ppt)
salinity.2 CA induction is believed to be under transcriptional regulation, as increases in CA mRNA
expression were shown to immediately precede increases in CA protein-specific activity during the
acute phase of low salinity adaptation.3 The overwhelming majority of studies on CA induction have
been done at only one low salinity value, 10 ppt. It is not known, therefore, whether CA induction is
regulated with any degree of precision by differences in the magnitude of salinity reductions and
whether any such putative regulation occurs at the transcriptional or post-translational level.
In order to test the effects of intermediate salinity reductions on CA activity and expression, green
crabs, acclimated to 32 ppt, were directly transferred to one of the following salinities: 25, 20, 15, or
10 ppt. Crabs were allowed to acclimate for 7 days, the time needed for fully acclimated levels of CA
activity to be reached after low salinity exposure. At that time, crabs were anesthetized in crushed ice,
hemolymph samples were taken from the infrabranchial sinus at the base of the walking legs, and crabs
were killed by exsanguination. Anterior, respiratory gills (G4) were used as control tissue, and
posterior, ion transporting gills (G8) served as the experimental tissue. G4 and G8 from the right side
of each crab were assayed for CA activity immediately upon dissection, and the corresponding gills
from the left side of each crab were used for total RNA extraction. The RNA was reverse transcribed,
and gene-specific primers for CA were used with the cDNA template for determination of mRNA
expression by quantitative PCR (Stratagene MX 4000). The sample with the highest level of CA
Figure 1. CA activity (µmol CO2
mg protein-1 min-1) in anterior
(G4, black bars) and posterior
(G8, gray bars) of green crabs
vs acclimation salinity.
Mean + SEM (N = 5-6).
59
activity was used to construct an internal standard curve against with other samples were compared.
Figure 2. CA mRNA relative
expression in anterior (G4, black bars)
and posterior (G8, gray bars) of green
crabs vs acclimation salinity. Mean +
SEM. (N = 5-6).
Anterior gills did not show any increase in CA activity in response to low salinity and thus served
as a control tissue (Fig. 1). CA activity in posterior gills, however, increased progressively with
decreasing salinity from 32 to 15 ppt, at which there was an approximate 8 fold induction.
Interestingly, CA activity then decreased in crabs transferred directly to 10 ppt. This is in contrast to
another, more euryhaline crustacean, Callinectes sapidus, in which CA activity in the posterior gills
continues to increase from 35 to 5 ppt.4 C. maenas is less euryhaline than C. sapidus, being found in
nature down to only 8 ppt, as opposed to 0 ppt for C. sapidus.
CA mRNA expression followed the same pattern as that seen for CA activity. There were no
significant changes in mRNA expression in anterior gills (Fig. 2), but mRNA expression in posterior
gills increased with decreasing salinity. As with CA activity, CA mRNA expression peaked at 15 ppt
and then decreased in crabs acclimated to 10 ppt.
The general pattern appears to be that acclimation salinity controls the level of CA mRNA
expression, and that, in turn, determines the level of CA activity. CA activity appears to be dependent
on the synthesis of different levels of the enzyme. It is also interesting to note that the maximum level
of CA expression occurs at or near 15 ppt. Below that value, the induction mechanism appears to
become less responsive to any further decrease in salinity. It may be, therefore, that the lower limit of
salinity tolerance is set by the value at which the induction mechanism becomes refractory and can no
longer increase the expression of the critical transport proteins. Green crabs are considered to be
moderately euryhaline and are rarely found in nature in salinities below 10 ppt, so it is possible that
these crabs were close to their physiological limit. It will be interesting to compare the pattern of CA
expression in a more euryhaline species, such as Callinectes sapidus, which can survive down to 0 ppt.
Supported by NSF IBN 02-30005 and by an award from the Thomas H. Maren Foundation.
1. Henry, R.P. Multiple functions of gill carbonic anhydrase. J. Exp. Zool. 248:19-24. 1988.
60
2. Henry, R.P., Garrelts, E.E., McCarty, M.M., and Towle, D.W. Differential induction of branchial carbonic anhydrase
and Na+/K+ ATPase in the euryhaline crab, Carcinus maenas, in response to low salinity exposure. J. Exp. Zool.
292:595-603. 2002.
3. Henry, R.P., Gehnrich, S., Weihrauch, D., and Towle, D.W. Salinity-mediated carbonic anhydrase induction in the
gills of the euryhaline green crab, Carcinus maenas. Comp. Biochem. Physiol. 136A:243-258. 2003.
4. Henry, R.P., and S.A. Watts. Early carbonic anhydrase induction in the gills of the blue crab, Callinectes sapidus,
during low salinity acclimation is independent of ornithine decarboxylase activity. J. Exp. Zool. 289:350-358. 2001.
61
A hemolymph-borne carbonic anhydrase repressor is effective at the level of CA mRNA
expression in the euryhaline green crab, Carcinus maenas
Katherine Smith1,3 & Raymond Henry2,3
1
Department of Biology
University of New Hampshire, Durham, N.H.
2
Auburn University, Auburn, AL 36849
3
The enzyme carbonic anhydrase (CA) is a central molecular component of the mechanism of active
ion uptake in the gills of euryhaline crustaceans. In the posterior, ion transporting gills of the
euryhaline green crab, Carcinus maenas, CA activity is induced by about 6 fold in response to transfer
from 32 ppt to 10 ppt salinity.1 This induction is under transcriptional control: low salinity results in
an increase in CA expression, as measured by an increase in CA mRNA, and that, in turn, leads to the
synthesis of new enzyme.2 CA gene expression, in turn, appears to be under the control of a repressor
compound found in the major endocrine complex of the crab, the eyestalk. Present in the eyestalk of
crabs acclimated to high salinity, this compound keeps CA expression (and therefore, CA activity) at
low, baseline levels. When crabs are exposed to low salinity, the repressor is down-regulated, thus
allowing for increased CA expression, which results in the induction of CA activity.3,4 Recently, it
was shown that the putative CA repressor is also found in the hemolymph of green crabs and that it
inhibits normal salinity-mediated induction of CA activity.5 As with the results from studies on the
repressor from the eyestalks, the CA inhibitor is present and effective in the hemolymph of crabs that
are acclimated to 32 ppt but absent from crabs treated with eyestalk ablation or from crabs acclimated
to low salinity. It is believed that the hemolymph is the route of transport from the eyestalk to the
target tissue, the gill. This report tests the hypothesis that the hemolymph-borne CA repressor acts at
the level of gene expression in the posterior gills, preventing the salinity-stimulated increase in CA
mRNA and thus preventing CA induction.
Green crabs were collected locally and held at 32 ppt salinity. One subset of crabs was transferred
directly to 10 ppt for 4 days with no other treatment. A second subset of crabs was transferred to 10
ppt and given twice daily injections of 2 mL of hemolymph taken from crabs acclimated to 32 ppt, a
salinity at which the CA repressor is known to be present.5 The gills were then dissected out and used
in the following manner. Anterior (G4) and posterior (G8) gills from the left side of the crab were
assayed for CA activity, and G4 and G8 from the right side of the crab were used for total RNA
extraction. The RNA was then reverse transcribed, and gene specific primers were used to monitor CA
mRNA levels through quantitative PCR (Stratagene MX 4000). The sample with the highest level of
CA activity was used to construct an internal standard curve against which the other samples were
compared.
For the 32 ppt acclimated crabs, CA activity was uniformly low in anterior and posterior gills (Fig.
1). There was a 3-fold induction of CA activity in the posterior gills in crabs transferred to 10 ppt for 4
days (174 + 22 to 575 + 51 µmol CO2 mg protein-1 min-1). This level of induction was reduced by 84%
(239 µmol CO2 mg protein-1 min-1; Fig 1) by the hemolymph injections. CA activity in the anterior
gills, the control, non-ion transporting tissue, was unaffected by either low salinity or hemolymph
injection.
62
Figure 1. Carbonic anhydrase
activity (µmol CO2 mg protein-1
min-1) in anterior (G4, black bars)
and posterior (G8, gray bars) gills
of C. maenas given the following
treatments: 32 = crabs acclimated
to 32 ppt; 10 = crabs transferred
to 10 ppt for 4 days; 10inj = crabs
transferred to 10 ppt for 4 days
and given injections of 2 mL of
hemolymph
from
32
ppt
acclimated crabs twice daily.
Mean + SEM (N = 6).
The same pattern was seen for CA mRNA expression (Fig. 2). Levels were low and uniform in
anterior and posterior gills in crabs acclimated to 32 ppt, but there was a 4.5-fold increase in CA
mRNA expression in posterior gills after 4 days of exposure to 10 ppt. This increase in expression was
inhibited by 87% by the hemolymph injections.
Figure 2. Carbonic anhydrase
mRNA expression (in relative
units) in anterior (G4, black bars)
and posterior (G8, gray bars) gills
of C. maenas given the following
treatments: 32 = crabs acclimated
to 32 ppt; 10 = crabs transferred
to 10 ppt for 4 days; 10inj = crabs
transferred to 10 ppt for 4 days
and given injections of 2 mL of
hemolymph
from
32
ppt
acclimated crabs twice daily.
Mean + SEM (N = 6).
The CA repressor appears to function at the transcriptional level, inhibiting the increase in CA
mRNA that normally occurs in response to low salinity exposure.
Supported by NSF IBN 02-3005, the Thomas H. Maren Foundation, and an NSF REU Site Award to MDIBL.
63
1. Henry, R.P., Garrelts, E.E., McCarty, M.M., and Towle, D.W. Differential induction of branchial carbonic anhydrase
and Na+/K + ATPase in the euryhaline crab, Carcinus maenas, in response to low salinity exposure. J. Exp. Zool.
292:595-603. 2002.
2. Henry, R.P., Gehnrich, S., Weihrauch, D., and Towle, D.W. Salinity-mediated carbonic anhydrase induction in the
gills of the euryhaline green crab, Carcinus maenas. Comp. Biochem. Physiol. 136A:243-258. 2003.
3. Henry, R.P. Suppression of branchial carbonic anhydrase induction by a compound in the eyestalk of the green crab,
Carcinus maenas. Bull. Mt. Desert Island Biol. Lab 40:35-36. 2001.
4. Henry, R.P. A branchial carbonic anhydrase repressor is down-regulated in the eyestalks of the euryhaline crab,
Carcinus maenas, after acclimation to low salinity. Bull. Mt. Desert Island Biol. Lab 42:60-62. 2003.
5. Smith, K., and Henry, R.P. A carbonic anhydrase repressor is found in the hemolymph of the euryhaline green crab,
Carcinus maenas. Bull. Mt. Desert Island Biol. Lab 43:108-109. 2004.
64
The efficiency of wire minnow traps in assessing populations of Fundulus heteroclitus
Kevin M. Kocot1, Jamie L. Baldwin1, Christopher W. Petersen2, Robert L. Preston1 and
George W. Kidder3
1
Dept. of Biological Sciences, Illinois State Univ., Normal, IL 61790
2
College of the Atlantic, Bar Harbor, ME 04609
3
Mt. Desert Island Biological Lab, Salisbury Cove, ME 04672
Wire minnow traps are routinely used for catching Fundulus heteroclitus for laboratory use. These
traps have also been used in an attempt to assess the population of this species. The usual assumption
has been that once fish enter the trap, they will remain inside until released. This assumption has been
challenged by Kneib and Craig1, who "seeded" traps similar to ours with marked fish, and showed that
a large number of fish left the trap in the course of a several-hour deployment. Layman and Smith2
also observed but did not enumerate F. heteroclitus leaving a trap under laboratory conditions. One
might assume that the rate at which fish leave a trap would be roughly proportional to the number in
the trap at that time, but the prior experimental designs did not allow testing this hypothesis, since they
had no way of knowing the number of fish in their traps over time.
To monitor fish movement through the trap entrances directly, we mounted two underwater
cameras on a trap to view each entrance hole, as shown in Figures 1 and 2. We wired a solid
aluminum plate to the back of each entrance to aid in discriminating between fish moving behind the
entrance and through the entrance. Each camera was connected to a video recorder powered by a 12
volt battery, which imaged fish entering and leaving the trap through each entrance separately. The
trap was deployed in Northeast Creek in the pool above the broken dam near the Rt. 3 bridge. At this
site, the water depth fluctuation with tide is attenuated to 10-20 cm. The unbaited traps were deployed
Figure 1. Schematic diagram of half of a trap
with camera mounted. The other half was
similarly equipped.
Figure 2. A single frame from one camera, showing a fish
exiting a minnow trap, with other fish in view.
2 hours before high tide and recovered around 2 hours after high tide. The resulting 8 hours of video
data per experiment were analyzed by watching the tapes and noting the time each fish entered or
exited the trap, using an event-recording program written for this purpose. The data were classified
into 10 minute time blocks relative to predicted high tide, with the block time assigned to the
beginning of the block. From this analysis, the number of fish in the trap at the end of the run could be
computed and compared to the number found in the trap, as a check on the accuracy of the counting.
The error rate was computed as (E – L – O)/(E + L), where E was the total number of fish seen to enter
65
400
A
A
350
Number in Trap
No. entering/10 min
120
80
40
300
250
200
150
100
50
0
-120
0
-120
0
60
B
100
80
40
-60
0
60
120
Minutes from High Tide
120
No. leaving/10 min.
No. leaving/10 min
120
-60
B
80
60
40
20
0
0
0
-120
-60
0
60
120
50
100
150
200
250
300
350
400
Number Present
Minutes from High Tide
Figure 3. Fish entry (A) and exit (B) per 10 minute
analysis period, as a function of tide stage. Eleven
experiments, plotted with different point shapes.
Figure 4. A. Trap contents increase around high tide.
B. Number leaving trap is roughly proportional to
number in trap – best fit line is (# leaving) = -2.59 +
0.151 · (# present), r2 = 0.655
the trap, L the total number leaving the trap, and O the number observed in the trap when it was
removed from the water. The number of fish found in the trap per deployment varied widely from 233
to runs in which no fish were caught, which were excluded from the analysis. The number of
individual events (E + L) in the 11 analyzed records likewise varied from 2947 to 11, and the error rate
varied from 0 to 15.15% with an overall rate of 3.99%. As expected, runs with fewer fish gave more
accurate results. These wide variations are consistent with trapping records at this site.
With these data we can test the hypothesis that the number of fish leaving the trap is proportional to
the number of fish in the trap, which is essentially a diffusion hypothesis. As seen in Figure 4B, there
is a rough agreement with this hypothesis at higher densities. Up to about 100 fish present, there are
few escapes; at higher trap contents the rate of leaving increases considerably. The slope of the line is
significantly different from zero. Overall, for 3353 fish observed entering a trap, 2194 or 65% were
observed to leave.
While there is considerable variability between runs, it is clear that while these traps are useful in
obtaining Fundulus for various purposes, the trap contents do not provide an unbiased sample of the
population of the creek, and should not be used for this purpose.
Supported by NSF C-RUI 0111860. We thank Stephen Senci for the analysis program used.
1. Kneib, R. T., and A. H. Craig. 2001. Efficacy of minnow traps for sampling mummichogs in tidal marshes. Estuaries
24:884-893
2. Layman, C. A., and D. E. Smith. 2001. Sampling bias of minnow traps in shallow aquatic habitats on the eastern
shore of Virginia. Wetlands 21:145-154
66
Expression of a crustacean hyperglycemic hormone isoform in the shore crab,
Pachygrapsus marmoratus, during adaptation to low salinity
1
Céline Spanings-Pierrot1 , Lucien Bisson2 and David W. Towle 3
Université Montpellier II, UMR 5171 GPIA-AEO, 34095 Montpellier cedex 05, France
2
Carrabassett Valley High School, ME 04947
3
In Crustacea, neurosecretory cells that produce crustacean hyperglycemic hormone (CHH) have
been located in different endocrine and neuroendocrine tissues like the X-organ/sinus gland complex
(XO/SG) in the eyestalk, the pericardial organ (PO) around the heart and the gut. Aspects of the
isolation, structural characterization, and functions of the neuropeptide CHH have been extensively
studied in various species of Crustacea 1, 11 . Initially known as a hyperglycemic factor, that led to the
appellation of the peptide, CHH is also involved in inhibition of ecdysteroid synthesis (i.e. in the
molting process) and of methyl farnesoate synthesis, in stimulation of oocyte development, in lipid
metabolism, and in secretion of digestive enzymes. Interestingly, studies have emphasized that CHH
also plays a direct or indirect role in crustacean osmoregulation 7 .
In crabs, different CHH sequence isoforms resulting from a splicing process have been described
in neurohemal organs like the sinus gland and the pericardial organ 2, 9 . One of these isoforms, the
so-called PO-type or unspliced form of CHH, exhibits the addition of about one hundred base pairs
in the cDNA sequence compared to the classical SG-type CHH. This longer isoform could
contribute to the control of hydromineral regulation 10 . In the crab Carcinus maenas, the PO-type
CHH indeed appears to regulate neither hemolymph glycemia nor ecdysteroid synthesis, that are
known functions of CHH 2 , but could play a role in osmoregulating mechanisms 10 . These
observations prompted us to examine whether PO-type CHH mRNA expression might change in
relation to environmental variation of salinity. This study was conducted on the hyperhyporegulating crab Pachygrapsus marmoratus during short-term acclimation from 2 to 48h to low
salinity, using real- time quantitative PCR. This work intends to contribute to the knowledge on the
neuroendocrine control of osmoregulation in crustaceans, and more especially on the involvement of
CHH isoforms.
XO/SG complex and PO/heart were isolated from crabs adapted to seawater (SW, 36ppt) and to
diluted seawater (DSW, 10ppt) and they were immediately preserved in RNAlater (Ambion). These
dissections were conducted on two sets of organs collected in 2003 and in 2004. Total RNA was
purified from both type of organs isolated from 4 animals per sample with the RNAgents Total RNA
Isolation System (Promega). The pur ified RNA was then analyzed for integrity and quantified using
the Agilent Technologies 2100 Bioanalyzer. A normalized quantity of poly-A mRNA was reverse
transcribed using oligo-dT primer and the SuperScript II reverse transcriptase (Invitrogen). The
resulting cDNAs were then amplified by polymerase chain reactions performed with specific
primers designed according to the sequence of the unspliced PO-type CHH 9 . PCR products were
purified and prepared for direct sequencing on an ABI Prism 3100 automated sequencer (MDIBL).
In real- time quantitative PCR (RT-QPCR), PO-type CHH cDNAs, analyzed in 1- µl triplicate
aliquots, were amplified in the presence of Stratagene Brillant SYBR Green Master Mix using the
Startagene MX4000 Multiplex Quantitative PCR System. To quantify relative mRNA expression,
one of the test samples expected to yield high mRNA levels was used as the basis for comp arison.
An internal control (i.e. a “housekeeping” gene), arginine kinase, was amplified in the same
templates using identical cond itions of RT-QPCR.
67
Figure 1 represents the kinetics of mRNA expression of the CHH unspliced form (PO-type) in
the PO/heart and the XO/SG complex from crabs adapted for a short-term to 10ppt. In XO/SG
complex, results indicated that the expression of CHH unspliced form increased rapidly 2h after
tranfer to low salinity. It has more than doubled after 4h and it was very significantly enhanced 6h
after transfer in both 2003 and 2004 XO/SG samples (p<0.0001, ANOVA and Fisher’s multiplerange LSD post hoc test). The expression returned to the initial level in SW (t=0) 2 days after
transfer. We can notice that the PO-type CHH is really less expressed in PO/heart samples compared
to the XO/SG complex samples. However, the PO-type CHH expression in PO/heart increased very
significantly 6h after transfer from seawater to dilute medium in 2003 sample s (from 0.04 ± 0.03 to
1.08 ± 0.17, p<0.0001) as well as in 2004 sample s (from 0.19 ± 0.03 to 1.15 ± 0.23, p<0.0001), but
PO/heart data in 2004 appears less impressive because of the scale of the graph due to the dramatic
increase in XO/SG data. Moreover, the graphics indicate similar profile of results between the two
groups of samples from 2003 and 2004 demonstrating reproducible experiments.
2003
relative CHH expression
5
PO/heart
4
XO/SG
3
2
1
0
0
2
4
6
24
48
6
24
48
time (h)
2004
20
PO/heart
relative CHH expression
18
16
XO/SG
14
Fig. 1. Relative abundance of PO-type
CHH mRNA from XO/SG complex
and PO/heart samples from 2003 and
2004 at various intervals following
transfer of the crab Pachygrapsus
marmoratus from seawater (t=0) to
diluted seawater of 10ppt (t=2 to 48h).
Organs from 4 animals were pooled for
each time point and relative mRNA
expression was measured by real-time
quantitative PCR in triplicate. cDNA
transcribed from RNA of PO/heart at
6h exposure to 10ppt salinity served as
the reference standard. Results are
expressed as mean ± S.D. and
statistically analyzed with analysis of
variance and Fisher’s multiple-range
least significant difference post hoc
test.
12
10
8
6
4
2
0
0
2
4
time (h)
The mRNA expression of arginine kinase remained very stable and the variations were not
significantly different from 2h to 48h after transfer to low salinity. The relative mRNA level varied
between 0.75 ± 0.07 at 2h to 0.83 ± 0.02 at 6h and to 0.62 ± 0.01 at 48h in PO/heart samples and
was at 0.60 ± 0.03 in SW, while it stayed constant from 0.11 ± 0.01 at 2h to 0.13 ± 0.01 at 48h in
XO/SG samples and was at 0.10 ± 0.01 in SW.
68
In crabs, it has initially been reported that PO extracts can stimulate Na+/K +-ATPase activity and
Na+ uptake in isolated gills 3, 5 . Purified CHH from the sinus gland, containing the spliced and
unspliced forms of CHH, was also shown to stimulate Na+ influx in perfused gills of the crab P.
marmoratus 8 . We have mentioned above that PO-type CHH seems not involved in functions such
as glucose regulation or ecdysteroid synthesis, but the present results indicate a relation between
PO-type CHH and salinity adaptation as the expression of this isoform of CHH strongly increases
following tranfer to low salinity. In Astacidae Crustacea, including lobster and crayfish, a CHH
polymorphism has been reported resulting from the post-translational isomerization of the
phenylalanine residue on the third position of the N-terminus end from a L- to a D- configuration 6 . It
has recently been demonstrated that the D-CHH isomer is more particularly effective on
osmoregulatory parameters in this group of Crustacea 4 . These observations combined with the
present result, suggest a differential specificity of CHH according to the isoforms and the groups of
Crustacea.
This work was supported by a National Science Foundation grant (IBN-0340622) to DWT and by
the MDIBL High School Research Fellowship Program.
1.
Böcking, D., H. Dircksen, and R. Keller. The Crustacean Neuropeptides of the CHH/MIH/GIH family :
structures and biological activities. In The Crustacean Nervous System. Eds Wiese, K., Springer, Berlin,
Heidelberg, New York: 84-97, 2002.
2. Dircksen, H., D. Böcking, U. Heyn, C. Mandel, J. S. Chung, G. Baggerman, P. Verhaert, S. Daufeldt, T.
Plösch, P. P. Jaros, E. Waelkens, R. Keller, and S. G. Webster. Crustacean hyperglycaemic hormone (CHH)like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab,
Carcinus maenas, are putatively spliced and modified products of multiple genes. Biochem. J. 356: 159-170, 2001.
3. Kamemoto, F. I. Neuroendocrinology of osmoregulation in crabs. Zool. Sci. 8: 827-833, 1991.
4. Serrano, L., G. Blanvillain, D. Soyez, G. Charmantier, E. Grousset, F. Aujoulat, and C. Spanings-Pierrot.
Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of
osmoregulation in the crayfish Astacus leptodactylus. J. Exp. Biol. 206: 979-988, 2003.
5. Sommer, M. J., and L. H. Mantel. Effect of dopamine, cyclic AMP, and pericardial organs on sodium uptake and
Na/K-ATPase activity in gills of the green crab Carcinus maenas. J. Exp. Zool. 248: 272-277, 1988.
6. Soyez, D., J.-Y. Toullec, C. Ollivaux, and G. Géraud. L to D amino acid isomerization in a peptide hormone is a
late post-translational event occurring in specialized neurosecretory cells. J. Biol. Chem. 275: 37870-37875, 2000.
7. Spanings-Pierrot, C. Osmoregulation: Morphological, Physiological, Biochemical, Hormonal, and
Developmental Aspects. Part B: Neuroendocrine Control of Osmoregulation. In Treatise on Zoology: Anatomy,
Taxonomy, Biology. Vol. 2. Eds K. Brill, Academic Publishers, Leiden, Netherlands: in press, 2005.
8. Spanings-Pierrot, C., D. Soyez, F. Van Herp, M. Gompel, G. Skaret, E. Grousset, and G. Charmantier.
Involvement of Crustacean Hyperglycemic Hormone in the control of gill ion transport in the crab Pachygrapsus
marmoratus. Gen. Comp. Endocrinol. 119: 340-350, 2000.
9. Spanings-Pierrot, C., J.-Y. Toullec, and D. W. Towle. Identification of two different forms of crustacean
hyperglycemic hormone (CHH) in sinus glands of the euryhaline crab Pachygrapsus marmoratus. Bull. Mt. Desert
Island Biol. Lab. 42: 49-51, 2003.
10. Townsend, K., C. Spanings-Pierrot, D. Hartline, S. King, R. Henry, and D. W. Towle. Expression of
crustacean hyperglycemic hormone (CHH) mRNA in neuroendocrine organs of the shore crab Carcinus maenas.
Bull. Mt. Desert Island Biol. Lab. 41: 54-55, 2002.
11. Van Herp, F. Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family.
In Recent Advances in Arthropod Endocrinology. Eds G. M. Coast and S. G. Webster, Cambridge Univ. Press,
Cambridge, U.K.: 53-70, 1998.
69
Cardiovascular effects of NO and Urotensin II in
longhorn sculpin, Myoxocephalus octodecimspinosus
David H. Evans & Sara Takeuchi
Department of Zoology
University of Florida, Gainesville, FL 32611
Although both NO and Urotensin II are vasoactive in isolated teleost blood vessels, it is not clear
what role these substances may play in gill hemodynamics1. To delineate more clearly specific cardiac
vs. gill an/or systemic resistance effects, we have utilized the afferent and efferent branchial arterycannulated sculpin preparation4 to examine the effect of ventral aortic infusion of either sodium
nitroprusside (SNP, NO donor; 10 µM) or Urotensin II (UTII; 0.1 µM). These nominal plasma
concentrations were chosen because they had produced vascular tension changes in published studies.
Longhorn sculpin were prepared as described previously4 and either SNP or UTII (in teleost
Ringers) was infused into the afferent branchial cannula after an initial, control Ringers infusion of the
same volume as a control. In both cases, an equivalent volume of blood was removed before the
infusion to avoid volume effects. Measurements of cardiac output (CO) and ventral and dorsal aortic
pressure (PVA, PDA), and calculations of gill resistance ((PVA-PDA)/CO) were made as described
previously3.
SNP produced no change in CO but it did stimulate bimodal alteration in both PVA and PDA, with
a significant (and equivalent: ca. 30%) decline (p = 0.01 and p = 0.04, respectively) in both parameters
(N = 4) within 5 minutes, followed by return to control levels (p > 0.50) within another 10 minutes. In
contrast, UTII produced a significant (and equivalent; ca. 12%) increase (p = 0.1 and p = 0.049) in both
parameters (N = 3-5) within 5 minutes, followed by a return to control levels (p > 0.25) within another
10 minutes. UTII did not alter CO. Since neither CO nor PVA-PDA was altered by either compound,
these preliminary data suggest that both NO and UTII can produce significant vasoactive responses in
the longhorn sculpin, but they appear to be secondary to alterations in systemic rather than branchial
resistances. The bimodal responses to both SNP and UTII suggest either neuroendocrine, homeostatic
reflexes or possibly the presence of receptor-specific, vasoconstrictory vs. vasodilatory pathways, as
have been described recently for UTII in mammalian systems2,5. (Supported by NSF IBN-0089943 to
DHE).
1. Evans DH, Piermarini PM, and Choe KP. The multifunctional fish gill: Dominant site of gas exchange,
osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Revs 85: in press, 2005.
2. Douglas SA, Dhanak D, and Johns DG. From 'gills to pills': urotensin-II as a regulatory of mammalian cardiorenal
function. Trends Pharmacol Sci 25: 76-85, 2004.
3. Giesbrandt K and Evans DH. The cardiovascular and branchial perfusion effects of endothelin in the longhorn
sculpin (Myoxocephalus octodecimspinosus). Bull Mt Desert Isl Biol Lab 43: 89, 2004.
4. Giesbrandt K and Evans DH. Preparation of the longhorn sculpin (Myoxocephalus octodecimspinous) for
simultaneous cardiovascular and videomicroscopic studies. Bull Mt Desert Isl Biol Lab 42: 98-99, 2003.
5. Maguire JJ and Davenport AP. Is urotensin-II the new endothelin? Br J Pharmacol 137: 579-588, 2002.
70
Does Acartia hudsonica exhibit an enzymatic stress response to toxic Alexandrium spp.?
R. Patrick Hassett & Elizabeth L. Crockett
Ohio University, Athens, OH 45701
In the Gulf of Maine marine copepods, the primary component of the zooplankton, are regularly
exposed to blooms of toxic Alexandrium spp.12. Copepods are known to accumulate toxins6,11 and pass
them up the food chain13. Ingestion rates decline with cell toxicity, a physiological effect and not
selection6. Alexandrium can produce rapid heart beat and “retching” behavior in copepods10. Ingestion
of toxic Alexandrium may also reduce fecundity in Acartia, a common genus of nearshore waters3. Not
all copepods, however, are affected by toxicity. A. hudsonica readily ingests Alexandrium, with both
survival and oxygen consumption unaffected, while two other copepods, Calanus finmarchicus and
Metridia lucens, avoid ingesting Alexandrium5. The variable response of copepods to toxic
Alexandrium raises the question of whether some copepods have defense mechanisms, such as
detoxifying enzymes, that allow them to safely ingest Alexandrium.
During experiments in which plasma membranes were prepared from Acartia hudsonica2 specific
activities of Na+/K+-ATPase (NKA) and NADPH-dependent cytochrome c reductase (NCCR) (used as
enzyme markers of biological membranes) were found to be nearly 2-3 times lower following 5 days
of acclimation to the diatom Thalassiosira weissflogii than they were in animals recently sorted and
held overnight in large tanks (Fig. 1). NCCR has been used as a biomarker of exposure to toxins in the
euphausid Meganyctiphanes norvegica4. NKA has been found to increase in fish gill in response to
environmental stress7. Since toxic algal species, including Alexandrium, are frequently found in the
Gulf of Maine and Frenchman Bay during the summer, the observed decline in these two enzymes
during the acclimation treatment may be the result of removing the animals from an environment
containing toxic algal species in the phytoplankton. In the present study we test the hypothesis that the
presence of toxic Alexandrium spp. induces higher activities of these enzymes.
Fig. 1. Activities of A.
Na+/K+ ATPase (NKA) and
B. NADPH-dependent
cytochrome C reductase
(NCCR) of Acartia
hudsonica on freshly caught
animals (24 h after
collection) and following 5 d
acclimation to the diatom
Thalassiosira weissflogii.
Acartia hudsonica were collected from Frenchman Bay with a 202µm ringnet towed at 2-3 m
below the surface. Contents of the net cod end were added to a 20g cooler and copepods were
71
presorted by attracting Acartia to a lighted corner of the cooler. Adult females were then sorted by
microscope and held in 4 l containers for experimental treatments. Algal stocks were obtained from the
Provasoli-Guillard Center for the Culture of Marine Phytoplankton (CCMP), Bigelow Laboratory for
Marine Sciences (Boothbay Harbor, ME) and maintained in L2 culture media (also from CCMP).
Thalassiosira weissflogii, toxic Alexandrium fundyense CCMP1719, and non-toxic Alexandrium
tamarense CCMP115 were used as food sources. Stocks were incubated at 18°C on a 14h light/10h
dark cycle. Copepods were acclimated for 2 days on T. weissflogii at 1000 cells ml-1, with
concentrations monitored by in vivo fluorescence (Turner handheld fluorometer). Following the initial
acclimation period CCMP1710 and CCMP115 were added to two of the 3 treatments at concentrations
of 10 cells ml-1, comparable to densities observed during Alexandrium blooms. T. weissflogii was
maintained at 1000 cells ml-1 in all 3 treatments during this period. After an additional 2 days copepods
were sorted from the treatments and prepared for enzyme analysis. Feeding activity was confirmed by
changes in fluorescence in the containers as well as by cell counts of Alexandrium and appearance of
Alexandrium in copepod guts (apparent by changes in color).
Animals from each treatment were homogenized in a Ten-Broeck ground-glass homogenizer using
25 mM Hepes/1 mM EDTA (pH 7.6) and stored in liquid nitrogen until enzymatic assays were
performed. NKA and NCCR were assayed according to Barnett1 and Masters et al. 8, respectively.
Enzymes were assayed in duplicate at 25oC. To obtain protein-specific activities of the enzymes,
protein contents were determined by the bicinchoninic acid method9.
Ingestion of Thalassiosira weissflogii, as measured by change in fluorescence, was comparable in
all 3 treatments. Ingestion of Alexandrium was evident from low concentrations remaining in the
containers as well as the color of copepod guts and fecal pellets, although the low concentrations used
in the acclimations precluded an accurate assessment of ingestion rates between the two Alexandrium
treatments. Neither activity of NKA nor NCCR changed significantly with either of the two
Alexandrium treatments (Fig. 2).
Fig. 2. Activities of A. Na+/K+ ATPase (NKA) and B. NADPH-dependent cytochrome C reductase
(NCCR) of Acartia hudsonica following 2 d acclimation to the diatom Thalassiosira weissflogii, and the
dinoflagellates Alexandrium fundyense CCMP1719 (A.f. 1719) and the non-toxic strain Alexandrium
tamarense CCMP115 (A.f. 115).
In conclusion, there is no evidence for an effect of Alexandrium (at concentrations typically seen
during blooms of this dinoflagellate) on activities of NKA and NCCR in Acartia hudsonica. Activities
72
of NKA and NCCR following exposure to all 3 food treatments were comparable to activities observed
the previous year following acclimation (5 days) to Thalassiosira weissflogii. Alexandrium densities
even 10-fold higher do not affect oxygen consumption rates of A. hudsonica5, and given the probable
importance of NKA in the animals’ energy budget, it is unlikely that NKA is affected at these high cell
densities. Oxygen consumption of A. hudsonica and other copepod species is elevated for several hours
after handling before declining to a level that remains stable for at least 4-7 days5. The high enzyme
activities observed initially (measured on copepods that had been held overnight) may be a transient
stress response to collection.
This research was supported by NSF OCE 0117132.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Barnett, R. E. Effect of monovalent cations on the ouabain inhibition of the sodium and potassium ion activated
adenosine triphosphatase. Biochem. 9: 4644-4648, 1970.
Crockett, E. L. and R. P. Hassett. A cholesterol-enriched diet enhances egg production and egg viability without
altering cholesterol content of biological membranes in the copepod Acartia hudsonica. Physiol. Biochem. Zool. 78: in
press.
Dutz, J. Repression of fecundity in the neritic copepod Acartia clausi exposed to the toxic dinoflagellate Alexandrium
lusitanicum: relationship between feeding and egg production. Mar. Ecol. Prog. Ser., 175: 97-107, 1998.
Fossi, M. C., J. F. Borsani, R. Di Mento, L. Marsili, S. Casini, G. Neri, G. Mori, S. Ancora, C. Leonzio, R.
Minutoli and G. Notarbartolo di Sciara. Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential
early indicator of health status of the Mediterranean "whale sanctuary." Mar. Environ. Res. 54: 761-767, 2002.
Hassett, R. P. Effect of toxins of the 'red-tide' dinoflagellate Alexandrium spp. on the oxygen consumption of marine
copepods. J. Plank. Res. 25: 185-192, 2003.
Ives, J. D. Possible mechanisms underlying copepod grazing responses to levels of toxicity in red-tide dinoflagellates.
J. Exp. Mar. Biol. Ecol. 112: 131-145, 1987.
Lappivaara J, J. Mikkonen, M. Soimasuo, A. Oikari, A. Karels, A. Mikkonen. Attenuated carbohydrate and gill
Na+, K+ -ATPase stress responses in whitefish caged near bleached kraft mill discharges. Ecotoxicol. Environ. Saf.
51: 5-11, 2002.
Masters, B. S., S. Williams, C. H. Kamin and H. Kamin. The preparation and properties of microsomal TPNHcytochrome c reductase from pig liver. In: Methods in Enzymology, vol. 10 (M.E. Pullman and R.W. Estabrook). pp.
565-573. New York, London, Academic Press. 1967.
Smith,P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N.
M. Goeke, B. J. Olson, and D. C Klenk. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 7685, 1985.
Sykes, P. F., and M. E. Huntley. Acute physiological reactions of Calanus pacificus to selected dinoflagellates: direct
observations. Mar. Biol. 94: 19-24, 1987.
Teegarden, G. J., and A. D. Cembella. Grazing of toxic dinoflagellates, Alexandrium spp., by adult copepods of
coastal Maine: Implications for the fate of paralytic shellfish toxins in marine food webs. J Exp. Mar. Biol. Ecol. 196:
145-176, 1996.
Townsend, D. W., N. R. Pettigrew and A. C. Thomas. Offshore blooms of the red tide organism, Alexandrium spp.,
in the Gulf of Maine. Cont. Shelf Res. 21: 347-369, 2001.
White, A. W. Marine zooplankton can accumulate and retain dinoflagellate toxins and cause fish kills. Limnol.
Oceanogr. 26: 103-109, 1981.
73
Regulation of Adult Neurogenesis in Decapod Crustaceans
Barbara S. Beltz1, Jeannie L. Benton1, Maria C. Genco1,
DeForest Mellon2, David C. Sandeman1 and Jeremy M. Sullivan1
1
Department of Biological Sciences, Wellesley College, Wellesley MA 02481
2
Department of Biology, University of Virginia, Charlottesville, VA 22901
The primary goal of our experiments at MDIBL was to test the feasibility of using different
crustacean species in our research. For many years, laboratory-reared juvenile lobsters (Homarus
americanus) have been the primary subject of our research on the regulation of life-long neurogenesis15,12
. However, during the summer of 2004, we wanted to investigate whether other crustacean species
indigenous to Mt. Desert Island might be useful models for studying neurogenesis. Adult neurogenesis
has been documented in Carcinus maenas, for example9, but environmental factors that regulate this
process have not been examined in this organism. Our initial hypothesis was that mechanisms
regulating neurogenesis would be very similar among decapod crustacean species, and that we could
therefore use lobsters, crabs or crayfish to answer our questions and expect to get similar answers.
The very important take-home message from our summer’s work is that environmental factors
regulating neurogenesis, as well as the cell cycle dynamics leading to the production of new neurons,
can be very different even in closely related species. Characteristics of the cell cycle and regulatory
mechanisms must therefore be evaluated independently in each organism. We believe these
differences may be related to the varied lifestyles and life spans of these species.
Most of our efforts at MDIBL were directed at exploring aspects of neurogenesis in the shore crab,
Carcinus maenas. We asked two primary questions: (1) Do tidal and circadian influences regulate the
timing of neurogenesis? This study was based on research in the lobster that demonstrated circadian
control of neurogenesis4; the idea that tides might also play a regulatory role in crabs emerged from
their lifestyle as an intertidal species. (2) Can we estimate the length of the cell cycle resulting in the
production of new neurons? Our interest in this question grew from our desire to understand circadian
influences on neurogenesis; in order to study these phenomena, ideally we need a model organism
where the S phase is relatively short and the complete cell cycle is less than 24 hours. To address
these questions we utilized bromodeoxyuridine (BrdU) labeling of cells in S phase, in the cluster of
olfactory projection neurons (cluster 10) in the midbrain of the crab. To examine tidal and circadian
influences, we collected crabs on days when sunset and high tide (SSHT), sunrise and high tide
(SRHT), sunset and low tide (SSLT), or sunrise and low tide (SRLT) were coincident. The idea here
was to take advantage of a presumed circadian rhythm in neurogenesis and look at the peak (dusk) and
trough (dawn) periods in neurogenesis that were observed in lobsters4, and examine this circadian
rhythm with an overlay of tidal influences; we therefore hoped to observe the levels of neurogenesis
for this species under natural conditions.
Animals were collected from Seawall and the rate of neurogenesis was assessed over the
subsequent 24-hour period. Groups of crabs (n=5-10 per group) were placed in BrdU in seawater
(2mg/ml) for 4 hours at 4 hour intervals. Following the 4-hour incubation in BrdU, crabs were killed
and their brains dissected and fixed. Immunocytochemical methods were used to detect the presence
of BrdU in cells in cluster 101. The presence of BrdU labeling indicated those cells that were in S
phase during the period of BrdU incubation. Labeled cells were counted for each time point, statistical
measures applied, and means and standard errors were graphed (Fig.1). Results indicate that there
may be a subtle influence of tides on the period of neurogenesis, but there were no statistically
significant differences between the peaks and troughs in the rate of neurogenesis throughout the 24hour periods sampled. There was no indication that the day/night cycle influenced the rate of
neurogenesis.
74
1
Fig. 1. Crabs (Carcinus maenas) were incubated in
BrdU for 4 hours at six time points during a 24-hour
cycle from June 29-30, 2004. n=4 for 12:00 and
0400 samples; n=5 for all other points. Sunset and
high tide (SSHT) were coincident on the day of this
study (sunset, 20:21; sunrise, 04:51; high tide, 08:18
and 20:37; low tide, 02:09 and 14:21). The tidal
cycle is indicated by the wave patterns, and the light
conditions by the shading on the graph. The
sun/moon symbols mark sunrise and sunset. on the
sampling day.
Since returning to our lab at Wellesley College where we have controlled lighting in the animal
care facilities, we have tested whether neurogenesis in Carcinus maenas is regulated by the light/dark
cycle. We found that there is no difference in the rate of neurogenesis relative to time of day, when the
crabs are removed from their tidal influences and maintained in laboratory tanks for at least 1 month
with a 12/12 L/D cycle. This finding, therefore, points out a critical difference in regulatory
mechanisms controlling neurogenesis between C. maenas and H. americanus, where the light cycle
entrains the period of neurogenesis4. With this in mind, we therefore will re-examine tidal influences
next summer from a different perspective. Instead of examining days when tides, sunset and sunrise
are coincident, we plan to examine neurogenesis during 24-hour periods when tides are at their
extremes (e.g., during a full moon).
While working with C. maenas, we also became curious about the duration of the S phase and cell
cycle, because it appeared that regardless of the length of the BrdU incubation period (3 to >24 hours),
approximately the same numbers of neurons were labeled. This was not consistent with our experience
using juvenile lobsters, where we observed differences in the numbers of cells labeled as a result of
relatively minor changes in BrdU incubation times. We therefore did a comparative study using both
crabs and lobsters, to ask how many neurons would label with BrdU over 3, 6, 9 and 12-hour
incubation periods. The results of this study demonstrate a critical difference in the dynamics of
neurogenesis between these two species (Fig. 2). While increases in the numbers of labeled neurons
are seen with increasing length of BrdU incubation in lobsters, there are no differences seen in the
numbers of labeled neurons in crabs over these time periods. This suggests that the S phase is
comparatively long in C. maenas relative to H. americanus. These data and the finding that
neurogenesis in crabs is not under circadian control demonstrate that there are significant differences in
cell cycle dynamics and regulatory controls between these species. Further, these findings indicate that
Carcinus would not be a favorable model for examining circadian control mechanisms.
Fig. 2. Graph of the numbers of BrdU-labeled cells in the
brains of lobsters (blue broken line) and crabs (pink solid
line) during a 12-hour period. The numbers of labeled cells
were assessed after incubation in BrdU for 3, 6, 9 and 12
hours. Labeling in the crab brains following a 3-hour BrdU
incubation resulted in weak labeling in cluster 10 neurons;
these faint cells could not be counted accurately, and
therefore this time point is not included in the graph for C.
maenas. This observation is consistent with the suggestion
that the cell cycle is longer in C. maenas than in H .
americanus.
2
75
In addition, while at MDIBL, we pursued three other lines of research related to our goal of
understanding neurogenesis and its regulation:
DeForest Mellon explored neurogenesis in the fiddler crab, Uca pugilator, another intertidal
species indigenous to Frenchman’s Bay. Its behavior is strongly influenced by the tides; periods of
locomotory activity are entrained by the tidal cycle and are maintained for several weeks even when
crabs are placed in laboratory conditions without tidal signals8. This persistent locomotory rhythm is
unlike the situation in Carcinus maenas, which lose their tidal rhythmicity after only a few days in
laboratory conditions7. We therefore hypothesized that comparing neurogenesis among these species
might provide an opportunity to examine tidal regulation of this process in organisms (U. pugilator, C.
maenas and H. americanus) that have very different life styles relative to the tides. We therefore
conducted pilot studies in adult fiddler crabs, to determine the extent of BrdU labeling over various
incubation periods. The principal result of these experiments is that very few (~4-6) olfactory
projection neurons label even over a 12-hour BrdU incubation period, as compared to 35-50 in C.
maenas and >100 in H. americanus. This result is not encouraging in terms of using U. pugilator as a
model for exploring circadian or tidal regulation of neurogenesis, as the phenomenon is not robust
enough for experimental manipulation over the course of a 24-hour period.
Jeannie Benton and Jeremy Sullivan developed immunocytochemical methods for detection of
proliferating nuclear cell antigen (PCNA). Although the use of the BrdU labeling method in our lab
has been highly successful as a marker for proliferating cells in crustacean species, this technique has
the disadvantage of requiring the incorporation of this thymidine analogue over a period of hours.
Therefore, while the label is incorporated during the S phase, the stage of the cell cycle when the label
is observed is not restricted to the S phase because many of the labeled cells will have advanced
through G2 and M phases by the time the tissue is fixed. PCNA is an enzyme expressed only in
mitotically active (S-phase) cells and its presence can therefore be used to define cells in S phase at a
specific time6. Utilization of the antibody against PCNA requires antigen retrieval techniques that have
been used in only a few non-mammalian species10. A novel version of a high temperature, low pH
pretreatment method to recover the antigenicity of tissue sections that had been masked by formalin
fixation was successful in whole brains of H. americanus, C. maenas and Cragnon cragnon.
Jeremy Sullivan also localized pigment dispersing hormone (PDH) in the brains of larval, juvenile
and adult lobsters using immunocytochemical methods. These studies were done in order to identify
neurons potentially involved in regulating the circadian rhythm of neurogenesis observed in the lobster
brain. PDH is a neuropeptide identified as an important component of the circadian pacemaker in the
brains of insects11. These studies identified an extensive network of PDH-immunoreactive neurons in
the brain of H. americanus, a number of which have arbors that overlap with those of extraretinal
photoreceptors which are thought to be important inputs to the circadian pacemaker. In addition, the
distribution of PDH-immunoreactivity was found to differ between larval, juvenile and adult lobsters
providing evidence of substantial changes in the organization of the brain circadian pacemaker during
the development of the lobster.
1.
2.
3.
4.
5.
76
Beltz, B. S., J. L. Benton, and J. M. Sullivan. Transient uptake of serotonin by newborn olfactory projection neurons
may mediate their survival. Proceedings of the National Academy of Science 98: 12730-12735, 2001.
Beltz, B. S. and D. C. Sandeman. Regulation of life-long neurogenesis in the decapod crustacean brain. Arthropod
Structure and Development 32: 39-60, 2003.
Benton, J. L. and B.S. Beltz. Patterns of neurogenesis in the midbrain of embryonic lobsters are different from
proliferation in the insect and crustacean ventral nerve cord. Journal of Neurobiology 53: 57-67, 2002.
Goergen, E., L. A. Bagay, K. Reh, J. L. Benton, and B. S. Beltz. Circadian control of neurogenesis. Journal of
Neurobiology 53: 90-95, 2002.
Harzsch, S., J. Miller, J. Benton, and B. Beltz. From embryo to adult: Persistent neurogenesis and apoptotic cell death
shape the crustacean deutocerebrum. Journal of Neuroscience 19: 3472-3485, 1999.
3
6.
Kisielewska, J., P. Lu, and M. Whitaker. GFP-PCNA as an S-phase marker in embryos during the first and subsequent
cell cycles. Biology of the Cell [electronic publication prior to paper], December, 2004.
7. Naylor, E. Crab clockwork: the case for interactive circatidal and circadian oscillators controlling rhythmic locomotor
activity of Carcinus maenas. Chronobiology International 13: 153-61, 1996.
8. Palmer, J. D. Contributions made to chronobiology by studies of fiddler crab rhythms. Chronobiology International 8:
110-130, 1991.
9. Schmidt, M. Continuous neurogenesis in the olfactory brain of adult shore crabs, Carcinus maenas. Brain Research
762: 131-43, 1997.
10. Shi, S-R, R. J. Cote, and C.R. Taylor. Antigen retrieval techniques: Current perspectives. Journal of Histochemistry
and Cytochemistry 49: 931-937, 2001.
11. Stanewsky, R. Clock mechanisms in Drosophila. Cell and Tissue Research 309: 11-26, 2002.
12. Sullivan, J. M., J. L. Benton, and B. S. Beltz. Serotonin depletion in vivo inhibits the branching of olfactory projection
neurons in the lobster deutocerebrum. Journal of Neuroscience 20: 7716-7721, 2000.
Sponsored by National Science Foundation Grant numbers IBN-0344448 (BSB) and DBI-0139190 (MDIBL), Fiske and
Staley Awards from Wellesley College, and the Maren Foundation (MDIBL).
4
77
Renal gene expression in Squalus acanthius following hyposmotic stress
Thomas L. Pannabecker and William H. Dantzler
Department of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724
Reabsorption of urea from shark renal filtrate minimizes excretion of this important osmolyte.
One important transporter involved in this process is the urea transporter ShUT that is expressed in
bundle tubules1. Urea reabsorption via ShUT is likely driven by countercurrent transport although the
precise mechanisms are unknown. Experimental interventions that alter renal blood and tissue urea
content (eg. hyperosmotic and hyposmotic stress) may induce differentially-regulated expression of
fluid and solute transport pathways and regulatory factors in the kidney. A case in point: exposure to
dilute seawater has previously been shown to increase GFR, urine flow, and urea excretion in the skate
kidney2, 3, and to reduce gene expression of the homologous skate urea transporter, SkUT, in skate
kidney homogenates4.
For much of its length, the nephron of marine elasmobranchs lies in the venous portal system
(mesial zone); however, a relatively short length lies within a closed peritubular sheath (bundle zone).
Capillary-like vessels and five structurally- and functionally-defined nephron segments forming 2
hairpin loops exist within the bundle zone. This arrangement of nephron segments and vessels
resembles the renal countercurrent architecture of worms, leeches, birds, and mammals3, 5. Studies of
countercurrent transport in all these animals – and particularly from intense investigations in
mammalian systems – indicate that multiple membrane transport pathways in contiguous epithelia and
endothelia play unified roles to accomplish countercurrent transport.
One of our goals is to establish a method for quantitating gene expression in isolated shark
tubules, and then to compare expression levels of genes encoding membrane transporters and
regulatory proteins following experimental intervention. This information can then be used to
formulate potential models of countercurrent transport. In this study we have used conventional and
real-time RT-PCR to assess relative quantities of transport-related genes expressed in kidney
homogenates following hyposmotic stress.
Figure 1. Expression of shUT (498 bp) in 4 of the
5 different tubule segments present in two
separate bundles from a single shark kidney.
Early distal tubules are identified with an
asterisk. Remaining segments were not identified.
Effects of hyposmotic stress were tested through gradual dilution of seawater. Freshwater was
added to a 4 ft. diameter tank containing continuous-flow, 100% seawater so that sharks experienced 2
days at ~85% seawater, 4 days at ~80% seawater, and 1 day at ~75% seawater. Animals were
sacrificed on day 7, kidneys were removed, and tissue was processed or stored at –80˚ C. Tissue urea
was determined by the method of Rahmatullah and Boyde6. Reverse transcription (RT) of messenger
RNA and amplification of first strand cDNA by conventional polymerase chain reaction (PCR) were
carried out as previously reported1. Real time RT-PCR was carried out with SYBR-green reporter
(Stratagene reagents and MX4000 instrumentation). The reference dye ROX was used to normalize
reporter dye signals. Target gene expression levels in each sample were normalized to β-actin7, whose
expression appeared unaffected by seawater dilution. In order to control for genomic contamination,
reverse transcriptase was omitted during the cDNA synthesis step in additional reactions and these
78
showed no reaction product. Following amplification, dissociation plots showed all primer pairs
produced single amplification peaks.
Analysis of gene expression in 6 kidneys with conventional RT-PCR showed the early distal
tubule (diluting segment) and several additional bundle segments exhibit strong shUT expression.
Expression of shUT in multiple tubule segments obtained from each of two separate bundles from a
single animal is shown in Fig. 1. Interestingly, the urea transporter from the dogfish Triakis scyllia
(87.5% protein homology) has been immunolocalized only to a single bundle segment, the bundle
collecting tubule8. We observed no ShUT expression in mesial segments adjacent to bundles.
Table 1. Renal tissue urea content and renal expression levels* of ShUT, NKCC1, and PLMS for sharks immersed
in 100% or 75% seawater.
100% seawater
75% seawater
Tissue Urea Content
(mmoles/kg wet wt)
ShUT
NKCC1
PLMS
303.2 ± 4.8
267.2 ± 8.8†
1.00
1.43 ± 0.67
1.00
1.41 ± 0.39
1.00
0.97 ± 0.40
*Relative gene expression levels reported as fold-change compared to 100% seawater (mean ± se, N = 3). No significant
changes were observed.
†
Significantly different from 100% seawater, Student’s 2 sample t-test, P < 0.05. N = 3 for each treatment.
We investigated tissue urea levels and expression levels of shUT, NKCC1, and shark
phospholemman (PLMS, a putative regulator of Na-K-ATPase) in kidneys from sharks immersed in
100% seawater or 75% seawater (Table 1) as described above. A significant decrease in urea content
was observed for animals in 75% seawater. A small but statistically insignificant increase was
observed in whole-kidney expression levels for shUT and NKCC1 expression, and no change was
observed for PLMS expression. This contrasts with a near 3-fold decrease in renal skUT following
immersion of skates in 50% seawater for 5 days4. An absence of change in shark renal shUT
expression with 75% seawater would suggest that non-genomic regulation of urea reabsorption may
exist. In order to more fully understand elasmobranch countercurrent transport, it will be informative to
assess expression of these and other transporters in individual tubule segments, as well as the nephron
permeabilities for urea, NaCl, and water, before and following variable degrees of osmotic stress.
This work was supported by an MDIBL New Investigator Award to Thomas Pannabecker and by
NSF grant IBN 9814448 to William Dantzler. We thank Dr. David Towle for helpful advice on realtime RT-PCR methodology.
1.
2.
3.
4.
5.
6.
7.
8.
Pannabecker TL and Dantzler WH. Dogfish shark renal urea transporter (ShUT) mRNA expression in bundle zone
nephrons of Squalus Acanthius. Bulletin MDIBL 42:58-59, 2003.
Goldstein L and Forster RP. Osmoregulation and urea metabolism in the little skate Raja erinacea. Am. J. Physiol.
220:742-746, 1971.
Schmidt-Nielsen B and Bankir L. Dilution of urine through renal fluid secretion: Anatomo-functional convergence in
marine Elasmobranchs and Oligochaetes. Bulletin MDIBL 42:2-9, 2003.
Morgan RL, Ballantyne JS, and Wright PA. Regulation of a renal urea transporter with reduced salinity in a marine
elasmobranch, Raja erinacea. J. Exp. Biol. 206:3285-3292, 2003.
Pannabecker TL, Abbott DE, and Dantzler WH. Three-dimensional functional reconstruction of inner medullary
thin limbs of Henle’s loop. Am. J. Physiol (Renal Physiol) :286: F38-F45, 2004.
Rahmatullah M and Boyde, TRC. Improvements in the determination of urea using diacetyl monoxime; methods
with and without deproteinisation. Clin. Chim. Acta 107:3-9, 1980.
Pfaffl, MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: 45-50,
2001.
Hyodo S, Katoh F, Kaneko T, and Takei Y. A facilitative urea transporter is localized in the renal collecting tubule
of the dogfish Triakis scyllia. J. Exp. Biol. 207: 347-356, 2004.
79
The Ciona intestinalis branchial sac and its microbiota
H. Rex Gaskins, Joel E. Thurmond, Gerardo M. Nava & Roderick I. Mackie
University of Illinois at Urbana-Champaign, Urbana, IL 61801
Ascidians, larvaceans, and thaliceans form a discrete group of advanced invertebrate species, the
urochordates, in the phylum Chordata (Fig. 1). Ciona intestinalis is a member of the class Ascidacea,
commonly known as tunicates because of their flexible, tough outer covering or "tunic". Although
sessile adult tunicates bear little resemblance to typical chordates, their larvae exhibit the four
fundamental characteristics of this phylum: i) a dorsal tubular nerve cord, ii) a notochord, iii)
pharyngeal gill slits, and iv) a postnatal tail.
Their critical evolutionary position as basal
chordates and the simplicity of their embryogenesis
have attracted developmental and evolutionary
biologists since the turn of the 20th century14.
Because of this, the genomes of C. intestinalis as
well as its close relative C. savignyi have been fully
sequenced recently2. The availability of whole
genome sequence enables the power of a
comparative genomic approach to pursue our longterm goal of determining whether the support by C.
intestinalis of a bacterial metagenome that
contributes to detoxification has influenced, over
evolutionary time, its complement of detoxification
Fig. 1. Phylogeny of the urochoradate C. intestinalis, a
member of the class Ascidiacea.
genes.
These animals are filter feeders and live attached to submerged substrates in the littoral zone of
marine waters where they encounter high concentrations of complex polyphenols, halogenated
aromatics, methylated sulfides, and some heavy metals. Little is known about the mechanisms by
which tunicates detoxify or otherwise tolerate these natural toxins in the marine environment. We are
addressing the hypothesis that filter-feeding tunicates rely on bacterial symbionts associated with the
branchial sac for this purpose.
The branchial sac precedes the intestine and is an
enlarged pharynx whose wall is perforated by numerous
tiny gill slits (Fig. 2). It is both a respiratory organ and
filter-feeding device. Water flows into the incurrent
siphon at approximately twenty milliliters per minute
and enters together with food particles the branchial sac.
The water passes through the gill slits and then out the
excurrent siphon while food remains in the gut and
passes posteriorly to be digested. Waste products are
secreted from the anus and out the excurrent siphon. The
present report describes studies that used molecular
ecological as well as conventional cultivation-based
microbiological approaches to characterize the
predominant microbes associated with the branchial sac
of C. intestinalis.
Fig. 2. Schematic of typical anatomy of solitary
tunicates. The large branchial sac or pharynx that
precedes the intestine is both a filter feeding and
respiratory organ.
The branchial sac was dissected from replicate specimens of C. intestinalis that colonize sediment
trays preceding the running seawater system at the University of Maine Darling Marine Center
(http://server.dmc.maine.edu/) or from specimens collected from Cobscook Bay by Gulf of Maine
80
Marine Life Supply Company (http://www.gulfofme.com) staff and shipped to MDIBL. Animals were
maintained in running sea water tanks at MDIBL prior to dissection. DNA was isolated from finely
minced tissue using an UltraCleanTM Soil DNA Kit (Mo Bio Laboratories; Carlsbad, CA;
http://www.mobio.com). 16S-V3 rDNA PCR-DGGE and community structure analyses, and the cloning
and sequencing of individual amplicons were performed as described previously9,10
Individual 16S-V3 rDNA amplicons were excised from DGGE gels and cloned and sequenced to
phylogenetically identify common branchial sac bacteria of C. intestinalis. Amplicons found in a
minimum of 3 animals were selected for sequencing. Bacteria from 5 major clades were identified
(Table 1). The closest matches for the two branchial sac alphaproteobacteria (class in phylum
Proteobacteria) sequences were each putative symbionts, one of sponge5 and the other from a deep sea
vent gutless annelid that depends on its endosymbionts for sulfide detoxification3.
Table 1. 16S-V3 rDNA sequences cloned from the branchial sac microbiota of C. intestinalis
Functional genes or
IdentitiesB
Taxonomy (Class)
Closest TaxonA
notable phenotypeC
P450 & GST genes
164/170
Unclassified Alphaproteobacteria; marine
Uncultured bacterium TK97
(96.5%)
isolate from sponge
P450 & GST genes
161/169
Olavius losiaeendosymbiont 3 Unclassified Alphaproteobacteria; from
(95%)
gutless marine oligochete (Annelida)
P450 & GST genes
189/194
Gammaproteobacteria; from marine sediment
Shewanella sp. HAW-EB5
(97%)
P450 genes
176/189
Flavobacteria
Tenacibaculum maritimum
• pigmented
(93%)
P450 genes
174/188
Reichenbachia agariperforans Sphingobacteria
• pigmented
(92%)
P450 & GST genes
193/194
Gammaproteobacteria; from Artic sea ice
Psychrobacter glacincola
(99%)
strain ANT9276b
Verrucomicrobia
173/182
Uncultured bacterium clone
Verrucomicrobia sp.
endo- (nematode) &
(95%)
VERRUCO1from Salmonid gill
ecto- (marine ciliate)
186/196
Uncultured Verrucomicrobia bacterium clone
Verrucomicrobia sp.
symbionts described
(94%)
PI_4z12f;from Plum Island Sound estuary
A
Taxon and corresponding accession number of the closest 16S-V3 rDNA sequence match to the cloned amplicons (bands)
as determined via a BLAST search of the Entrez Nucleotides database (http://www.ncbi.nlm.nih.gov/ BLAST/).
B
Percent similarity of sequenced 16S-V3 PCR-DGGE amplicons to sequence of closest taxon.
C
Some of the 16S rDNA sequences belong to a restricted group of bacterial taxa that possess GST and P450 genes, which
are poorly characterized in prokaryotes but are key biotransformation enzymes in eukaryotes. Others belong to bacterial
taxa for which marine members possess carotenoid pigments.
In fact, numerous alphaproteobacterial endosymbionts have been described; classic examples being
the nitrogen-fixing rhizobia of plant legumes12 and the ultimate prokaryote endosymbiont, the
mitochondrian of eukaryotic cells whose genome is thought to be an alphaproteobacterial descendent7.
Two additional sequences were most closely related to uncultured Verrucomicrobia species. Two
exceptionally intriguing cases of symbioses have been described for members of this division, one
being an ectosymbiont of marine hypotrich ciliates (genus Euplotidium)13 and another in which
obligate intracellular Verrucomicrobia species are specifically associated with ovary wall and gut
epithelia of Xiphinema nematodes15. One of the 16S-V3 rDNA sequences exhibited 97% similarity
with Shewanella sp. HAW-EB5. Shewanella are gammaproteobacteria noted for their ability to
enzymatically reduce and thereby precipitate metals such as uranium, technetium and chromium,
leading to considerable interest in its potential for bioremediation of subsurface sediments and
groundwater contaminated with heavy metals and radionuclides11. Finally, two of the bacterial 16S-V3
rDNA sequences associated with the C. intestinalis branchial sac belong to classes whose marine
members are characterized, in part, by the carotenoid pigments they produce. Orange pigmentation is a
81
phenotypic characteristic of the Ciona branchial sac, and we’ve observed animal-to-animal variation in
this trait, consistent with the possibility that the branchial sac pigmentation is bacterial in nature.
With this working hypothesis in mind, we isolated bacteria from branchial sac enrichment cultures
with pigmentation as a primary selection criterion. This led to the isolation in pure culture of a pinkish
red bacterium that was identified by cloning and sequencing of full length 16S rDNA as a
Methylobacterium species (Fig 3). The genus Methylobacterium is comprised of a group of strictly
aerobic, facultatively methylotrophic, gram-negative, rod-shaped bacteria noted, in part, for their
production of pink to bright orange-red carotenoid pigments4.
Methylotrophs are also unique in their ability
to grow on one-carbon compounds more reduced
than carbon dioxide as sole carbon and energy
sources,
including
methanethiol
and
dimethylsulfide (DMS), which are important
intermediates in the biogeochemical sulfur
cycle4,16. Important sources of DMS in marine
environments include phytoplankton, macroalgae,
and coastal vascular plants, which use dimethyl
sulphoniopropionate (DMSP), a precursor of
DMS, as an osmolyte17. Interestingly, these
organic sulfur compounds are typically present in
Fig. 3. Phylogeny (A), TEM (B), and SEM (C)
high concentrations in shallow marine waters of
photomicrographs of the Methylobacterium isolate from C.
the littoral zone where tunicates thrive6.
intestinalis branchial sac. The full length 16S rRNA gene
Transmission electron microscopy was
shares 99% sequence identity with the uncultured
performed1 to further examine possible niches
Methylobacteriaceae clone 10-3Ba02, a molecular species
occupied by the putative branchial sac symbionts.
derived from a soil library18.
The TEM photomicrographs revealed sheath-like
structures in the stromal region underlying the
branchial sac epithelium containing structures of a
similar size and morphologically resembling
bacteria (Fig. 4). These sheaths appeared to be
adherent to the epithelial basement membrane but
lacked host cell nuclei. The positive identification
of the content of these sheath-like structures
requires additional investigation. Efforts are now
focused on determining: i) with domain and group
specific fluorescence in situ hybridization (FISH)
probes whether or not Bacteria and specifically
Methylobacteria are found in the stromal region of
the C. intestinalis branchial sac, and ii) if the
Methylobacterium isolate can grow on DMS and
particularly its precursor, the algal osmolyte
DMSP.
Fig. 4. TEM photomicrograph of C. intestinalis
branchial sac reveling sheath-like structures possibly
containing bacteria in the stromal region underlying the
epithelium.
These data will further guide efforts to determine if bacterial detoxification of the methylated
sulfides serves as a basis for stable association of Methylobacteria with the Ciona branchial sac and if
and how this has impacted host genes involved in sulfur metabolism and detoxification.
82
Supported by a New Investigator Award (HRG) with funds from MDIBL'
s NIEHS Center for
Membrane Toxicity Studies (ES03828-19). Michelle Diamond (University of Illinois) is acknowledged
for her assistance with 16S rDNA cloning as are staff of the W. M. Keck Center for Comparative and
Functional Genomics (University of Illinois) for sequence analysis. Thanks to Gary King, University
of Maine Darling Marine Center, for providing Ciona specimens and for useful discussions regarding
microbial biogeochemistry.
1.
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1919-1925, 1990.
Dehal P, Satou Y, Campbell RK, et al. The draft genome of Ciona intestinalis: insights into chordate and vertebrate
origins. Science 298: 2157-2167, 2002.
Dubilier N, Amann R, Erseus C, Muyzer G, Park SY, Giere,O and Cavanaugh CM. Phylogenetic diversity of
bacterial endosymbionts in the gutless marine oligochete Olavius loisae (Annelida). Mar Ecol Prog Ser 178: 271-280.
1999.
Green PN. Methylobacterium. In: The Prokaryotes:an Evolving Electronic Resource for the Microbiological
Community, 3rd edn. release 3.5. edited by M. Dworkin, S. Fallow, E. Rosenberg, K-H. Schleifer and E. Stackebrandt.
New York, USA: Springer-Verlag, 2001 [WWW document] URL http://link.springerny.com/link/service/books/10125/.
Hentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, Hacker J and Moore BS. Molecular evidence for a
uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68: 4431-4440, 2002.
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phytoplankton in estuarine and coastal water. Limnol Oceanogr 34: 53067, 1989.
Kurland CG and Andersson SG. Origin and evolution of the mitochondrial proteome. Microbiol Mol Biol Rev 64:
786-820, 2000.
Lueders T, Wagner B, Claus P and Friedrich MW. Stable isotope probing of rRNA and DNA reveals a dynamic
methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil. Environ Microbiol 6:
60-72, 2004.
McCracken VJ, Simpson JM, Mackie RI, and Gaskins HR. Molecular ecological analysis of dietary and antibioticinduced alterations of the mouse intestinal microbiota. J Nutr 131: 1862-1870, 2001.
Muyzer G, Brinkhoff T, Nubel U, Santegoeds C, Schafer H and Wawer C. Denaturant gradient gel electrophoresis
in microbial ecology. In: Molecular Microbial Ecology Manual, Vol. 3.4.4, edited by A. Akkermans, J.D. van Elsas
and F. de Bruijn. Boston, MA: Kluwer Academic Publishers, 1998, pp. 1-27.
Nealson KH, Belz A and McKee B. Breathing metals as a way of life: geobiology in action. Antonie Van
Leeuwenhoek 81: 215-222, 2002.
Patriarca EJ, Rosarita Tate R, and Iaccarino M. Key role of bacteral NH metabolism in Rhizobium-plant
symbiosis. Microbiol Mol Biol Rev 66: 203-222), 2002.
Petroni G, Spring S, Schleifer KH, Verni F, Rosati G. Defensive extrusive ectosymbionts of Euplotidium
(Ciliophora) that contain microtubule-like structures are bacteria related to Verrucomicrobia. Proc Natl Acad Sci USA
97: 1813–1817, 2000.
Satoh N, Satou Y, Davidson B, and Levine M. Ciona intestinalis: an emerging model for whole-genome analyses.
Trends Genet 19: 376-381, 2003.
Vandekerckhove TT, Coomans A, Cornelis K, Baert P and Gillis M. Use of the Verrucomicrobia-specific probe
EUB338-III and fluorescent in situ hybridization for detection of "Candidatus Xiphinematobacter" cells in nematode
hosts. Appl Environ Microbiol 68: 3121-3125, 2002.
Visscher PT, Kiene RP and Taylor BF. Demethylation and cleavage of demethylsulfoniopropionate in marine
intertidal sediments. FEMS Microbiol Ecol 14:179–190, 1994.
Yoch DC. Dimethylsulfoniopropionate: its sources, role in the marine food web, and biological degradation to
dimethylsulfide. Appl Environ Microbiol 68: 5804-5815, 2002.
83
Osmoregulation in Fundulus heteroclitus oocytes and embryos
measured by sedimentation pycnometry.
Robert L. Preston1, Michael E. Gille1, Daniel M. Richmond1,
Lauren B. Sliga1, Christopher W. Petersen2 and George W. Kidder3
1
Department of Biological Sciences, Illinois State University, Normal, Illinois 61790,
2
College of the Atlantic, Bar Harbor, Maine 04609,
3
Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672
In teleost fish, maturing oocytes are in contact with maternal fluids which are isosmotic with the
maternal blood3,5. After spawning and fertilization, most teleost eggs become much less permeable to
water which allows them to maintain osmotic balance during development. Killifish, Fundulus
heteroclitus, spawn in estuaries at intermediate salinities and the embryos, which ordinarily require
about 14 days to develop, are exposed to large changes in salinity due to periodic tidal inflow of
seawater (SW) and subsequent flushing by fresh water (FW) outflow. In addition, killifish embryos
are capable of full development aerially in moist environments2. Guggino3,4 showed that the water
permeability of killifish embryos is sufficiently high that active osmoregulatory mechanisms are
necessary and this is consistent with our previous findings that embryos showed increasing expression
of cystic fibrosis transmembrane regulator (CFTR) mRNA as development proceeds6. We also
showed that unfertilized oocytes were responsive to osmotic changes in the external medium using
sedimentation pycnometry (SP) a method that measures oocyte density changes as an index of water
gain or loss6,7. In this study, we extend our SP measurements to killifish embryos to test the hypothesis
that killifish embryos should show increased regulatory capability compared with unfertilized oocytes,
Oocytes were manually expressed from females adapted to SW (922 mOsM) and fertilized in vitro
in 10 o/oo medium (325 mOsM). The embryos were held at 20o C in air at 100% humidity. Oocyte
and embryo density was measured by SP as follows7: Freshly collected oocytes or embryos (stages 29
through 36)1 were placed in SW or diluted SW of the following osmolarities: 10 mOsM, 296 mOsm
and 917 mOsM. After incubation periods of one and four hours, single oocytes or embryos were
transferred to 1.5 ml microfuge tubes containing 0.3 ml of the appropriate SW and 0.5 ml of phthalate
mixtures. By varying the quantities of two phthalates of differing densities, phthalic acid diethyl ester
and phthalic acid bis (3,5,5-trimethylhexyl) ester, a 12-member density step gradient was formed with
densities ranging between 1.03 g/ml and 1.06 g/ml. The tubes were centrifuged at 14,000 x g and the
tubes inspected for floating or sedimented oocytes. A modification of our earlier procedure7 was used
to assure consistent sedimentation behavior with embryos. Tubes containing the oocytes or embryos
plus phthalates were centrifuged in additive increments for 5, 15, 30, 45, 60 and 300 sec and
sedimentation or lack thereof was noted at each time. It was concluded that the best representative
total centrifugation period was 50 sec (5+ 15 + 30 sec increments). Each measurement was done at
least 6 times.
Figure 1 shows the apparent osmotic behavior of killifish embryos (stages 29-36) compared with
that of unfertilized oocytes. The embryos or oocytes were exposed to hypotonic (10 mOsM), isotonic
(296 mOsM) or hypertonic media (917 mOsM) for one and four hours and then the density change was
determined by SP. The data show that as the osmotic pressure of the medium changes the embryos
gain water (at 10 mOsM) or lose water (at 917 mOsM) compared with the isotonic condition (296
mOsM). It is apparent that there is a greater change in density in unfertilized oocytes compared with
embryos, a result that is consistent with the hypothesis that active osmoregulatory mechanisms are
being expressed as embryos develop. The amount of density change when comparing FW (10 mOsM)
84
Density at which oocyte sinks
1.06
1.055
1.05
1.045
1.04
1.035
1.03
1.025
and SW (917 mOsM) is
two to three times greater
in unfertilized oocytes
than embryos (Table 1).
These results support our
hypothesis that increasing
osmoregulatory capacity
is to be expected during
killifish
embryonic
development.
1.02
1.015
In other experiments,
we
measured
the
10
296 917
quantitative expression of
Embryos
Oocytes
Na/K
ATPase
alpha
subunit
1a
mRNA
using
Figure 1: Mean minimum densities measured by sedimentation pycnometry for single
real-time PCR, which
Fundulus heteroclitus oocytes incubated in 10, 296, and 917 mOsm/kg SW solutions
for one hour (solid bars) or four hours (open bars). Data are displayed as means ±
increases as development
standard errors, n=6. Significant differences are present between the densities at the
proceeds.
Although
three salinities in each experimental group, p<0.05 (t-test).
preliminary in nature, the
results are consistent with our earlier findings of increasing CFTR mRNA expression during killifish
embryonic development6. It has also been noted that chloride cells appear early in development in the
yolk sac of embryos before their appearances in embryonic gills4. These data support the hypothesis
that killifish embryos
Table 1: Change in density of Fundulus embryos and oocytes after one hour and four
actively
osmoregulate
hour exposure to FW (10 mOsM), isotonic (296 mOsM) and hypertonic (917 mOsM)
during development and
medium. The density changes were calculated as follows: (ρ917 – ρ10)/(917 mOsM –
10 mOsM), where ρ917 and ρ10 are the apparent oocyte densities in 917 mOsM and 10
that they do not simply
mOsM medium respectively.
resist osmotic stress by
remaining
completely
One hour incubation
Four hour incubation
impermeable to water and
Density change
Density change
solute transfer.
g cm-3 osmole-1 x 10-6
g cm-3 osmole-1 x 10-6
Embryos
7.39
7.28
Supported by NSF C-RUI
Oocytes
22.5
13.9
0111860.
1.01
1 Armstrong, P. B., and Child, J. S. 1965. Stages in the normal development of Fundulus heteroclitus.
Biol. Bull.
128:143-168.
2. Baldwin, J.L., C.E. Goldsmith, C. W. Petersen, R. L. Preston and G. W. Kidder. Synchronous hatching in
Fundulus heteroclitus embryos: Production and properties. Bull. Mt. Desert Isl. Biol. Lab. 43: 25-27, 2004.
3. Guggino, W.B. Water balance in embryos of Fundulus heteroclitus and F. bermudae in seawater. Am. J. Physiol. 238:
R36-R41, 1980.
4. Guggino, W.B. Salt balance in embryos of Fundulus heteroclitus and F. bermudae adapted to seawater. Am. J. Physiol.
238: 42-R49, 1980.
5. Preston, R. L., R. J. Clifford, A. K. Guy, N. B. Richards, C. W. Petersen and G. W. Kidder. Preliminary studies of
salinity adaptation in Fundulus heteroclitus and apparent CFTR mRNA expression in gill tissue and oocytes. Bull. Mt.
Desert Isl. Biol. Lab. 42: 68-70, 2003.
6. Preston, R. L., R. J. Clifford, J.A. Thompson, D.L. Slager, C. W. Petersen and G. W. Kidder. CFTR mRNA
expression in developing Fundulus heteroclitus embryos. Bull. Mt. Desert Isl. Biol. Lab. 43: 25-27, 2004.
7. Preston, R. L., L.F. Hartema and S. A. Miller. Cell density measurement as an index of cell volume change in red
blood cells exposed to hypo-osmotic media. Bull. Mt. Desert Isl. Biol. Lab. 31: 138-140, 1992.
85
Bile duct obstruction and drainage modulates Bsep and Ostα gene expression in Leucoraja
erinacea, the little skate
Shi-Ying Cai1, Shuhua Xu1, Ned Ballatori2,3, and James L. Boyer1,3
Liver Center, Yale University School of Medicine, New Haven, CT 06520
2
Dept. of Environmental Medicine, Univ. of Rochester School of Medicine, Rochester, NY 14642
3
1
Bile salts play an important role in maintaining normal body functions not only because they
facilitate food digestion and nutrient absorption, but also because they serve as key signaling
molecules in the enterohepatic circulation. Bile salts activate nuclear receptors, specifically FXR, and
regulate the expression of a number of genes in mammals1. Several key genes which are involved in
bile salt synthesis in the liver and their enterohepatic circulation are regulated by FXR, including
Cyp7A1 (the rate limiting enzyme converting cholesterol to bile salts) via regulation of Shp, and Bsep
(Abcb11), the bile salt export pump. In addition, a bile acid response element in the human BSEP
promoter region has been identified2. We have previously cloned and functionally characterized a
Bsep orthologue from the liver of the small skate, Leucoraja erinacea. However it is not known how
this gene is regulated 3. More recently, we have identified Ostα (organic solute transporter alpha) as a
heteromeric partner for the basolateral bile acid efflux transporter in ileum4,5. Ostα is highly expressed
in skate liver, but whether it undergoes adaptive regulation is not known. To address these questions,
we performed bile duct ligation and biliary drainage in the small skate, procedures that increase and
decrease bile retention in the liver and circulation.
Sham, common bile duct ligation (CBDL), and bile drainage (BD) were carried out for 7 days in
four skates for each group. Skates were caught by trawl from the coast of Maine. Total RNA was
extracted with Trizol from liver, kidney, and intestine and further treated with Qiagen RNeasy kit. A
set of primers and TaqMan probe were designed with Primer Express Software (Applied Biosystem
Inc.) and synthesized by IDT (Integrated DNA Technology) for skate Bsep, Ostα, and β-actin genes,
respectively. Quantitative real-time RT-PCR was performed on an ABI 7700 DNA Sequence
Detection System. To minimize variations in gene expression between individual animals, equal
amount of total RNA from each sample were pooled together for each group. β-Actin was used as a
reference and data from CBDL and BD groups were compared with the sham animals. The results
suggest that Bsep is up-regulated in CBDL liver by 41% and downregulated in BD liver by 53% (Fig.
1A). These findings are consistent with observations obtained from mammalian liver and HepG2 cell
lines2,3, and suggest that skate Bsep may also be regulated by FXR in response to the retention or
depletion of the bile salt (bile alcohol) pool. Similar to Bsep in liver, Ostα expression in kidney was
up 48% in the CBDL group and down 74% in the BD group (Fig. 1C). But Ostα was down-regulated
in both CBDL and BD intestines by 51% and 39% respectively (Fig. 1D). These results suggest that
the expression of skate Ostα gene in kidney and intestine is also regulated by the systemic retention of
bile. However no significant difference was observed for Ostα gene among sham, CBDL, and BD
treatment in liver (Fig.1B). We also measured the relative abundance of skate Ostα gene expression in
liver, kidney and intestine by using β-actin as a reference. These results show that the Ostα is most
abundant in liver, since the mRNA ratio was 800:150:1 liver>kidney> intestine, consistent with our
previous report4. Taken together, our findings indicate that bile salts (e.g. skate symnol sulfate),
modulate gene expression in the small skate, presumably via FXR, which we have also recently
characterized from skate liver. These studies were supported by National Institutes of Health Grants
ES03828, ES01247, DK34989, and DK25636.
86
B. Liver Ost alpha
1.41
1.5
1
1
0.47
0.5
0
sham
CBDL
mRNA (fold)
mRNA (fold)
A. Liver Bsep
1
1
0.26
0
sham
CBDL
CBDL
BD
1
0.5
D. Intestine Ost alpha
1.48
0.5
1.32
1
sham
BD
mRNA (fold)
mRNA (fold)
1.5
1.22
0
BD
C. Kidney Ost alpha
2
1.5
1.5
1
1
0.49
0.61
CBDL
BD
0.5
0
sham
Figure 1. Skate Bsep and Ostα expression in sham, CBDL, and BD treated liver, kidney and intestine by Q-RT-PCR. A,
skate Bsep expression in liver from pooled samples; B, skate Ostα expression in liver from pooled samples; C, skate Ostα
expression in kidney from pooled samples; D, skate Ostα expression in intestine from pooled samples.
1
2
3
4
5
Xu G, Pan LX, Li H, Forman BM, Erickson SK, Shefer S, Bollineni J, Batta AK, Christie J, Wang TH, Michel
J, Yang S, Tsai R, Lai L, Shimada K, Tint GS, Salen G. Regulation of the farnesoid X receptor (FXR) by bile acid
flux in rabbits. J Biol Chem. 277:50491-6, 2002.
Ananthanarayanan M, Balasubramanian N, Makishima M, Mangelsdorf DJ, Suchy FJ. Human bile salt export
pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J Biol Chem. 276(31):28857-65, 2001.
Cai SY, Wang L, Ballatori N, Boyer JL. Bile salt export pump is highly conserved during vertebrate evolution and
its expression is inhibited by PFIC type II mutations. Am J Physiol Gastrointest Liver Physiol. 281:G316-22, 2001.
Wang W, Seward DJ, Li L, Boyer JL, Ballatori N. Expression cloning of two genes that together mediate organic
solute and steroid transport in the liver of a marine vertebrate. Proc Natl Acad Sci USA. 98:9431-6, 2001.
Dawson PA, Hubbert M, Haywood J, Craddock AL, Zerangue N, Christian WV, Ballatori N. The heteromeric
organic solute transporter alpha-beta, Ostalpha -Ostbeta, is an ileal basolateral bile acid transporter. J Biol Chem. 2004
Nov 24; [Epub ahead of print]
87
The organic anion transport protein 1d1, Oatp1d1, mediates hepatocellular uptake of phalloidin
in the little skate, Leucoraja erinacea
Fabienne Meier-Abt1, Bruno Hagenbuch1, Ned Ballatori2, and James L Boyer3
Clinical Pharmacology and Toxicology, University Hospital, CH-8091 Zurich, Switzerland
2
Dept. of Environmental Medicine, Univ. of Rochester School Medicine, Rochester, NY 14642
3
Liver Center, Department of Medicine, Yale Univ. School of Medicine, New Haven, CT 06520
1
Organic anion transporting polypeptides (animals, Oatps; human, OATPs) of the OATP/SLCO
superfamily of solute carriers mediate multispecific substrate uptake in various organs of vertebrate
animal species 3. In mammalian liver, members of the OATP1 family (i.e. mouse and rat liver:
Oatp1a1, Oatp1a4, Oatp1b2; human liver: OATP1A2, OATP1B1, OATP1B3) mediate sodiumindependent uptake of bile salts, organic dyes, steroids and steroid conjugates, thyroid hormones,
prostaglandins, various oligopeptides and numerous drugs 2. Interestingly, the substrate spectrum of
the hepatic Oatps/OATPs also include endogenous and exogenous linear and cyclic oligopeptides such
as the endothelin receptor antagonist BQ-123, the thrombin inhibitor CRC220, the opioid receptor
agonists DPDE and deltorphin II and the enterohepatic hormone cholecystokinin 8 (CCK-8) 2,3. More
recently, the hepatic Oatps/OATPs have also been shown to be responsible for the uptake of the
hepatotoxic exogenous cyclic peptide phalloidin into rat and human livers 4.
In the liver of the little skate Leucoraja erinacea an evolutionary ancient Oatp has been identified
at the basolateral (sinusoidal) plasma membrane domain of hepatocytes 1. This skate Oatp or
Oatp1d1/Slco1d1 (for classification and nomenclature see reference 3) shares 41.2-43.3% amino acid
sequence identities with the rat liver Oatp1b2 (also called Oatp4) and the human liver OATP1B1 (also
called OATP-C, OATP2, LST-1) and OATP1B3 (also called OATP8) 1. Since the mammalian liver
Oatps/OATPs have most probably arisen from gene duplication after species divergence 2,3, the present
study examined whether the ancient skate Oatp1d1 shares the phalloidin transport properties with the
mammalian liver Oatps/OATPs.
Skate hepatocytes were isolated as previously described 8, and uptake of [3H]-demethylphalloin 7, a
biological equivalent of phalloidin 4,5,6, was measured in freshly isolated cell suspensions. As a control
for transport competent skate hepatocytes, [3H]-taurocholate uptake was determined in parallel 9. In
addition, Oatp1d1 was expressed in Xenopus laevis oocytes and phalloidin uptake activity was
compared with that of another skate liver organic solute transporter Ostα-Ostβ 10. As a control for
active Oatp1d1 and Ostα-Ostβ expression, uptake of [3H]-estrone-3-sulfate was also determined.
As previously demonstrated 9 and as illustrated in figure 1, isolated skate hepatocytes showed
predominant sodium-independent taurocholate uptake. Approximately 60% of total taurocholate
uptake at 60 min were observed within the first 10 min of incubation (Fig. 1A). Similar sodiumindependent uptake was also seen for demethylphalloin, although its uptake into skate hepatocytes was
slower (uptake at 10 min: ~ 40% of uptake at 60 min) as compared to taurocholate (Fig. 1B).
Furthermore, after an initial rapid uptake phase, time-dependent demethylphalloin uptake did not level
off, but continued to rise steadily up to 60 min.
Initial linear demethylphalloin uptake rates (1 min uptake values) showed clear saturability with
increasing substrate concentrations with an apparent Km value of 0.38 ± 0.06 µM and a Vmax-value of
10.1 ± 0.3 fmol/mg protein x min-1 (data not shown).
88
12 0
0
A) Taurocholate
80
sodium
sodium
choline
choline
40
0
0
10 20 30 40 50 60
% uptake
of 60 min uptake
% uptake
of 60 min uptake
These data strongly indicate active carrier-mediated uptake of phalloidin into skate hepatocytes.
Interestingly, the affinity of the skate liver uptake system for phalloidin is 15-40-fold higher than the
phalloidin affinities of the rat and human liver uptake systems 4. This high affinity of the phalloidin
uptake system in skate liver is somewhat surprising, since phalloidin is a mushroom toxin, which
marine skates are hardly exposed to. Nevertheless, the data demonstrate that skate liver is equipped
with an uptake system for the bicyclic peptide phalloidin and perhaps structurally related marine
compounds.
B) Demethylphalloin
120
80
sodium
sodium
choline
40
0
0
10
5
60
3 40 50
0
0 time (min)
Incubation
20
Incubation time (min)
(min)
(min)
Fig. 1: Time course of taurocholate (A) and demethylphalloin (B) uptake in skate hepatocytes. Skate hepatocytes
were incubated with labeled taurocholate [10 µM] (A) or demethylphalloin [1 µM] (B). For sodium-independent uptakes all
sodium in the elasmobranch Ringer solution 8 was replaced by choline. Substrate uptakes were determined at the indicated
time intervals. Data represent the mean ± SE of three separate cell preparations.
Because demethylphalloin has been shown to be taken up into rat and human livers by members of
the OATP/SLCO superfamily of solute carriers 3,4, and because an ancient Oatp (Oatp1d1) has been
identified in skate liver 1, additional studies examined whether skate Oatp1d1 is involved in phalloidin
transport. For this purpose the inhibition pattern of demethylphalloin uptake in skate hepatocytes was
examined using typical substrates and/or inhibitors of skate Oatp1d1 1 and/or of mammalian liver
Oatps/OATPs 2,3. The strongest inhibitor of demethylphalloin uptake was phalloidin, which is
consistent with demethylphalloin being a structural and biological equivalent of phalloidin (data not
shown). Approximately 40-60% inhibition was found for the well-known Oatp substrates estrone-3sulfate, taurocholate and related bile salt derivatives, the organic dye sulfobromophthalein and the
organic anion transport inhibitor probenecid. The inhibitory effect of cyclosporine A (~40%) is
consistent with its strong inhibition of rat and human liver Oatps/OATPs 2,3. This spectrum of
inhibitory substances indicated that Oatp1d1 may be involved in the uptake of phalloidin into skate
hepatocytes.
To test this hypothesis, Xenopus laevis oocytes were injected with skate Oatp1d1 cRNA and uptake
of demethylphalloin and estrone 3-sulfate was measured (Fig. 2). Only oocytes injected with Oatp1d1
cRNA exhibited higher demethylphalloin uptake than water injected oocytes (Fig. 2A). Oocytes
injected with cRNAs for the skate liver organic anion transporter Ostα-Ostβ 10 did not show increased
demethylphalloin uptake, although these oocytes demonstrated markedly increased uptake of the
known substrate estrone 3-sulfate (Fig. 2B).
These data support the observations in skate hepatocytes and show that Oatp1d1 represents a
phalloidin uptake system in skate liver. Thus, the results obtained in skate hepatocytes and cRNA
injected Xenopus laevis oocytes demonstrate that the skate liver Oatp1d1 can mediate transport of the
cyclic peptide phalloidin. These data support the assumption that Oatp1d1 represents an ancient
precursor of the mammalian liver Oatps/OATPs and of the other members of the OATP1 family 3.
89
While it is now clear that the skate Oatp1d1 shares numerous substrates with the mammalian liver
Oatps/OATPs it remains to be investigated whether the same is also true for the closest human
Oatp1d1 homologue, OATP1C1 3.
A) 3H-Demethylphalloin
B) 3H-Estrone-3-sulfate
300
0
600
fmol/oocyte
fmol/oocyte
800
400
200
0
water
Oatp1d1
Ostα/β
200
100
0
water
Oatp1d1
Ostα/β
β
Fig. 2: Uptake of demethylphalloin (A) and estrone-3-sulfate (B) in water or cRNA (Oatp1d1, Ostα-Ostβ)
injected Xenopus laevis oocytes. Oocytes were injected with water or with 5ng Oatp1d1-cRNA 1 or 2 ng Ostα-OstβcRNA 10. After 3 days in culture, uptakes of [3H]-demethylphalloin (4 µM) or [3H]-estrone-3-sulfate (50 nM) were
measured at 25°C for 30 min. Data represent the means ± SE of eight uptake measurements in one representative out of two
oocyte preparations.
These studies were supported by National Institutes of Health Grants ES03828, ES01247,
DK34989, DK25636, DK48823, and by NSF-REU DBI0139190.
1. Cai S.Y., W. Wang, C.J. Soroka, N. Ballatori, and J.L. Boyer. An evolutionarily ancient Oatp: insights into
conserved functional domains of these proteins. Am J Physiol 282:G702-G710, 2002.
2. Hagenbuch, B., and P.J. Meier. The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta
1609:1-18, 2003.
3. Hagenbuch, B., and P.J. Meier. Organic anion transporting polypeptides of the OATP/SLC21 family: phylogenetic
classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties. Pflugers Arch – Eur
J Physiol 447:655-665, 2004.
4. Meier-Abt, F., H. Faulstich, and B. Hagenbuch. Identification of phalloidin uptake systems of rat and human liver.
Biochim Biophys Acta 1664:64-69, 2004.
5. Münter, K., D. Mayer, and H. Faulstich. Characterization of a transporting system in rat hepatocytes. Studies with
competitive and non-competitive inhibitors of phalloidin transport. Biochim Biophys Acta 860:91-98,
6. Petzinger, E. Competitive inhibition of the uptake of demethylphalloin by cholic acid in isolated hepatocytes. Evidence
for a transport competition rather than a binding competition. Naunyn-Schmiedeberg’s Arch Pharmacol 316:345-349,
1981.
7. Puchinger, H., and T. Wieland. [3H]-Desmethylphalloin, Liebigs Ann Chem 725:238-240, 1969.
8. Smith, D.J., M. Grossbard, E.R. Gordon, and J.L. Boyer. Isolation and characterization of a polarized isolated
hepatocyte preparation in the skate Raja erinacea. J Exp Zool 241:291-296, 1987.
9. Smith, D.J., M. Grossbard, E.R. Gordon, and J.L. Boyer. Taurocholate uptake by isolated skate hepatocytes: effect
of albumin. Am J Physiol 252:G479-G484, 1987.
10. Wang, W., D.J. Seward, L. Li, J.L. Boyer, and N. Ballatori. Expression cloning of two genes that together mediate
organic solute and steroid transport in the liver of a marine vertebrate. Proc Natl Acad Sci USA 98:9431-9436, 2001.
90
Lectins as markers for tubule regions in the kidney of Squalus acanthias, with further
observations on SGLT2 immunohistochemistry
Thorsten Althoff, Hartmut Hentschel and Rolf K. H. Kinne
Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund, Germany
After presenting the first results of immunohistochemical studies on the localization of the Na+/Dglucose cotransporter (SGLT) in the kidney of the spiny dogfish (Squalus acanthias) in last year's
bulletin3, we can now refine these results with more details. During analysis of the tissue sections we
encountered some problems in identifying individual tubule segments. Here we present a new valuable
approach to identify distinct nephron regions in tissue sections in a reproducible manner.
For immunohistochemistry we used frozen sections of shark kidney preferentially from female
animals (simply because the kidneys are easier to prepare). The tissues were fixed during preparation
by a drip of ice cold 4% paraformaldehyde (in 3x PBS, pH6.8), cut into small pieces and then further
fixed over night at 4°C. After rinsing the samples in PBS the tissue was embedded in Tissue-Tek OCT
compound (Sakura Finetek U.S.A., Inc., Torrance, CA) and frozen in liquid nitrogen. Cryosections of 6
µm thickness (obtained with a Leica Microsystems cryostat) on slides were blocked for one hour in 3%
dry skim milk (in PBS) and subsequently incubated for another hour with specific primary antibodies
(diluted 1:300 in PBS + 1% Triton-X-100). The primary antibodies were detected with fluorescentlabelled secondary antibodies (Alexa-Fluor 488 or Alexa-Fluor 555 anti rabbit IgG, Molecular Probes),
diluted as described above. The DNA stain 4',6-Diamidino-2-phenylindole (DAPI) was used to
counterstain the nuclei. To label the endogenous alkaline phosphatase, a marker for the brush border
membrane (BBM), the Vectastain ABC-AP kit (Vector Laboratories) was applied according to the
manufacturers manual. Fluorescein-labeled lectins, also obtained from Vector Labs, were used at 1:150
dilution in 1x PBS and incubated for 20 min at room temperature. In detail these were Sophora
japonica agglutinin (SJA), Griffonia simplicifolia lectin I (GSL I), Ricinus communis agglutinin I
(RCA I), Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Ulex europaeus agglutinin
(UEA I), peanut agglutinin (PNA) and Concanavalin A (Con A). The stained sections were mounted
with ProLong antifade solution (Molecular Probes). Microscopy was performed on an Axiovert 200M
microscope equipped with Apotome and CCD camera Axiocam MR (Carl Zeiss, Göttingen, Germany)
with Plan-Neofluar lenses 25x/0.8 and F-Fluar 40x/1.3.
The complex renal architecture of Squalus acanthias, and other marine cartilaginous fish, involves
a distinct zonation of the tissue (for review see Elger et al.2). Thus cross sections through the excretory
opisthonephric kidney generally reveal three characteristic regions: (1) The zone of lateral
countercurrent bundles, (2) the mesial tissue zone, and (3) a region between the two zones, where
glomeruli abound (Figure 1).
An individual nephron displays the following segments: neck segment NS, proximal tubule segments
PI and PII, intermediate segment IS, early distal tubule segment EDT, and late distal tubule segment
LDT. According to microdissection combined with histology, spiny dogfish display a specific
subdivision of the proximal tubule segment PI and PII (PIa in the bundle, PIb in the region of the
glomeruli, PIIa and PIIb exclusively in the mesial tissue) (Elger and Hentschel1). The early distal
tubule EDT, which is present exclusively in the lateral bundle zone, is contiguous with the late distal
tubule, which thereafter performs numerous bends in mesial tissue. The late distal tubule LDT is
present in mesial tissue, where it courses along the pathway of PIIa tubules.
91
Table 1: Lectin binding to renal structures of Squalus acanthias and localization of SGLT2. Results were obtained from 2
animals. As we did not attempt semi-quantitative evaluation, scoring was done with an arbitrary scale with:
- no signal; +/- very weak signal; + weak signal; ++ medium signal; +++ strong signal; ++++ very strong signal
SJA
GSL I RCA I
SBA
DBA
PNA
UEA I
SGLT2
Glomerulus
++
+
+ to ++
Neck segment
+
Proximal tubule segment
PIa
+
++
+
+ to ++
PIb
+
+++
+++
+++
++ to ++++
PIIa
+
+
(+)
PIIb
+
+++
+
(+)
++
++ to +++
Intermediate segment
+
+
+
++
(+)
Early distal tubule
+++
++
+
(+)
+/Late distal tubule
++
++
(+)
++ to +++
+++
Collecting tubule/collecting duct
++
++
++ to +++
+ to ++
+++
After incubation with the fluorescence-labelled lectins we found that individual lectins bound
preferentially to distinct regions of renal tubules. Three patterns of lectin-binding were observed: (1)
lectins labelled a variety of structures, glomeruli and various tubule segments (GSL-1, SBA), (2)
lectins bound preferently to certain tubular regions, proximal (PNA, RCA-I) or distal (DBA, UEA-1),
(3) lectins showed specificity for a distinct tubule segment (SJA, DBA) (Table 1 and Figure 1).
Fig.1. Lectin binding to renal structures. A. Cross sections through lateral bundles. Several tubular profiles including early
distal tubule (EDT) are labeled by Griffonia simplicifolia lectin (GSL I).B. Selective staining of proximal tubule segment
PIb near glomerulus (GL) by Peanut agglutinine (PNA) fluorochrome. C. Mesial tissue with large profiles of proximal
tubule segment PIIa, small profiles of late distal tubule (LDT) and collecting tubule (CT). LDT and CT bind Ulex europaeus
agglutinin (UEA-I). D. Staining by Sophora japonica agglutinin (SJA) at the apex of epithelial cells of intermediate segment
(IS) is highly specific. Nuclear counterstain DAPI. Bar equals 50 µm. Shown are typical images from 2 animals.
92
Anti-SGLT2 antibody labelled specific nephron subsegments and the collecting tubule-collecting
duct system in all regions of dogfish kidney: lateral bundle zone (LB), glomerular region (GL) and
mesial tissue (MT) (Figure 2).The PIa segment in the lateral bundle zone displayed apical SGLT2
fluorescence signal within the region of its brush border. The PIb segment, which performs a tortuous
course with many bends in the region of the glomeruli showed a very strong immunoreactivity in the
apical cell region, including the brush border. The PIIa and PIIb segments of the proximal tubule
present in mesial tissue diplayed markedly different patterns of anti SGLT2 labeling characteristics.
PIIa cells exhibited no SGLT2 immunolabel. In contrast, PIIb cells consistently displayed specific
SGLT2 immunoreactivity. The apical cell membrane of EDT epithelial cells was weakly labeled by
anti-SGLT2 antiserum. LDT cells showed strong SGLT2-labeling at the apical zone. Significant antiSGLT2 immunoreactivity was observed in the collecting tubules and collecting ducts. SGLT2 antibody
binding was confined to the region of the apical cell membrane and its adjacent cytoplasmic zone (see
Table 1). In summary, we consistently found SGLT2 labeling in nephron segments PIa, PIb, PIIb and
CT.
Fig. 2. Cross section through renal tissue of
Squalus acanthias showing binding sites of
anti-SGLT2 antibody. Frozen section (6µm)
was incubated with rabbit anti-shark SGLT2
L13c antibody (1:300) and Alexa Fluor-488 anti
rabbit secondary antibody. Shown is a typical
image as seen in 2 animals.
GL = glomerular region; PIb = proximal tubule;
LB = lateral bundle zone; MS = mesial tissue.
The knowledge about which lectin stains a specific segment of the nephron provides a powerful
tool for further studies on transporter localization. Moreover, the anti-SGLT2 antibody has binding
characteristics, which are useful to reveal epithelial cells of proximal tubule segment PIb and collecting
tubule in tissue preparations of Squalus acanthias.
This research was supported by NIEHS 1-P30-ESO 3828 to RK.
1.
2.
3.
Elger, M., Hentschel, H. Microdissection of renal tissue and isolated tubules in kidney of Squalus acanthias, with
emphasis on tissue zonation and renal tubular segmentation. Bull. Mt Desert Isl. Biol. Lab. 32: 23-27, 1993
Elger, M., H. Hentschel, M. Dawson and J.L. Renfro. Urinary Tract. Microscopic Functional Anatomy. In:
Handbook of experimental animals. (Eds.) G. Bullock and D.E. Bunton. The laboratory fish, edited by G.K.
Ostrander, Academic Press, San Diego, 2000, p 385-413.
Scharlau, D., Althoff, T., Hentschel, H., and Kinne, R. Immunohistochemical studies of Na+/D-glucose
cotransporters in the intestine and kidney of Squalus acanthias and Leucoraja erinacea. Bull. Mt Desert Isl. Biol.
Lab. 43: 18-21, 2004
93
Identification and cloning of a unique somatostatin receptor in the brain of the spiny dogfish
shark, Squalus acanthias
1
Carolina Klein1 and J.N. Forrest, Jr.1,2
Department of Internal Medicine, Yale University, School of Medicine, New Haven, CT 06510
2
Mt. Desert Island Biological Laboratory, Salisbury Cove, ME 04672
Somatostatin receptors (sst, SSR) are present in numerous tissues, where their activation inhibits a
wide variety of exocrine and endocrine secretions. They belong to the rhodopsin superfamily of Gprotein coupled receptors, all of which contain seven transmembrane domains. Somatostatin receptors
contain no introns. To date, five SSR subtypes have been described, each having specific agonist
affinity, mechanisms of action, tissue distribution and function. There is a 39-57% amino acid
sequence identity among the subtypes, with a highly conserved sequence in the seventh transmembrane
domain1. In 1999, Florian Plesch in our laboratory obtained partial sequence of four somatostatin
receptors in shark tissues (rectal gland and brain)2. We report here the full length cloning and analysis
of one of these receptors from shark brain which we have designated as SSR3/5.
Tissue was extracted from adult S. acanthias brain, and total RNA was obtained by the standard
TRIzol (Gibco BRL, Carlsbad, CA) method. To perform RACE PCR (Clontech BD Biosciences
Marathon RACE PCR, Palo Alto, CA), messenger RNA was extracted and double stranded cDNA was
produced using AMV reverse transcriptase (Super Script-II, BD Biosciences Clontech, Palo Alto, CA)
followed by a Second-Strand Enzyme cocktail containing E. coli DNA polymerase I and E. coli DNA
ligase. Finally, an adaptor was ligated to the double strand cDNA using a T4 DNA ligase. Degenerate
primers were designed from our sst partial sequence using Codehop and gene specific primers were
designed using DNA Star software.
The
following
degenerate
primers
were
used
on
brain
cDNA:
5’GGGATTCTTTATTCCATTTACTATTATTTGTYTNTGYTAYHT-3’ and 5’-TGAAAAATCCCATCAC
AAAAGTGTANAYNAYRAA-3’. A gradient PCR was run with initial denaturation at 95˚ for 2 min
followed by 35 cycles of a denaturating step at 95˚ for 45 seconds, annealing temperature of 61˚ for 1
min- gradient +/- 10˚ (Gradient Mastercycler, Eppendorf), extension at 72˚ for 2 minutes. The receptor
was isolated using gene specific primers that were designed based on a 522 bp known segment of the
receptor2: for sst 3/5, 5’-GCCCCGGATAGCCAAGATG-3’ and 5’-TAAAACGGGAGCCAGCAAATC-3’
followed by 5’-CCCGCTGTTAATAATATGCCTCTG-3’ and 5’-CGTAAACCAGATGACTTGAC
TTTGAT-3’.
These primers yielded 5’ 719 and 3’ 522 bp fragments that were extracted from a 1% agarose gel
(QIAquick Gel Extraction Kit, QIAGEN Sciences, Valencia, CA) and sequenced by the MDIBL
sequencing facility and identified as known somatostatin receptors. Start to stop primers-5’CAATGGACATGAGTACAGTTTTGTAGAGA -3’ and 5’- TGCAGTAAACGCT CATATTTAGCC
-3’ were used to amplify both reactions using the same cycle program, and sequence on both strands
was confirmed. These were cloned into the Plac lacZ site of the pCR II-TOPO 4.0 kb (TOPO TA
Cloning, Invitrogen, Carlsbad, CA) and sequenced again.
RACE PCR gave multiple bands (not shown). Nested PCR reaction using RACE PCR as substrate
resulted in a 522 bp 3’ band and a 719 bp 5’ band. Both overlapped with our known partial sequences.
Start to stop primers were designed and a PCR reaction yielded a 1074bp sequence (Figure 1) that had
highest homology to both SSR3 and SSR 5 in other species.
94
Figure 1. Full length clone of the somatostatin 3/5 receptor
(1074 bp) using shark brain as cDNA template.
The longest open reading frame predicted a 366 aa protein sequence, with molecular weight of
41117. The translated protein contains 7 TM domains. The highly conserved region in the 7th TM
domain is conserved in this receptor as well (YANSCANPI/VLY). (Figure 2).
Figure 2. Viseur diagram of amino
acid sequence showing the 7
transmembrane domains of the shark
3/5 receptor. Solid circles represent
shark unique sequence compared to
sequences shown in the phylogenetic
tree below.
As expected, the highest homology to mammalian receptors was in the transmembrane domains
where only 14 of 140 (10%) amino acids were unique. In constrast, 33% of the amino acids in the N
and C termini were unique in shark. This protein had highest homology with SSR from Takifugu
95
rubripes (75% homology, 62% identity). Compared to mammalian SSRs, the highest homology was
with SSR3 from human (58%), followed by SSR5 from rat (57%), SSR5 from human (56%, SSR3
from rat (49%) and SSR3 from mouse (47%).
Figure 3: The shark somatostatin receptor
roots tree of SSR 3 and 5 subtypes.
A phylogram (Figure 3) shows that this shark somatostatin receptor forms the root of the tree for
mammalian SSRs 3 and 5 and the fugu SSR.
This work was supported by NIH grants DK 34208 and NIEHS 5 P30 ES03828 (Center for
Membrane Toxicity Studies).
1. Lin, X., R. Peter, Somatostatins and their receptors in fish. Comparative Biochemistry and Physiology Part B 129: 543550, 2001.
2. Plesch, F., C. Smith, S. Aller, J.N. Forrest Jr. Identification and partial sequence of six somatostatin receptors
expressed in the shark rectal gland, shark brain, and skate kidney. Bull. Mt Desert Isl. Biol. Lab. 38: 107-109, 1999.
96
Biomechanical properties of fibers assembled from native sea cucumber (Cucumaria frondosa)
collagen fibrils
Thomas J. Koob, Magdalena M. Koob-Emunds and Douglas Pringle
Skeletal Biology Section, Shriners Hospitals for Children, Tampa, Fl 33612
Echinoderms employ mutable collagenous tissues (MCTs) for a variety of functions including
autotomy, locomotion, size and shape changes, energy-free postural control, defense, and feeding.
These tissues have in common the ability to undergo rapid and reversible changes in their mechanical
properties, alternating between stiff and compliant states over physiological time scales. Mutability
derives from cell-mediated changes in interactions between parallel arrays of spindle shaped collagen
fibrils: fibrils slide past one another in the compliant state, inter-fibril displacement is prevented in the
stiff state. While the specific mechanisms underlying natural changes in inter-fibrillar interactions are
not entirely understood, we have identified several proteins that are capable of acting on live dermis
specimens1 or isolated collagen fibrils4,5: these include cell-derived stiffening and plasticizing factors
that act in vivo on the intact dermis MCT, stiparin, a glycoprotein that aggregates purified collagen
fibrils, and stiparin-inhibitor, a sulfated polygalactose containing glycoprotein that prevents stiparin
from aggregating purified fibrils. We have two objectives for the experiments reported here: the first
is to develop a system to examine the endogenous MCT parameters that regulate inter-fibrillar
interactions and thereby biomechanical properties in the sea cucumber dermis; the second is to explore
the potential of utilizing fibers formed from native sea cucumber collagen fibrils for biomedical
applications.
Intact, native collagen fibrils were isolated from the inner dermis from the two ventral
interambulacra of Cucumaria frondosa as previously described3 by washing 36 g of minced specimens
three times for 30 min each in 250 ml deionized water, treating the washed specimens with 250 ml 4
mM ethylenediamine tretraacetic acid (EDTA) in 50 mM Tris-HCl, pH 8.0 for 24 hr, washing the
chelated specimens three times for 30 min each in 250 ml deionized water, then slowly stirring the
specimens in 500 ml deionized water for 24 hr; all steps were performed at 4oC. The fibril preparation
was centrifuged at 160 x g for 30 min and the resulting suspension of fibrils was separated from the
tissue by decanting the upper two thirds off the tissue pellet. Fibers were produced from the fibril
preparation by dialyzing 10 ml of the suspension in 6.4 mm diameter SpectraPor 2 dialysis tubing
against 0.5 M CH3COOH for 24 hr at 4oC. The gel formed during dialysis was extruded into water,
one end was gripped with a plastic hemostat, and the gel was slowly drawn out of the water and
allowed to dry at 16oC for 16 hr. The mechanical properties of fibers were measured with uniaxial
tensile tests to failure. Approximately 4 mm of the mid-portion of the fiber segment was hydrated in
deionized water, the dry ends were secured to the materials testing system with compression clamps,
and the segment was pulled until it ruptured. The force in Newtons (N) was recorded during the test
and the maximum force achieved was taken as the tensile strength. For some experiments, the
diameter of the hydrated portion of the fiber was measured microscopically before the test; the force at
failure was normalized to the calculated cross-sectional area to compute the material strength in
Pascals (Pa = N/m2). The hydrated fibers were on average 0.38 mm in diameter. Analysis of proteins
and proteoglycans associated with these fibers, as well as extracted fibers described below, was
performed by extracting dried segments of the fibers in SDS/PAGE gel sample buffer (1 cm of
fiber/100 µl buffer) and electrophoresing the extract directly on Novex 4-20% linear Tris-glycine
PAGE pre-cast gels. Gels were stained with Coomassie brilliant blue for proteins and Alcian blue for
proteoglycans and glycosaminoglycans.
97
The tensile strength of fibers produced directly from the fibril preparation by dialysis against acetic
acid at pH 2.5 averaged 7.48 +/- 1.10 N (three separate experiments, n = 15). The material strength of
these fibers was 101.6 +/- 14.7 MPa. The latter is similar to the tensile strength of tendon fibers at 100
MPa and that of artificial tendons produced from bovine molecular type I collagen polymerized with
nordihydroguaiaretic acid at 90 MPa2.
The basis for the cucumber fiber tensile strength was hypothesized to derive from the ionic
interaction between positively charged collagen fibrils (pK of 3.5) and negatively charged chondroitin
sulfate chains (pK of 1.0) covalently bound to the intact native fibrils. To examine this hypothesis, dry
fibers formed in acetic acid were hydrated in solvents of varying composition for 16 hr at 16oC, they
were then dried as described above and subjected to tensile tests after hydrating the mid-portion of the
fiber in deionized water. The results are shown in Fig 1. Treating the fibers a second time in acetic
acid had no effect on their properties. Incubating the fibers in deionized water lowered the tensile
strength by 55%. Sterile filtered sea water lowered the tensile strength by 83%. The greatest reduction
was caused by neutral phosphate buffer (0.1 M, pH 7.4) which lowered the tensile strength by over
95%. These data indicate that elimination of the positively ionized groups with sea water and
phosphate buffer significantly lowered the tensile strength, thereby supporting the hypothesis that the
basis for interfibrillar binding is the ionic interaction caused by the differential ionization of the
collagen fibrils and constituent chondroitin sulfate chains.
Tensile strength (MPa)
120
80
40
0
no
treatment
acetic acid de-ionized
water
sea water
phosphate
buffer, pH
7.4
Figure 1. Tensile strength of fibers treated in the indicated reagents, dried and then tested with
uniaxial tensile tests to failure. Values shown are means of 5 specimens +/- S.D.
Further support of this hypothesis was gained by adding purified chondroitin sulfate to the fibril
preparation before fiber formation. Chondroitin 6-sulfate (Sigma Chemical Company, St. Louis, MO)
was added to the fibril preparation at concentrations ranging from 0.05 to 3.0 mg/ml, the mixtures were
allowed to equilibrate with gentle stirring for 1 hr, they were then loaded into dialysis bags and
dialyzed against acetic acid to form fibers. Chondroitin sulfate caused a concentration dependent
decrease in the tensile strength of the fibers (Fig. 2). Lowest tensile strength was achieved at a
concentration of 0.5 mg/ml. The capacity of chondroitin sulfate to reduce the tensile strength of the
fibers suggests that it competes with the endogenous, fibril-bound glycosaminoglycan for binding sites,
and thereby lowers the number of effective cross-links that are responsible for the fiber’s tensile
strength. Further experiments using unsulfated chondroitin as the competitor will determine whether
the sulfate group is responsible for cross-linking the fibrils. In addition, experiments examining the
effects of related glycosaminoglycans such as dermatan sulfate, keratin sulfate and hyaluronic acid
98
may provide evidence for the role of the composition of the glycosaminoglycan disaccharide in
mediating interfibrillar interactions.
140
105
70
35
0
0.0 cs .05 cs 0.1 cs 0.5 cs 1.0 cs 3.0 cs
chondroitin s ulfate (m g/m l)
Figure 2. Tensile strength of fibers produced in the presence of varying concentrations of
chondroitin 6-sulfate shown on the abscissa. Values are means of 5 specimens +/- S.D.
Production of mechanically testable fibers facilitated experiments to assess the potential role of
matrix and cell derived protein factors on the interactions of collagen fibrils in the sea cucumber MCT.
The initial approach was designed to examine the effects of removing non-fibrillar macromolecules
from the assembled fibers. Fibers were incubated in solvents of varying composition, they were then
dried and their tensile strengths were measured. Segments of the same fibers were extracted with
SDS/PAGE gel sample buffer and the extract was electrophoresed on 4 – 20% Tris-glycine gels to
determine the nature and extent of the proteins remaining in the fibers. Figure 3 shows the proteins
remaining in the fibers after incubation in 0.5 M acetic acid, deionized water, sterile filtered sea water,
0.1 M phosphate buffer, pH 7.4, or Hanks balanced salt solution (HBSS) modified with addition of
salts for sea water conditions. Acetic acid and deionized water did not appreciably affect the relative
amount or composition of the proteins associated with the fibers. Sea water and phosphate buffer
removed a substantial amount of these proteins. Hanks balanced salt solution treated fibers retained
the least amount of fiber associated proteins.
MW kDa 1
2
3
4
5
6
1 – no treatment
2 – 0.5 M acetic acid
3 – deionized water
185
98
Stiparin
4 – sea water
5 – 0.1 M phosphate, pH 7.4
52
6 – Hanks balanced salt solution
31
19
17
Stiparin
inhibitor
11
6
3
Figure 3. SDS/PAGE analysis of proteins remaining in the fibers after incubation in the
indicated solutions.
99
The tensile properties of the fibers incubated in the solutions described above are shown in Fig. 4.
The tensile strength was not affected by any of the incubations, despite the reduction in amount of
associated proteins in the fibers as shown above. These results suggest that the basis for the
intermolecular interactions responsible for the biomechanical properties of the fibers is likely the
collagen fibrils and covalently bound chondroitin sulfate.
160
120
80
40
0
no treatment 0.5M acetic
acid
sea w ater
deionized
w ater
phosphate
buf f er, pH
7.4
Hanks
balanced
salt solution
Figure 4. Tensile strength of fibers incubated in the solutions noted on the abscissa. Following
incubation, the fibers were dried , re-equilibrated in acetic acid, then dried again. Values shown
are means +/- S.D. for 5 specimens.
Taken together the experiments described here establish that a model system can be fabricated to
examine the parameters that mediate specific interactions between native collagen fibrils that are
involved in determining the tensile properties of echinoderm MCTs and artificial fibrous materials
intended for biomedical applications. For MCTs, it will be important first to ascertain the ionic
conditions that best replicate the in situ milieu, particularly with respect to divalent anions that might
participate in interactions between chondroitin sulfate chains. Future experiments will investigate the
effects of including purified extracellular macromolecules, such as stiparin and stiparin inhibitor, and
cell derived stiffening and plasticizing factors on the biomechanical properties of the fibers.
The tensile strength of these fibers is particularly intriguing from a biomedical perspective since
they are as strong as tendon and ligament fibers without the need for exogenous cross-linking agents.
Mimicking the chemistry responsible for the fiber’s tensile strength while at the same time formulating
it to be stable at neutral pH could prove beneficial for designing biologically based artificial fibrous
materials for a variety of applications. Funded by Shriners Hospitals for Children, award 8610.
1.
2.
3.
4.
5.
100
Koob, T.J., M.M. Koob-Emunds and J.A. Trotter. Cell-derived stiffening and plasticizing factors in sea cucumber
(Cucumaria frondosa) dermis. J. Exp. Bio. 202: 2291-2301, 1999.
Koob, T.J. and D. J. Hernandez. Material properties of polymerized NDGA-collagen composite fibers: development
of biologically based tendon constructs. Biomaterials 23: 203-212, 2002.
Trotter, J.A., F.A. Thurmond and T.J. Koob. Molecular structure and functional morphology of echinoderm
collagen fibrils. Cell Tiss. Res. 275: 451-458, 1994.
Trotter, J.A., G. Lyons-Levy, D. Luna, T.J. Koob, D.R. Keene and M.A.L. Atkinson. Stiparin: A glycoprotein
from sea cucumber dermis that aggregates collagen fibrils. Matrix Biol. 15: 99-110, 1996.
Trotter, J.A., G. Lyons-Levy, K. Chino, T.J. Koob, D.R. Keene and M.A.L. Atkinson. Collagen fibril
aggregation-inhibitor from sea cucumber dermis. Matrix Biol. 18: 569-578, 1999.
Retinal sensitivity in the fiddler crab Uca pugilator: evidence of a circadian rhythm and influence
of melatonin
Jocelyn LeBlanc1, Emily Hand2, Eric Luth1, Rharaka Gilbert1, Catherine Downing1, Amanda Shorette3,
& Andrea R. Tilden1
1
Department of Biology
2
Mount Desert Island High School, Mt. Desert, ME 04660
3
Winslow High School, Winslow, ME 04901
Melatonin’s influence on vertebrate circadian rhythms has been extensively studied, and recent
attention has focused on the localized production and signaling of melatonin in the vertebrate retina3.
Specifically, melatonin entrains dark-adapting responses of the retina. Our lab has previously shown
that melatonin is produced in the crustacean eyestalk2, and we suspect phylogenetically conserved roles
of melatonin across phyla.
The crustacean eye responds to changes in illumination by adjusting light sensitivity through
several mechanisms1 including the migration of extra-photoreceptor shielding pigments, such that the
retina is shielded during the day, and changes in sensitivity and neural activity of the photoreceptors
themselves. Some of these changes are direct responses to light, similar to the vertebrate pupillary
response. Other changes are the result of circadian changes in cell physiology that persist in the
absence of light.
Fiddler crabs were acclimated to an ambient photoperiod of 16L:8D for two weeks and were then
placed in constant darkness during the experiment. Electroretinograms (ERGs) were recorded using a
chlorided silver wire in contact with the cornea through a saline bathing medium. Crabs were given
four brief flashes of light (approximately 40 msec each) from a camera flash every three hours
beginning three hours after they were placed in darkness at 1200h. Flashes were administered for a 72hr period. Data were recorded using an A-M Instruments model 1700 differential AC amplifier
interfaced with a PowerLab computer interface and Scope software. Data were recorded from 10
control crabs and 9 crabs that received an infusion of melatonin (5 ng/g) administered in the seawater
bathing the animals.
Currently, we have analyzed two components of the data at two time-points, mid-photophase
(1500h) and mid-scotophase (2400h) of the third day of recording: first, we measured the amplitude of
the ERG signal from the first flash of the series of 4 flashes. Second, we measured the duration of the
ERG response from the same first flash.
Table 1. Amplitude and duration of ERG response in fiddler crabs, controls and melatonin-treated, in response to a brief
pulse of light. Values represent the mean + SEM.
Time of Day
Amplitude (mV)
Duration (msec)
Control Mid-photophase
Control Mid-scotophase
Melatonin Mid-photophase
Melatonin Mid-scotophase
1.15 + 0.22
1.92 + 0.25
1.24 + 0.36
2.34 + 0.33
61 + 15
123 + 27
69 + 21
199 + 28
Control crabs had a significantly greater response to light during subjective darkness than
subjective light in both amplitude (t = 2.61; P < 0.01) and duration (t = 3.29; P < 0.01). Melatonin101
treated crabs did not differ significantly from controls during mid-photophase in amplitude or duration;
they did differ significantly from controls during mid-scotophase in duration (t = 2.24; P < 0.01) but
not in amplitude. These preliminary analyses indicate an endogenously-driven circadian rhythm of
retinal sensitivity in fiddler crabs that persists in conditions of constant darkness, and a potential role of
melatonin in dark-adaptation. The lack of responsiveness to melatonin during subjective photophase
suggests a circadian rhythm of sensitivity to melatonin.
Supported by Maine Biomedical Research Infrastructure Network (1-P20-RR16463-01), Hancock
County Scholars Program, a New Investigator Award, and the Clare Boothe Luce Program of the
Henry Luce Foundation. Our deepest gratitude to Dan Hartline for extensive assistance with
electrophysiological techniques.
1.
2.
3.
102
Garfias, A., L. Rodriguez-Sosa, H. Arechiga. Modulation of crayfish retinal function by red pigment concentrating
hormone. J. Exp. Biol. 198: 1447-1454, 1995.
Tilden, A.R., R. Brauch, R. Ball, A.M. Janze, A.H. Ghaffari, C.T. Sweeney, J.C. Yurek, R.L. Cooper.
Modulatory effects of melatonin on behavior, hemolymph metabolites, and neurotransmitter release in crayfish. Brain
Res. 992: 252-262, 2003.
Tosini, G., M. Menaker. Circadian rhythms in cultured mammalian retina. Science 272: 419-421, 1996.
Promoting Cross-Species Comparative Approaches to Environmental Health Research:
The Comparative Toxicogenomics Database (CTD)
Carolyn J. Mattingly1, Glenn T. Colby1, Michael C. Rosenstein1,
John N. Forrest, Jr.1,2, James L. Boyer1,2
1
Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672; 2Department of
Medicine, Yale University School of Medicine, New Haven, CT 06520
The etiology of most chronic diseases involves interactions between environmental factors and
genes that modulate important physiological processes (Olden and Wilson, 2000).4 We are developing
a publicly available database, the Comparative Toxicogenomics DatabaseTM (CTDTM;
http://ctd.mdibl.org/), to promote understanding about the effects of environmental chemicals on
human health.2,3 CTD identifies interactions between chemicals and genes and facilitates cross-species
comparative studies of these genes. The use of diverse animal models and cross-species comparative
sequence studies is critical for understanding basic physiological mechanisms and gene and protein
functions. These approaches are also being used to explore the molecular mechanisms of action of
environmental chemicals and the genetic basis of differential susceptibility.4
A prototype version
of CTD is available via
the World Wide Web
(Figure 1). Although
publicly
accessible,
improvements are in
progress
for
data
curation,
data
integration, and web site
usability.
Here, we
summarize the major
features (entities) of
CTD. There are seven
primary entities that
have been integrated in
CTD:
1) sequences
(nucleotide and protein
sequences
from
vertebrates
and
invertebrates),
2)
references
(reference
publications), 3) genes
(curated, cross-species
groups of nucleotide
Figure 1. CTD Home Page.
and protein sequences
for toxicologically important genes), 4) Gene Sets (sets of curated genes), 5) chemicals (hierarchical
vocabulary of chemicals or xenobiotic agents), 6) Gene Ontology terms (GO; hierarchical vocabulary
of biological processes, cellular components, and molecular functions), and 7) taxonomy (hierarchical
vocabulary of taxa representing taxonomic groups (Figure 2). Nucleotide sequences and annotations
are acquired from the National Center for Biotechnology Information. We include only Reference
Sequences (RefSeqs) for human (H. sapiens), mouse (M. musculus), rat (R. norvegicus), fruit fly (D.
melanogaster), and nematode (C. elegans). Amino acid sequences and annotations are acquired from
103
the European Bioinformatics Institute’s Swiss-Prot and TrEMBL databases. References are acquired
from PubMed®.
CTD data are linked to 26 other sequence, protein
domain, and toxicology databases (e.g., Swiss-Prot,
Pfam, and TOXNET®, respectively). We provide twodimensional chemical drawings and links to regulatory
and toxicology data for approximately 53,000 of the
chemical terms in our vocabulary.
We created
“vocabulary browsers” with detail pages in CTD that
allow users to navigate the hierarchical structures of
controlled vocabularies (chemicals, taxonomy, and
GO) to formulate queries or quickly access data
(Figure 3).
Figure 2. High-Level View of the Primary
Entities in CTD. Lines indicate a relationship
Genes and Gene Sets are manually curated in between two entities. Single- and double-headed
CTD to promote cross-species comparisons of arrows indicate one-to-many and many-to-many
toxicologically important genes and proteins. In relationships, respectively. Lines that meet at a
diamond indicate a multi-way association among
CTD, genes are defined by their constituent several entities.
nucleotide and protein sequences from vertebrates
and invertebrates and are presented in a cross-species context. We use sequence analysis methods in
combination with literature review to curate genes. We developed the concept of a Gene Set to group
closely related, curated genes, such as those that have undergone duplication events in specific species
(e.g., CYP1A4, CYP1A5) or are
members of large families (e.g., ABC
transporters). Gene Sets provide the
user with a broad perspective about
their gene of interest. For example,
the CYP1A Gene Set includes the
curated genes CYP1A, CYP1A1,
CYP1A2, CYP1A3, CYP1A4, and
CYP1A5. By combining these genes,
a user familiar only with mammalian
CYP1A1 and CYP1A2 genes is
introduced to the avian CYP1A4 and
CYP1A5 genes and associated
supplementary
information
(sequences, references, associated
chemicals, and GO terms). Multiple
alignments and phylogenetic trees are
constructed from sequences of curated
Gene Sets and may be downloaded
from Gene Set detail pages (Figure 4).
These files assist users in evaluating
conservation and divergence in
toxicologically important sequences
among diverse organisms and in
developing hypotheses about the
Figure 3 . CTD Chemical Browser Detail Page.
104
functions of these genes in modulating chemical actions.
From the CTD home page, users
can initiate searches with sequence
or reference query forms or by
browsing the chemical vocabulary.
Queries of varying complexity for
the novice and
experienced
molecular toxicologist are possible.
Examples of supported queries
include: Which chemicals interact
with mygene of interes? Which
genes are affected by my chemical
of interest? Which regions of my
favorite toxicologically important
protein are conserved in vertebrates
and
invertebrates?
Which
references report information about
a
particular
gene-chemical
interaction? In which organisms
has a particular gene-chemical
interaction been studied? Are the
proteins affected by my chemical of
interest involved in a particular
biological process (e.g., apoptosis)?
Priorities for future development
include expanding the set of
references in CTD, curating specific
types of gene-chemical interactions
described in the literature (e.g.,
Figure 4. AHR Gene Set Page. Member genes are indicated, with
protein “X” binds chemical “Y”) ,
links to sequence analysis results, sequences, and chemical and GO
and continuing to curate genes and
annotations.
Gene Sets.
The community is
encouraged to participate in CTD
development by providing feedback ([email protected]). Please contact us if you are interested in
submitting curated data sets for inclusion in CTD.
This project is funded by NIEHS ES11267 and is a component of the MDIBL CMTS.
1.
2.
3.
4.
Ballatori N., J.L. Boyer, J.C. Rockett. Exploiting genome data to understand the function, regulation, and
evolutionary origins of toxicologically relevant genes. Environ Health Perspect Toxicogenomics. 111: 61-5, 200
Mattingly, C.J., G.T. Colby, J.N. Forrest Jr., and J.L. Boyer. The Comparative Toxicogenomics Database (CTD).
Environ Health Perspect. 111: 793-795, 2003.
Mattingly, C.J., G.T. Colby, M.C. Rosenstein, J.N. Forrest Jr., and J.L. Boyer. Promoting comparative molecular
studies in environmental health research: an overview of the comparative toxicogenomics database (CTD).
Pharmacogenomics J. 4: 5-8, 2004.
Olden, K. and S. Wilson. Environmental health and genomics: visions and implications. Nat Rev Genet. 1: 149-153,
2000.
105
Resveratrol interacts with multiple transporters in killifish, Fundulus heteroclitus, renal
proximal tubules
Kai Swenson and David S. Miller
Laboratory of Pharmacology and Chemistry, NIH/NIEHS, Research Triangle Park, NC 27709
Resveratrol (trans-3,5,4′-trihydroxystilbene) is a constituent of grapes, berries and peanuts with
cardioprotective and anticancer properties1,7. Although there is some evidence that the compound can
enter cells by a mediated pathway, nothing is known about the transporters involved2. To determine
whether resveratrol interacts with xenobiotic transporters, we used killifish renal proximal tubules as a
test system to measure its effects on the transport of three fluorescent compounds: NBD-cyclosporine
A (NBD-CSA), a substrate for p-glycoprotein, fluorescein-methotrexate (FL-MTX), a substrate for
multidrug resistance-associated protein isoform 2 (MRP2), fluorescein (FL), a substrate for organic
anion transporter 1 and 3, (OAT1/3).
Renal proximal tubules were isolated from killifish and maintained in a teleost marine saline
medium (in mM: 140 NaCl, 2.5 KCl, 1.5 CaCl2, 1.0 MgCl2, 20 Tris, pH 8.0). For experiments, tubules
were transferred to confocal chambers containing medium with fluorescent compound without
(control) or with 1-50 µM resveratrol. After 60 min, the steady state distribution (epithelial cells and
tubular lumen) of the fluorescent compound was measured using confocal microscopy and quantitative
image analysis as described previously3.
250
B
75
50
25
150
% Control FL
Accumulation
100
% Control FL-MTX
Accumulation
% Control NBD-CSA
Accumulation
A
100
50
0
10
20
30
40
50
Resveratrol Concn, µM
60
Lumen
Cells
150
100
50
0
0
0
C
200
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Figure 1. Effects of resveratrol on transport of fluorescent xenobiotics in killifish renal proximal
tubules. Each point represents the mean value for data from 12-35 tubules from 2-6 fish; variability
is shown as SE bars. The broken lines indicate the control levels.
Initial experiments indicated that resveratrol concentrations as high as 50 µM caused no obvious
changes in tubular morphology. In spite of this, resveratrol did reduce the transepithelial transport of
all three fluorescent compounds (Fig. 1), indicating interactions with all of the transporters involved.
Resveratrol was a potent inhibitor of luminal NBD-CSA accumulation, with an IC50 value
(concentration causing 50% inhibition) of about 5 µM (Fig. 1A). In previous experiments carried out
under similar conditions, potent and specific p-glycoprotein inhibitors, e.g., PSC833, exhibit
submicromolar IC50 values4. In this assay, the effectiveness of resveratrol appeared to be comparable to
some of the Ca-channel blockers, which exhibit IC50 values in the 5-10 µM range4. Resveratrol was
106
clearly less effective as an inhibitor of organic anion (FL-MTX and FL) transport. With FL-MTX,
resveratrol did not alter cellular accumulation, but significantly reduced luminal accumulation at all
three concentrations tested (P<0.01; t-test: Fig. 1B). The IC50 was about 25 µM. This is substantially
higher than that found for leukotriene C4 and MK571, potent inhibitors of MRP-mediated transport4.
The pattern of effects for FL transport was more complex (Fig. 1C). FL transport across the tubular
epithelium involves two concentrative steps6. The first step is mediated by an OAT and the second step
by an as yet unidentified transporter. Cellular accumulation of FL was slightly stimulated by 10 µM
resveratrol and more than doubled by 25 µM resveratrol; cellular accumulation returned to control
levels with 50 µM resveratrol. Luminal accumulation was not reduced at 10 µM resveratrol, but was
significantly reduced (P<0.01; t-test) at 25 and 50 µM. For luminal FL accumulation the IC50 was
about 25 µM. Since no decrease in cellular accumulation of FL was observed it is unlikely that
resveratrol interacts with the basolateral OAT present in these tubules. This transporter mediates Nadependent, organic anion uptake and its ability to function is dependent on cellular metabolism6. Thus,
the lack of reduction in cellular FL accumulation suggest that resveratrol did not reduce NBD-CSA
and FL-MTX transport by inhibiting metabolism. The observed increase in cellular FL accumulation
may be secondary to blocked luminal efflux, a phenomenon seen previously5.
The present results for a comparative model system indicate that resveratrol interacts with high
potency with p-glycoprotein and with moderate potency with MRP2. It does not appear to interact with
the basolateral OAT present in the tubules, but is a moderate inhibitor of cell to lumen organic anion
transport. Obviously, inhibition of transport does not necessarily mean that the inhibitor is also
transported. Thus, additional studies will be needed to determine which transporters influence the
cellular accumulation of resveratrol and its distribution within the body. Supported in part by the
Maren Foundation and the MDIBL Center for Membrane Toxicity Studies.
1. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, and Takada Y. Role of resveratrol in
prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 24: 2783-2840, 2004.
2. Jannin B, Menzel M, Berlot JP, Delmas D, Lancon A, and Latruffe N. Transport of resveratrol, a cancer
chemopreventive agent, to cellular targets: plasmatic protein binding and cell uptake. Biochem Pharmacol 68: 11131118, 2004.
3. Masereeuw R, Russel FG, and Miller DS. Multiple pathways of organic anion secretion in renal proximal tubule
revealed by confocal microscopy. Am J Physiol 271: F1173-1182, 1996.
4. Miller DS. Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule. J
Pharmacol Exp Ther 299: 567-574, 2001.
5. Miller DS, Letcher S, and Barnes DM. Fluorescence imaging study of organic anion transport from renal proximal
tubule cell to lumen. Am J Physiol 271: F508-520, 1996.
6. Pritchard JB and Miller DS. Mechanisms mediating renal secretion of organic anions and cations. Physiol Rev 73:
765-796, 1993.
7. Shih A, Zhang S, Cao HJ, Boswell S, Wu YH, Tang HY, Lennartz MR, Davis FB, Davis PJ, and Lin HY.
Inhibitory effect of epidermal growth factor on resveratrol-induced apoptosis in prostate cancer cells is mediated by
protein kinase C-alpha. Mol Cancer Ther 3: 1355-1364, 2004.
107
Effects of cortisol and arsenic on seawater acclimation in killifish (Fundulus heteroclitus)
Joseph R. Shaw1, Lydia Durant2, Renee Thibodeau3, Roxanna Barnaby4, Katherine Karlson4,
Joshua W. Hamilton5 & Bruce A. Stanton4
1
Biology Department, Dartmouth College, Hanover, NH 03755; 2Colby College, Waterville, ME
04901; 3Whitman College, WallaWalla, WA 99362; 4Physiology and 5Pharm/Toxicology Departments,
Dartmouth Medical School, Hanover, NH 03755
The killifish, Fundulus heteroclitus, can withstand large changes in salinity, which require the gills
to rapidly switch between NaCl absorption (freshwater, FW) and secretion (seawater, SW) to maintain
salt balance. In SW, Cl secretion is accomplished by CFTR Cl channels. Relatively few studies have
focused on the mechanisms whereby CFTR increases Cl secretion when fish move from FW to SW1.
While Marshall et al.1 have shown that this transition causes a dramatic increase in plasma cortisol that
precedes increased CFTR mRNA, direct evidence for cortisol regulation of CFTR is lacking.
Nonetheless, Singer et al.2 have identified a consensus sequence of a putative glucocorticoid response
element in the regulatory region of the killifish CFTR gene. Recent studies in cell culture have
demonstrated that arsenic inhibits cortisol mediated transcriptional activation3. In killifish arsenic
increases plasma Cl during the move from FW to SW, which was also when they were most vulnerable
to arsenic toxicity4. These results are consistent with an inhibition of CFTR mediated Cl secretion.
Accordingly, the goals of our research were to test the hypotheses that 1) cortisol stimulates CFTR
gene expression in killifish and is required for FW to SW movement, and 2) arsenic inhibits cortisol
stimulated CFTR gene expression.
Killifish were gradually acclimated to FW5 and their response to SW challenge for 24-h was
studied (i.e., direct transfer, FW to SW). Treatments included cortisol (40 nmol/g) and a cortisolreceptor antagonist, mifepristone (100 nmol/g) injected IP, arsenic (8000ppb) dissolved in swimwater,
and controls (FW, SW, Sham/vehicle). CFTR transcript was quantified by real-time PCR5.
CFTR mRNA levels increased when FW fish were injected with cortisol. Likewise, CFTR mRNA
increased following SW challenge (which naturally increases cortisol levels) and this increase was
even greater in cortisol injected animals. Furthermore, the increase in CFTR mRNA expression was
blocked (with/without SW challenge) by mifepristone. If cortisol is essential for stimulating the
physiological changes occurring during the transition from FW to SW, then the combination of
mifepristone and SW challenge should be toxic. While this combination was not overtly toxic over 24h, almost all (>80%) fish were moribund and this was the only treatment that produced such effects.
These observations were confirmed with toxicity tests, as the same treatments produced 65% and 90%
mortality after 48-h and 96-h (controls <10% at 96-h). Arsenic blocked CFTR mRNA expression
following SW challenge (with/without cortisol). Decreased CFTR mRNA expression is consistent with
the decreases in Cl secretion5 and increases in plasma Cl4 that have been observed following similar
arsenic exposures. Future investigations will concentrate on defining the cellular mechanisms
responsible for the arsenic inhibition CFTR expression. (Supported by an MDIBL New Investigator
Award, Center for Membrane Toxicity Studies (NIEHS P30 ESO3828-18), Superfund Basic Research
Program (NIEHS ESO7373), NCRR MBRIN (1-P20-RP-6463-01) and NSF REU (DBI-0139190).
1.
2.
3.
4.
5.
108
Marshall, WS, Emberley, TR, Singer, TD, Bryson, SE, McCormick, SD. Time course of salinity adaptation in a strongly
euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach. J Exp Biol. 202:1535-1544, 1999.
Singer, TD, Hinton, MR, Schulte, PM, McKinley, RS. Teleost CFTR transcriptional regulation: a comparative approach. Comp
Biochem Physiol. 126B:S86, 2000.
Bodwell, JE, Kingsley, LA, Hamilton, JW. Arsenic at very low concentrations alters glucocorticoid receptor (GR)-mediated gene
activation but not GR-mediated gene repression: complex dose-response effects are closely correlated with levels of activated GR
and require a functional GR DNA binding domain. Chem Res Toxicol. 17(8):1064-1076, 2004.
Shaw, JR, Curtis-Burnes, J, Stanton, BA, Hamilton, JW. The toxicity of arsenic to the killifish, Fundulus hetertoclitus: Effects
of salinity. The Bulletin. Vol 43:134, 2004.
Stanton, C, Prescottt, D, Lankowski, A, Karlson, K, Mickle, J, Shaw, J, Hamilton, J, Stanton, BA. Arsenic and adaptation to
seawater in killifish (Fundulus heteroclitus). The Bulletin. Vol 42:117-119, 2003.
Trophic Transfer of Mercury in Intertidal Food Webs
Celia Y. Chen1 and Brandon M. Mayes1
1
Dartmouth College, Hanover, NH 03755
Mount Desert Island (MDI) is an important location to study Hg bioaccumulation in aquatic food
webs because Hg deposition in Northeastern coastal areas is known to be elevated, Hg levels in
aquatic biota on MDI are exceptionally high, and intertidal areas are important sites of Hg
methylation1,2. The objective of this research was to examine the bioaccumulation and trophic
transfer of Hg in resident and transient benthic, epibenthic, and nektonic species inhabiting the
intertidal and subtidal portions of estuaries. The food webs of several sites on MDI including
Northeast Creek, Salisbury Cove, Seal Cove, and Somes Sound were sampled in the summers of
2003-4 as part of a larger survey of sites in the Gulf of Maine including Great Bay NH.
We collected biotic samples of resident intertidal and transient subtidal nekton species for Hg
analysis at each site. Zooplankton and particulate samples were taken in deeper water near the
intertidal sites. Samples from each site were collected using minnow traps, Ponar dredge, pitfall traps,
and fyke nets and were handled using trace metal clean technique. We measured 15N and 13C
signatures for each taxa in order to determine whether Hg bioaccumulation was related to trophic
level or carbon source. Earlier studies have shown that 15N is preferentially enriched with increasing
trophic level and 13C is more depleted in organisms with pelagic vs. detrital food sources. Samples
from 2003 were analyzed for Hg, 15N, and 13C and samples from 2004 are being analyzed for Hg
speciation (inorganic and MeHg).
Hg concentrations in biota from MDI were positively related to 15N signature indicating
biomagnification. Also, there were higher Hg concentrations in fish (Fundulus heteroclitus, Menidia
menidia) and benthic epifauna (Littorina littorea, Mytilus edulis, amphipods) than in benthic infauna
(polychaetes, Mya arenaria). The latter indicated that organisms feeding at lower trophic levels and
in the sediments had lower Hg bioaccumulation. Although, as expected, the algal grazers (Mytilus
edulis and Menidia menidia) were more depleted in 13C than benthic infauna such as Mya arenaria
and polychaetes that feed on detrital carbon, there was no relationship between Hg and 13C
enrichment. Hg bioaccumulation at MDI contrasted greatly with Great Bay NH, a more contaminated
industrialized site. Although aqueous concentrations were similar at the two sites, Hg concentrations
in biota were 2-20X higher in Great Bay and did not increase with increasing 15N signature indicating
that the exposure to metals via sediments was a much more important vector for Hg bioaccumulation.
Thus, in intertidal sites with relatively uncontaminated sediments (like MDI), benthic infauna have
very low exposures and Hg is biomagnified from lower to higher trophic levels.
This work was supported by the MDIBL new investigator award (NIEHS-P30ES03828-18) and
the Salisbury Cove Research Fund, New Hampshire Sea Grant development funds, and the
Dartmouth Center for Environmental Health Sciences.
1.
2.
Bank, M.S., C.S. Loftin , R.E. Jung. Mercury bioaccumulation in two-lined salamanders from streams in the
northeastern US. Ecotoxicology (in press).
VanArsdale, A, L. Alter and G. Keeler. Mercury deposition and ambient concentrations in New England: Results
and plans for the four-site MIC-B Network. In: Proc. of the Conf. on Mercury in Eastern Canada and the Northeast
States edited by Burgess, N and Giguere, M.F. 1998.
109
Effects of phenolic acids on organic anion transport in killifish, Fundulus heteroclitus, renal
proximal tubules
Natascha A. Wolff1 and David S. Miller2
Institute of Systems Physiology, University of Goettingen, 37073 Goettingen, Germany
2
1
One function of vertebrate renal proximal tubules is the elimination of potentially toxic small
organic anions (OA), including drugs, dietary components and waste products of metabolism. This is
accomplished through potent, active secretory transport that involves two concentrative steps: tertiary
active uptake at the basolateral membrane of the tubular epithelial cells and efflux into the lumen by an
as yet undefined process. The first, rate-determining step in transport (uptake) is mediated by at least
one member of the organic anion transporter (OAT) subfamily. In teleosts, basolateral OA uptake is
likely to be mediated by only one OAT, possibly representing a common ancestral protein of
mammalian OAT1 and OAT3 (Aslamkhan, A. G. et al. The flounder organic anion transporter (fOat):
an ancestral ortholog of mammalian OAT1 and OAT3? Manuscript in preparation).
Animals are continuously exposed to OAT substrates, but it is not clear to what extent such
exposure can alter renal tubular OA transport. Candidate substrates of plant origin ingested by prey
species are various phenolic acids, either found as such in marine algae (caffeic acid, cinnamic acid)2,3,
or derived by degradation from other phenolic compounds, such as gallic acid from the brown algal
phlorotannins1. Initial experiments had shown that cinnamic acids as well as gallate potently inhibited
p-aminohippurate transport by the cloned OAT from flounder (fOAT)4 when expressed in Xenopus
laevis oocytes. Moreover, 4-hydroxycinnamic acid was found to be an fOAT substrate. Here we report
results of preliminary experiments designed to determine the effects of gallate and cinnamate exposure
on the transport of fluorescein (FL), a fluorescent OA, by isolated killifish renal tubules.
Renal proximal tubules were isolated from control killifish or from killifish exposed to phenolic
acids in seawater. Isolated tubules were transferred to teflon chambers containing teleost Ringer
solution (in mM: 140 NaCl, 2.5 KCl, 1.5 CaCl2, 1.0 MgCl2, 20 Tris, pH 8.0) with 1 µM FL. In some
experiments with tubules from control fish the medium also contained phenolic acids. After reaching
steady-state (1 h), cellular and luminal fluorescence levels were measured using confocal microscopy
(Zeiss) and quantitative image analysis (ImageJ 1.32, NIH).
Steady state FL accumulation in tubules from untreated fish was significantly altered by addition of
10 µM gallate or cinnamate to the medium. With 10 µM gallate, cellular accumulation decreased by 17
± 7% and luminal accumulation decreased by 33 ± 10% relative to gallate-free controls (mean ± SE,
n=3 animals). With 10 µM cinnamate, only luminal accumulation decreased significantly (35 ± 13%,
n=3 fish). Thus, when applied directly to isolated tubules, 10 µM gallate affected at least the basolateral
step in FL transport, while cinnamate appeared to affect predominantly the luminal step.
When killifish were exposed to 10 µM gallate for 30 h and FL transport measured in isolated
tubules, cellular accumulation was at control levels, but luminal accumulation was increased (32 ± 7%,
n=3 dosed and control fish each). Exposure of fish to 10 or 100 µM cinnamate for 24 h had variable
effects; these also suggested increased transepithelial transport. Thus, for gallate, we saw a consistent
picture. In vitro exposure inhibited FL transport, suggesting interaction with at least the basolateral
OAT. In vivo exposure appeared to increase transepithelial transport, but only at the luminal step.
Further studies are clearly needed to evaluate the effects on the individual carriers involved. Supported
in part by an MDIBL New Investigator Award to Natascha A. Wolff.
110
1.
2.
3.
4.
Arnold, T. M. and Targett, N. M. To grow and defend: lack of tradeoffs for brown algal phlorotannins. Oikos 100:
406-408, 2003.
Miranda, M. S., Cintra, R. G., Barros, S. B. M. and Mancini, J. Antioxidant activity of the microalga Spirulina
maxima. Brazilian Journal of Medical and Biological Research 31: 1075-1079, 1998.
Miranda, M. S., Sato, S. and Mancini-Filho, J. Antioxidant activity of the microalga Chlorella vulgaris cultered on
special conditions. Boll Chim Farm 140: 165-168, 2001.
Wolff, N. A., Werner, A., Burkhardt, S. and Burckhardt, G. Expression cloning and characterization of a renal
organic anion transporter from winter flounder. FEBS Lett 417: 287-291, 1997.
111
Regulation of organic anion transport in the choroid plexus of
spiny dogfish shark, Squalus acanthias
Carsten Baehr1, Kathleen D. DiPasquale1, David S. Miller2, Gert Fricker1
Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-Universität,
D-69120 Heidelberg, Germany
2
1
The choroid plexus epithelium forms the blood-cerebrospinal fluid (CSF) barrier, which along with
the blood-brain barrier, maintains the fluid environment of the brain. The choroid plexus not only secretes CSF but also transports potentially toxic xenobiotics and waste products of neural metabolism to
the blood for eventual clearance in the kidney and liver. In this regard, previous studies have shown
that choroid plexus, like kidney, actively transports organic anions4. For the large, fluorescent organic
anion, fluorescein-methotrexate (FL-MTX), similar systems appear to drive CSF to blood transport in
tissue from mammals and dogfish shark. Two carrier-mediated steps in series are involved, with uptake
at the apical plasma membrane (CSF-side) being Na-dependent and efflux at the basolateral membrane
(blood-side) being highly concentrative1,2. Although we are beginning to obtain a molecular level understanding of organic anion transport in choroid plexus4,5, little is known about how such transport is
regulated. The present study was initiated to identify cellular signals that modulate FL-MTX transport
in shark choroid plexus.
Transepithelial transport of FL-MTX across isolated lateral choroid plexus from dogfish shark was
studied using confocal laser scanning microscopy (Olympus) and quantitative image analysis (Scion
Image software) as described previously1-3. This procedure allowed us to measure the steady state (60
min) distribution of FL-MTX within the tissue and quantify levels in the epithelial cell and subepithelial/blood vessel compartments. In control tissue incubated in medium with 2 µM FL-MTX, cellular
fluorescence was somewhat lower than medium fluorescence, but subepithelial/blood vessel was about
5 times higher than both. FL-MTX accumulation in both compartments was abolished when probenecid, a potent inhibitor of organic anion transport, was added to the medium.
In renal proximal tubule, organic anion transport is stimulated by a mitogen-activated protein
kinase pathway and inhibited through activation of protein kinase C (PKC)6. In shark choroid plexus,
phorbol ester, which activates PKC, significantly reduced FL-MTX accumulation in both cellular and
subepithelial/blood vessel compartments; these effects were blocked by bisindolylmaleimide, a PKCselective inhibitor. In addition, forskolin, which activates PKA increased FL-MTX accumulation in
both cellular and subepithelial/blood vessel compartments by more than 50%; this effect was blocked
by H89, a specific inhibitor of PKA. Time course studies with forskolin showed rapid activation of
apical FL-MTX uptake followed by increased subepithelial/blood vessel accumulation. Thus, in shark
choroid plexus, protein kinase-based mechanisms are in place to both increase and decrease transport,
presumably in response to hormonal exposure. The hormones that actually act through these protein
kinases to modulate organic anion transport remain to be identified. This study was supported by grant
GF 1211/12-1 of the German Research Foundation (GF), the MDIBL Center for Membrane Toxicity
Studies and the Howard Hughes Medical Institute (KDD).
1. Baehr C, DiPasquale KD, Fricker G and Miller DS. Fluorescein-methotrexate (FL-MTX) transport in dogfish shark,
Squalus acanthias, choroid plexus. Bull MDIBL. 43:137-8, 2004.
2. Breen CM, Sykes DB, Baehr C, Fricker G, and Miller DS. Fluorescein-methotrexate transport in rat choroid plexus
analyzed using confocal microscopy. Am. J. Physiol., 287:F562-9, 2004.
112
3. Breen CM, Sykes DB, Fricker G, and Miller DS. Confocal imaging of organic anion transport in intact rat choroid
plexus. Am J Physiol. 282:F877-85, 2002.
4. Kusuhara H and Sugiyama Y. Efflux transport systems for organic anions and cations at the blood-CSF barrier. Adv
Drug Deliv Rev. 56:1741-63, 2004.
5. Sykes D, Sweet DH, Lowes S, Nigam SK, Pritchard JB, and Miller DS. Organic Anion Transport in Choroid Plexus
From Wild-Type and Organic Anion Transporter 3 (Slc22a8)-Null Mice. Am J Physiol. 286:F972-8, 2004.
6. Wright SH and Dantzler DH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol
Rev. 84:987-1049, 2004.
113
Transport of a fluorescent cAMP analog in killifish, Fundulus heterclitus, renal proximal tubules
Valeska Reichel1, Kathleen D. DiPasquale1, David S. Miller2, Gert Fricker1
1
Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg,
D-69120 Heidelberg, Germany
2
Two members of the multidrug resistance-associated protein sub-family of organic anion transporters
(ABCC2 and ABCC4: MRP2 and MRP4) are localized to the luminal plasma membrane of mammalian renal
proximal tubule2,4. These two ATP-driven xenobiotic export pumps have somewhat overlapping specificities.
However, cAMP appears to be selectively transported by MRP4, which also transports the adenine
derivative adefovir (PMEA) and monophosphorylated nucleoside analogs such as azidothymidinemonophosphate (AZT-MP)4. Since nucleoside analogs are used in HIV treatment, a possible role of
MRP4 in the disposition of these drugs may be clinically relevant. Recent studies indicate that killifish
renal proximal tubules express both MRP2 and MRP4 and that MRP2-mediated transport of
fluorescein-methotrexate (FL-MTX) is regulated by endothelin (ET) acting through an ETB receptor,
nitric oxide synthase and protein kinase C (PKC); PKA was not involved1,3. Here we show that the
concentrative, cell to tubular lumen transport of a fluorescent cAMP analog (fluo-cAMP) is not
mediated by MRP2, but possibly by MRP4.
Transepithelial transport of fluo-cAMP and of FL-MTX across isolated killifish renal proximal
tubules was studied using confocal laser scanning microscopy (Olympus) and quantitative image
analysis (Scion Image software) as described previously1,3. Tubules incubated in medium containing 1
µM fluo-cAMP rapidly accumulated the fluorescent compound in the tubular lumen. Luminal
accumulation was blocked by inhibitors of metabolism and by the non-selective MRP inhibitors,
probenecid and MK571 (concentration causing 505 inhibition, I50=7 µM), but not by the organic anion
transporter (Oat) inhibitor, p-aminohippurate. Importantly, PMEA (I50<1 µM) and cAMP (I50=10 µM)
were potent inhibitors of fluo-cAMP transport, but not of FL-MTX transport. As before1, we found that
1-100 nM ET-1 rapidly decreased MRP2-mediated luminal accumulation of FL-MTX as did PKC
activation by phorbol ester. In contrast, neither 1-10 nM ET-1 nor 10-100 nM phorbol ester had any
effect on fluo-cAMP transport. Fluo-cAMP transport was inhibited by forskolin, a phosphodiesterase
inhibitor that increases cAMP levels; this effect was not attenuated by H-89, a PKA inhibitor. The
MRP4 inhibitor dipyridamole4, had a strong inhibitory effect on luminal fluo-cAMP accumulation
(I50=10 µ) . However, at the same concentrations we also saw reduced FL-MTX transport. Thus, in
killifish tubules, dipyridamole may inhibit MRP2-mediated transport as well.
Taken together these findings indicate different cell to lumen transport mechanisms for FL-MTX
and fluo-cAMP. This conclusion is based on differences in inhibitor profiles and in signals that alter
short-term regulation. Since both adefovir and cAMP block apical efflux of fluo-cAMP, involvement
of a killifish form of MRP4 is likely. This study was supported by a grant from the Boehringer Ingelheim
Foundation (V.R.), grant GF 1211/12-1 of the German Research Foundation (GF), the MDIBL Center for
Membrane Toxicity Studies and the Howard Hughes Medical Institute (KDD).
1.
2.
114
Masereeuw R, Terlouw SA, van Aubel RA, Russel FG, and Miller DS. Endothelin B receptor-mediated regulation
of ATP-driven drug secretion in renal proximal tubule. Mol Pharmacol 57: 59-67, 2000.
Russel FG, Masereeuw R, and van Aubel RA. Molecular aspects of renal anionic drug transport. Annu Rev Physiol
64: 563-594, 2002.
3.
4.
Terlouw SA, Graeff C, Smeets PH, Fricker G, Russel FG, Masereeuw R, and Miller DS. Short- and long-term
influences of heavy metals on anionic drug efflux from renal proximal tubule. J Pharmacol Exp Ther 301: 578-585,
2002.
van Aubel RA, Smeets PH, Peters JG, Bindels RJ, and Russel FG. The MRP4/ABCC4 gene encodes a novel apical
organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am
Soc Nephrol 13: 595-603, 2002.
115
Differential effects of permeant and non-permeant chelators of mercuric chloride on chloride
secretion in the in vitro perfused rectal gland and cultured cell monolayers
of the spiny dogfish shark (Squalus acanthias)
Martha Ratner1, Sarah Decker2, Catherine Kelley3, and John N. Forrest Jr1,2.
1
2
3
Skidmore College, Saratoga Springs, NY 12866
Mercuric chloride inhibits chloride secretion in the shark rectal gland4. Mercury is known to alter
protein action by binding to cysteinyl sulfhydryl (SH) groups and disrupting disulfide bonds. Previous
work in this laboratory has suggested that the site of this action is the apical membrane protein
CFTR3,5. We sought to examine the effects of two chelating agents, the cell permeant dithiothreitol
(DTT), and the non-permeant glutathione (GSH), on mercuric chloride inhibition of stimulated
chloride secretion. These chelators were tested in both the in vitro perfused shark rectal gland and in
Isc measurements of cultured monolayers of shark rectal gland cells.
Freshly excised rectal glands were perfused in vitro as previously described2. HgCl2 and the
chelating agent were perfused throughout the experiment. At t=30 min, the secretagogues forskolin (1
µM) and IBMX (100 µM) were added to the perfusate for the remainder of the experiment. Rectal
gland tubular cells were cultured and grown on collagen coated nylon membranes, and Cl- secretion
was measured as Isc in intact monolayers, as described previously1.
Figure 1. A) Prevention of the inhibitory effects of 10 µM HgCl2 by 500 µM DTT. Both HgCl2 and DTT were added to
the perfusate at t=0 min (n=10 for control perfusions without mercury; n=4 with DTT and HgCl2; n=5 with HgCl2 alone).
B) Prevention of the inhibitory effects of 10 µM HgCl2 by 1 mM GSH. Both HgCl2 and GSH were added to the perfusate
at t=0 min (n=10 for control perfusions without mercury; n=3 with GSH and HgCl2; n=5 with HgCl2 alone. All values are
mean ± SEM.
In the perfused gland, both DTT and GSH added concurrently with mercury significantly prevented
HgCl2 inhibition of chloride secretion. (Figure 1, Panels A and B). In Isc measurements of cultured
rectal gland cell monolayers, both DTT and GSH added prior to the addition of HgCl2 also prevented
inhibition (Figure 2, Panels A and C, Figure 3, Panel A).
When DTT was added to maximally inhibited cells, it reversed the inhibition by 53% (Figure 2,
Panel B, Figure 3, Panel B). In contrast, the addition of GSH to maximally inhibited cells did not
significantly reverse the inhibitory effects of inorganic mercury (Figure 2, Panel D; Figure 3, Panel B).
116
Figure 2. Representative Isc
tracings with DTT and GSH
added to shark rectal gland
monolayer cultures stimulated
with forskolin (10 µM) and IBMX
(100 µM) A) DTT (500 µM)
added to the apical surface 17 min
before the addition of HgCl2 to
the apical membrane prevents
HgCl2 inhibition. B) DTT (500
µM) added to the apical solution
during maximum inhibition by
inorganic mercury reverses the
inhibition by 53%. C) GSH (1
mM) added to the apical surface
approximately 17 min before the
addition of HgCl2 to the apical
membrane
prevents
HgCl2
inhibition.
D) GSH (1 mM)
added to the apical solution
during maximum inhibition by
inorganic mercury minimally
reverses the inhibition.
Figure 3. Mean percent prevention and reversal by
DTT and GSH of the effects of mercury on Isc. In all
experiments shark rectal gland monolayer cultures
were stimulated with forskolin (10 µM) and IBMX
(100 µM). All values are mean ± SE. Both DTT and
glutathione completely prevent the effects of
mercury when added prior to the addition of HgCl2
but only the membrane permeant chelator DTT
significantly reverses these effects. A) Percent
prevention of the effects of mercury by DTT (500
µM) and GSH (1 mM) added apically 17 min before
apical HgCl2 (10 µM) (n=4 for DTT; n= 9 for GSH).
B) Percent reversal of the effects of HgCl2 by DTT
(200-500 µM) and GSH (1 mM) added apically
during maximal inhibition by HgCl2 (10 µM)
(p<0.0001 for DTT, p=0.34 for GSH, n=10 for DTT;
n= 8 for GSH). Percent prevention was calculated as
(100-(IscFOR+IBMX+DTT - IscHgCl2) / (Isc FOR+IBMX+DTT) x
100). Percent reversal was calculated as [(IscDTT IscHgCl2) / (IscFOR+IBMX - IscHgCl2) x 100].
117
Both DTT and GSH, cell permeant and non-permeant chelators, respectively, completely prevented
the inhibitory effects of mercury when added simultaneously with HgCl2, indicating that both agents
prevented mercury from reaching its site of action. In contrast, only the permeant chelator was able to
reverse the effects of mercury when added under conditions of maximal inhibition. The ability of
DTT, but not GSH, to reverse inhibition by HgCl2 suggests that the site(s) at which inorganic mercury
inhibits the CFTR chloride channel are intracellular cysteinyl residues.
1. Aller, S.G., I.D. Lombardo, S. Bhanot S. JN Forrest Jr. Cloning, characterization, and functional expression of a
CNP receptor regulating CFTR in the shark rectal gland. Am. J. Physiol. 276:C442-9, 1999.
2. Kelley, G.G., E.M. Poeschla, H.V. Barron, J.N. Forrest Jr. A1 adenosine receptors inhibit chloride transport in the
shark rectal gland. Dissociation of inhibition and cyclic AMP. J. Clin. Invest. 85 (5): 1629-36,1990.
3. Ratner, M., G. Weber, C. Smith, S. Aller, K. Rizor, D. Dawson, J.N. Forrest, Jr. Polarity of mercury toxicity in the
shark (Squalus acanthias) rectal gland: apical chloride transport and shark CFTR channels expressed in Xenopus
oocytes are highly sensitive to inorganic mercury. Bull. Mt Desert Isl. Biol. Lab. 37: 20-22, 1998.
4. Silva, P., F. H. Epstein, R. J. Solomon. The effect of mercury on chloride secretion in the shark (Squalus acanthias)
rectal gland. Comp. Biochem. Physiol. C. 103 (3): 569-575, 1992.
5. Sirota, J.C., G.H. Weber, S.G. Aller, D.C. Dawson, and J.N. Forrest, Jr. Shark and human CFTR expressed in
Xenopus oocytes have different sensitivities to inhibition by the thiol-reactive metals mercury and zinc. Bull. Mt.
118
A full-length farnesoid X receptor (FXR) from Leucoraja erinacea, the little skate
Shi-Ying Cai1, Ned Ballatori2,3, and James L. Boyer1,3
1
Liver Center, Yale University School of Medicine, New Haven, CT 06520
2
Dept. of Environmental Medicine, Univ. of Rochester School of Medicine, Rochester, NY 14642
3
FXR (NR1H4) is a member of group I of the nuclear receptor superfamily, which consists of 48
members in humans. It was originally identified as a binding protein of retinoid X receptor (RXR)
from yeast two-hybrid screening, and classified as an “orphan receptor” 1. FXR was subsequently
found to be specifically activated by bile acids2,3. Chenodeoxycholic acid (CDCA) binds with the
highest affinity, whereas hydrophilic bile acids are less active. Several key genes are activated by FXR,
including genes for enzymes which are involved in bile acid synthesis in the liver (Cyp7A1, the rate
limiting enzyme for converting cholesterol to bile acids), as well as those related to the transport of bile
acids into and out of the liver (Ntcp/Slc10a1, the sodium-dependent taurocholate co-transporting
polypeptide, and Bsep/Abcb11, the bile salt export pump), and uptake in the ileum (Abst/Slc10a2, the
apical bile salt transporter, and Babp, the ileal bile acid binding protein).
FXR orthologs have been cloned and characterized from several other species (Table 1). These
orthologues share significant sequence identity, especially in the DNA binding domain (more than
85%). However, ligand specificity is quite diverse. CDCA is the most potent endogenous ligand for
human, rat, and mouse FXR alpha form but does not activate Xenopus laevis FXR. Lanosterol, a
precursor of cholesterol, but not CDCA, binds to the beta form of FXR. Recently, the crystal structure
of the rat FXR alpha ligand-binding domain has been solved, and binding residues to 6-ethyl-CDCA
have been identified4. However, the structure/function relationship of the ligand binding domain and
its promiscuous ligand specificity are still unknown. To further elucidate the receptor/ligand structure
relationship of FXR, we took an evolutionary approach by identifying an FXR from the evolutionarily
primitive little skate, whose predominate bile salt is scymnol sulfate, a sulfated bile alcohol.
Table 1. Sequence comparison of FXR from skate and other species with mouse beta and human alpha.
%
Human Rat
Mouse Chicken Tetraodon Zebrafish Skate Xenopus Chicken
alpha alpha
alpha
alpha
alpha
beta
Identity alpha
Mouse 43
44
40
46
46
45
52
54
58
beta
Human 100
90
93
79
63
64
57
47
51
alpha
Mouse
beta
100
43
Degenerate primers were designed to the most conserved DNA binding domain and a DNA
fragment was amplified by PCR. DNA sequencing confirmed that this fragment was a part of skate
FXR. A full-length 2365 bp sequence was obtained by 5’ and 3’ RACE from skate liver. The fulllength skate FXR cDNA contains 186 bp at the 5’ UTR, 1557 bp for the coding region, 722 bp at the
3’ UTR, and an AATAAA sequence at 11 bp upstream of the polyA sequence. A Genbank BLAST
search indicates that this skate FXR shares homology with all known FXRs. Protein alignment analysis
showed that sFXR has 132AA for the AF1 (A/B) domain, 66AA for the DNA binding (C) domain as
for other FXRs, 52AA for the hinge (D) domain, and 268AA for the ligand binding (E) domain. The
phylogenetic tree is shown in figure 1. Table 1 illustrates sequence comparison of FXR from skate and
other species with the mouse beta form and the human alpha form. Of note, skate FXR shares 57%
amino acid identity with human FXR, which is lower than all alpha forms, but higher than all beta
forms. On the other hand, skate FXR is 52% identical to mouse FXR beta, which is higher than all
119
alpha forms but lower than all beta forms. Skate FXR contains two insertions in its ligand-binding
domain when compared to rat FXR (Fig. 2). These insertions also appear in FXR beta. Further protein
hydrophilicity analysis and secondary structure prediction have shown that the inserted portions are
highly hydrophilic and likely form coil-coil structures attached to the outer surface of the core helices
structure of the ligand binding domain. Because the alpha and beta forms of FXR have different ligand
affinities, and because skate FXR may be an intermediate between the alpha and beta forms, we
speculate that skate FXR may have an affinity for both CDCA and lanosterol. This hypothesis remains
to be tested. We are currently in the process of functionally characterizing skate FXR ligand binding
activity. These studies were supported by National Institutes of Health Grants ES03828, ES01247,
DK34989, and DK25636.
mouse alpha
rat alpha
human alpha
chicken alpha
tetraodon alpha
zebrafish alpha
skate ?
xenopus beta
chicken beta
mouse Beta
65.5
60
50
40
30
20
10
0
Figure 1. The phylogenetic tree of FXRs from different species.
RatLBD
SkateLBD
RatLBD
SkateLBD
RatLBD
SkateLBD
RatLBD
SkateLBD
RatLBD
SkateLBD
EKTELTVDQQTLLDYIMDSYSKQRMPQEITNKILKEEFSAEENFLILTEMATSHVQILVE
QRVEFTAEQQQLLDYIVEAHHKYRIPQEAARKYLFEPANPVEDFLRLSENATLQVEVLVE
::.*:*.:** *****:::: * *:*** :.* * * .. *:** *:* ** :*::***
H1
H2
H3
FTKRLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPAG----------------FAKRLPGFQTLDNEDQIALLKGSTVEVMFLHSAQLYNQSFTQSSHQYIQDTEHYSTLPGC
*:**********:**********:**.***:**:::*:.:. .
H4
H5
-HADLLEERI-------------------RKSGISDEYITPMFSFYKSVGELKMTQEEYA
CTNQCFEEHIPAVMMEQSNLDEISIVVPNRTLGIAEEFITPMFDFYRSMGELNVTDTEYA
: :**:*
*. **::*:*****.**:*:***::*: ***
H6
H7
LLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIYQPENPQHFACLLGRLTELRTF
LLSATTILFSDRPYLKNKTHVEKLQEPLLEILHKYSKIHHPESPQRFARLLGRLTQLRTL
**:* .** .** *:*:: *********::*:* .**::**.**:** ******:***:
H8
H9
H10/11
NHHHAEMLMSWRVNDHKFTPLLCEIWDVQ
NHNHSEVLMSWKMKDQKLTPLLCEIWDVQ
**:*:*:****:::*:*:***********
H10/11
H12(AF2)
Figure 2. Rat and skate FXR ligand binding domains (LBD) sequence alignment and comparison. Identical amino acids are
labeled by * underneath. The helices which bind to ligand are H3, H5, H7, H10/11, and H12.
1
2
3
4
120
Forman B.M., Goode E., Chen J., Oro A.E., Bradley D.J., Perlmann T., Noonan D.J., Burka L.T., McMorris T.,
Lamph W.W., Evans R.W., and Weinberger C. Identification of a nuclear receptor that is activated by farnesol
metabolites. Cell, 81: 687-693, 1995.
Makishima M., Okamoto A.Y., Repa J.J., Tu H., Learned R.M., Luk A., Hull M.V., Lustig K.D., Mangelsdrof
D.J. and Shan B. Identification of a nuclear receptor for bile acids. Science. 284:1362-1365, 1999.
Parks D.J., Blanchard S.G., Bledsoe R.K., Chandra G., Consler T.G., Kliewer S.A., Stimmel J.B., Willson T.M.,
Zavacki A.M., Moore D.D., and Lehmann J.M. Bile acids: Natural ligands for an orphan nuclear receptor. Science.
284:1365-1368, 1999.
Mi L.Z., Devarakonda S., Harp J.M., Han Q., Pellicciari R., Willson T.M., Khorasanizadeh S., and Rastinejad
F. Structural basis for bile acid binding and activation of the nuclear receptor FXR. Molecular Cell, 11:1093-1100,
2003.
Suppression subtractive hybridization (SSH) cDNA library construction from fuel oil treated
winter flounder (Pseudoplueronectes americanus)
Peter F. Straub, Mary L. Higham and William C. Phoel
Natural Sciences & Math
Richard Stockton College, Pomona, NJ 08240
Polycyclic aromatic hydrocarbons (PAH) are among the most prevalent pollutants associated with
urbanized aquatic ecosystems. Recent studies by Straub et al.1 have detected transcript differences in
the livers of marine bottom dwelling fish that appear to be related to the degree of anthropogenic
contamination in the natural system. The purpose of this study was to isolate transcripts that were
related directly to PAH exposure and compare these transcripts with those found in wild caught fish.
Young of the year winter flounder were caught in late summer and randomly assigned to 40 l
filtered seawater aquaria, kept at 18o C and fed each day with blue mussels and chopped baitfish. After
14 days, the flounder were exposed to 0.8% w/v fuel oil #6 based on 1 kg of clean dry sand per
aquaria. The fuel oil was mixed with 10 g of calcined clay, to adsorb the oil, and then added to
aquaria. Control tanks received only calcined clay. After a 10 day exposure, the fish were sacrificed
and their livers removed for RNA extraction. Two SSH cDNA libraries, forward and reverse, were
constructed from treated and control mRNA used alternatively as tester and driver with a PCR Select
(SSH) cDNA kit (BD-Clontech). The forward, or up-library, was enriched for transcripts
predominating in the fuel oil treated fish and the reverse, or down-library, was enriched for transcripts
predominating in the untreated control. The cDNA were cloned using a pGEM-T cloning kit
(Promega) and individual clones prepared for plasmid sequencing. Clones were sequenced using a
DCTS sequence kit and a CEQ 8000 genetic analyzer (Beckman-Coulter). Sequence was trimmed and
submitted for BLAST search at the NCBI website.
A total of 157 up- and 123 down-regulated transcripts were sequenced. These sequences are
entered into GenBank as accession numbers CO57806- CO576085 inclusive. Of the 280 sequenced
transcripts, 68% were presumptively identified by BLAST search using a cutoff E-value of 1x10-5.
Some transcripts common to the wild-caught pollution-impacted fish1 and up-regulated with oil were:
cytochrome P450 1A, complement component C-3, C-type lysozyme, fibrinogen, antifreeze protein,
gastrulation specific protein and ceruloplasmin. Specific to the up-regulated with oil treatment were:
defender against death cell protein, retinoid-x receptor-alpha, ribophorin1, GABA receptor associated
protein, complement components C-5 and C-8, Cytochrome P450 24A, hepatic glucose transporter,
antithrombin and cardiac morphogenesis protein ES/130. Unique down-regulated with oil treatment
transcripts found were: cytochrome P450 2D, mRNA for spermatogonial stem-cell renewal factor,
alpha-2-HS glycoprotein, saxitoxin-tetrodotoxin binding protein1, hemopexin-like protein,
chemotaxin, acidic chitinase and matrix metalloproteinase. These transcripts can be used to further
dissect the effects of specific environmental pollutants on the liver of model aquatic vertebrates.
1.
Straub, P.F, M.L. Higham, A. Tanguy, B.J. Landau, W.C. Phoel, L.S. Hales, T.K.M. Thwing. Suppression
Subtractive Hybridization cDNA Libraries to Identify Differentially Expressed Genes from Contrasting Fish Habitats.
Marine Biotechnology 6:386-399.
Funding sources, MDIBL-CMTS under NIEHS P30 ESO3828-18, NSF DBI-0116165 and NJ Sea
Grant under USDOC-NOAA, Sea Grant NA 16RG1047, publication # NJSG-05-580.
121
Up-regulation of Mrp2 expression and transport activity by dexamethasone in killifish
(Fundulus heteroclitus) renal proximal tubules
1
Femke M. van de Water1, Rosalinde Masereeuw1, Frans G.M. Russel1 and David S. Miller2
Department of Pharmacology and Toxicology, Radboud University Medical Centre/ Nijmegen Centre
for Molecular Life Sciences, Nijmegen, The Netherlands
2
Lab of Pharmacology and Chemistry, NIH/NIEHS, Research Triangle Park, NC 27709
The multidrug resistance-associated protein isoform 2 (Mrp2) is highly expressed at the luminal
membrane of renal proximal tubule cells, where it excretes xenobiotics and metabolic wastes into the
urinary space. Previous studies with isolated killifish tubules showed that Mrp2-mediated transport
activity is rapidly reduced by endothelin-1 (ET-1) acting through an ETB receptor, nitric oxide
synthase (NOS), cyclicGMP and protein kinase C (PKC)3,5,6. This signaling system is also activated by
a number of tubular nephrotoxicants8. In contrast, both ET-1 and nephrotoxicants increase Mrp2
transport activity and function 24 h after a transient exposure7, suggesting transcriptional regulation. In
mammalian liver, Mrp2 expression was shown to be transcriptionally regulated by ligand-activated
nuclear receptors; the pregnane X receptor (PXR), constitutive androstane receptor (CAR) and
farnesoid X receptor (FXR)1. Ligands for these receptors include both endogenous metabolites and
xenobiotics. A recent screen of the Fugu genome shows that teleost fish also express analogous ligandactivated nuclear receptors, including, FXR and PXR, but not CAR2. In addition, using the cloned
zebrafish receptor in a promoter assay, Moore et al.4 identified several ligands for teleost PXR. The
present experiments were designed to determine to what extent xenobiotics acting through these
nuclear receptors can alter Mrp2 expression in killifish tubules.
Freshly isolated killifish kidney tubules were exposed for 3-24 h to xenobiotics known to be
ligands for nuclear receptors. After exposure, tubules were incubated for 1 h in medium with 2 µM FLMTX to assay Mrp2-mediated transport. Cellular and luminal FL-MTX accumulation was measured
using confocal microscopy and quantitative image analysis (ImageJ software)3. In some experiments,
tubules were immunostained with a Mrp2 specific antibody as described previously3.
Percentage fluoresecence (%)
In initial screening experiments, the FXR ligand, chenodeoxycholic acid and zebrafish PXR
ligands4, dehydroisoandrosterone, n-propyl p-hydroxybenzoate, 15α-androstan 17β-ol, 5-β pregnane
3,20 dione, clotrimazole and pregnenolone-16-α-carbonitril, were tested at 1-50 µM. They had no
effect on Mrp2 mediated transport. In contrast, the synthetic glucocorticoid, dexamethasone, potently
increased Mrp2 mediated transport after 3 h exposure (Fig. 1). Furthermore, exposure to 1 µM
dexamethasone for 3 hours increased expression of Mrp2 on the luminal plasma membrane of the
tubular epithelial cells as determined by immunocytochemistry.
250
lumen
cell
200
150
100
50
0
0
0.25
0.5
1
2.5
5
Conc. dexamethasone (µ M )
122
10
Figure 1. The effects of 3 hours incubation with
dexamethasone on 2 µM FL-MTX transport. The
fluorescence in the lumen and cell are shown as a
percentage of the fluorescence intensity in the control
lumen. All concentrations of dexamethasone tested
significantly increased luminal FL-MTX transport
(p<0.01; t-test). Means ± SEM are shown for 48-231
tubules.
Although Mrp2-mediated transport and expression increased after tubules were exposed to
dexamethasone, it was not clear how this drug signaled the increases. Further experiments indicated
that blocking the ET signaling pathway at NOS did not reduce dexamethsone’s effects. Moreover, it
was unlikely that dexamethasone acted through PXR, since a number of other PXR ligands were
without effect (above). However, RU-486, a potent and specific blocker of the glucocorticoid receptor
(GR) substantially attenuated the effects of dexamethasone on Mrp2-mediated transport (Fig. 2). Thus,
Mrp2 expression in killifish renal proximal tubules appears to be transcriptionally regulated by GR, but
not by PXR or FXR. This work was supported by the Netherlands Organization for Scientific Research
(Zon-MW grant 902-21-227) and the MDIBL Center for Membrane Toxicity Studies.
Percentage fluorescence (%)
250
*
lumen
200
##
*
150
100
50
cell
Figure 2. The effects of dexamethasone (dex) and
RU-486 on FL-MTX transport. Tubules were
incubated for 3 hours without (control) or with 1 µM
dexamethasone, 1 µM RU-486 or both of these drugs.
The fluorescence in the lumen and cell are shown as a
percentage of the fluorescence intensity in the control
lumen. Means ± SEM are shown for 49-231 tubules. *
p<0.001 vs. control; ## p<0.001 vs. dex (one-way
ANOVA).
0
Control
1.
2.
3.
4.
5.
6.
7.
8.
Dex
RU-486
Dex + RU-486
Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, Tontonoz P, Kliewer S, Willson TM, and
Edwards PA. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X
receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem 277: 2908-2915, 2002.
Maglich JM, Caravella JA, Lambert MH, Willson TM, Moore JT, and Ramamurthy L. The first completed
genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily.
Nucleic Acids Res 31: 4051-4058, 2003.
Masereeuw R, Terlouw SA, van Aubel RA, Russel FG, and Miller DS. Endothelin B receptor-mediated regulation
of ATP-driven drug secretion in renal proximal tubule. Mol Pharmacol 57: 59-67, 2000.
Moore LB, Maglich JM, McKee DD, Wisely B, Willson TM, Kliewer SA, Lambert MH, and Moore JT. Pregnane
X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three
pharmacologically distinct classes of nuclear receptors. Mol Endocrinol 16: 977-986, 2002.
Notenboom S, Miller DS, Smits P, Russel FG, and Masereeuw R. Involvement of guanylyl cyclase and cGMP in
the regulation of Mrp2-mediated transport in the proximal tubule. Am J Physiol Renal Physiol 287: F33-38, 2004.
Notenboom S, Miller DS, Smits P, Russel FG, and Masereeuw R. Role of NO in endothelin-regulated drug
transport in the renal proximal tubule. Am J Physiol Renal Physiol 282: F458-464, 2002.
Notenboom S, Masereeuw R, Russel FG, and Miller DS. Upregulation of multidrug resistance-associated protein
(Mrp2) in renal proximal tubules from killifish (Fundulus heteroclitus). Bull Mt Desert Island Biological Lab 43:135136, 2004.
Terlouw SA, Masereeuw R, Russel FG, and Miller DS. Nephrotoxicants induce endothelin release and signaling in
renal proximal tubules: effect on drug efflux. Mol Pharmacol 59: 1433-1440, 2001.
123
Sensitivity of shark (Squalus acanthias) CFTR to mercury is mediated by cysteine residues
located in the first membrane spanning domain
1
Ali Poyan Mehr1, Sarah E. Decker1, and John N. Forrest, Jr.1,2
2
Previous studies in our laboratory by Sirota et al.1 have shown shark CFTR (sCFTR) to be much
more sensitive to inhibition by mercury than human CFTR (hCFTR). The present study investigated
the molecular site(s) responsible for this difference by constructing human/shark CFTR chimeric
proteins.
sCFTR has 7 unique Cys residues. Two are located in the first membrane spanning domain
(MSD1), two in the regulatory domain (RD), one in the second membrane spanning domain (MSD2)
and two in the C-terminus of the protein. One or more of these Cys residues likely mediates the
greater sensitivity of sCFTR to mercury. To elucidate which of these residues may be mediating this
sensitivity we first constructed a chimeric protein replacing the human MSD1 with shark MSD1.
Using Xenopus laevis oocytes and two electrode voltage clamping (TEVC), we investigated whether
the unique Cys residues in the MSD1 of sCFTR would confer mercury sensitivity to hCFTR.
A chimeric cDNA construct was prepared using PCR and restriction enzyme digestion. The MSD1
of shark was amplified using a sense primer with an overlapping sequence homologous to the Nterminal of hCFTR and an antisense primer with an introduced restriction site for BsiW I (sense:
AAAATCCTAAACTCATTAATGCACTTCGCCGATG, antisense: CACGTTCGTACGGCAGATG
GAAATTGTCTG). The N-terminal end of hCFTR was then amplified in a separate PCR (sense:
ATACGACTCACTATAGG, antisense: GCATTAATGAGTTTAGG).
Both fragments were
combined in a third reaction. This product was joined to the rest of hCFTR using Not I and BsiW I
restriction sites, and cloned in the pBlueskript KS(-) vector (Invitrogen).
Human/shark MSD1 chimera cRNA as well as the hCFTR and sCFTR cRNAs were each injected
into Xenopus laevis oocytes, and TEVC was performed after two to three days of incubation. After
establishing baseline conductance in ND96 solution, chloride conductance was activated by adding 10
µM forskolin and 1 mM isobutylmethylxanthine (IBMX) to the bath solution. Average conductance
during steady state was 199±23 µS for the MSD1 chimera 182±37 µS for sCFTR, and 411±66 µS for
hCFTR. After 30 min. of activation, HgCl2 (1 µM) was added to the bath solution for 30 min.
Figure 1 shows a representative experiment from each group. Human CFTR (Figure 1, panel A)
showed only partial inhibition of forskolin +IBMX stimulated chloride conductance by 1 µM mercuric
chloride. In contrast, sCFTR (panel B) and the human/shark MSD1 chimera (panel C) showed nearly
complete inhibition under identical conditions. Panel D illustrates the mean percent inhibition by 1
µM mercuric chloride of the three protein constructs: (44% vs. 89% and 90%, p<0.0001) comparing
hCFTR to both sCFTR and human/shark MSD1 chimera.
124
Figure 1. Differential effects of Hg on chloride conductance in hCFTR, sCFTR and human/shark MSD1chimera. Panel A:
representative experiment demonstrating modest inhibition of hCFTR by 1 µM Mercuric chloride. Panel B: representative
experiment demonstrating complete inhibition of sCFTR by 1 µM mercuric chloride. Panel C: representative experiment
demonstrating complete inhibition of human/shark MSD1 chimera by 1 µM mercuric chloride. Panel D: percent inhibition
of the three channels in 23 experiments (n= 9 for hCFTR, n=8 for sCFTR, n= 6 for human/shark MSD1 chimera. P<0.0001)
comparing both wild type shark and chimeric construct to human CFTR.
These results demonstrate that the 2 unique Cys residues in sCFTR MSD1 confer sensitivity to
mercury in the human CFTR channel and suggest that these residues are responsible for the greater
sensitivity of shark CFTR to mercury. Our findings do not exclude the possibility that other shark
specific cysteines may play a role in the differential sensitivities of shark and human CFTR to mercury.
This work was supported by NIH grants DK 34208 and NIEHS 5 P30 ES03828 (Center for
Membrane Toxicity Studies).
1. Sirota, J.C., G.H. Weber, S.G. Aller, D.C. Dawson, and J.N. Forrest, Jr. Shark and human CFTR expressed in
Xenopus oocytes have different sensitivities to inhibition by the thiol-reactive metals mercury and zinc. Bull. Mt.
125
Effect of lipopolysaccharides from Microcystis and Lyngbya on
metal toxicity in Fundulus heteroclitus
Emily G. Notch, Cassandra A. Patenaude, Danielle M. Miniutti, Amy N. Hicks, and Gregory D. Mayer
Biochemistry, Microbiology, and Molecular Biology
University of Maine, Orono, ME 04469
Cyanobacteria are prevalent in the freshwater environment and can reach abundant mass in
harmful algal blooms (HAB’s). These blooms are common in aquatic environments with high nutrient
influx and slow moving warm water, as found in the Florida Everglades1. Lipopolysaccharides (LPS’s)
are components of the cell walls of all Gram-negative bacteria and related Cyanobacteria. Often
referred to as bacterial “endotoxin” or pyrogens (i.e., fever inducers), they have been recognized as the
causative agent of sepsis and “toxic shock” associated with bacterial infection, and have more recently
received attention related to the environment in association with HAB’s. The concurrent exposure of
aquatic fauna to both LPS and metal is significant, as both are present in many aquatic environments,
including the Florida Everglades. Recently, Best, et al.2 described the effect of cyanobacterial LPS’s on
the inhibition of glutathione S-transferase (GST) activity in zebrafish (Danio rerio) embryos. GST is
an important detoxifying enzyme that catalyzes the conjugation of reduced glutathione to many
potentially toxic compounds, including metals. Additionally, GST activity is normally increased after
metal insult, and has been indicated as a first line of defense against Cd2+ toxicity before upregulation
of metallothionein synthesis occurs3. We investigated the combined effect of cyanobacterial LPS
extracts and CdCl2 exposure in the killifish, Fundulus heteroclitus, to delineate a possible role of
cyanobacterial blooms in the potentiation of metal toxicity.
Wild caught killifish from Northeast Creek, Mount Desert Island, ME were transferred to 10 liter,
high-density polyethylene tanks containing static, Salisbury Cove seawater. Tanks were immersed in
flow-through seawater to maintain constant temperature. Fish were exposed to several waterborne
concentrations of CdCl2, with and without addition of LPS’s from either Microcystis or Lyngbya, to
determine nominal LC50 values for Cadmium and examine the effects of LPS exposure on metal
toxicity. For these studies, lyophilized LPS’s from cyanobacterial isolates were prepared by the
method of Raziuddin et al.4, using the “hot phenol/water” extraction method, and added to the
appropriate experimental tanks at a concentration of 3.8 EU/L for Microcystis and 58.9 EU/L for
Lyngbya. LPS concentrations were based upon Limulus amoebocyte lysate assay results. Cadmium, as
CdCl2, was prepared in 5mg/L concentration increments from 35-75 mg/L for LC50 determination as
shown in Table 1. Concentrations of CdCl2 used were based upon prior research and LC50. Additional
fish were also exposed to the same concentrations of Cadmium and LPS and samples of gill and liver
taken at a variety of time points to measure glutathione S-transferase activity. A sample graph of
hepatic glutathione S-transferase activity for Fundulus exposed to Cadmium and Microcystis or
Lyngbya is shown in Figure 1. GST activity was measured using the glutathione S-transferase kit from
Cayman chemicals and normalized to protein using the BCA protein assay kit from Pierce
Biotechnology Inc. All kits were used to analyze sample per manufacturer’s directions.
Table 1. 96 hour LC50 values for killifish exposed to CdCl2 in static seawater with semi-daily renewal. Values are reported
as the mean ± standard deviation of probit analysis outcomes. For all measurements n=6, with 3 experimental repeats.
Probit analysis was used for data determination.
69.008 ± 1.029 mg/L
LC50 for CdCl2
LC50 for CdCl2 with the addition of 3.8EU/L Microcystis LPS
68.244 ± 9.021 mg/L
LC50 for CdCl2 with the addition of 58.9EU/L Lyngbya
>75 mg/L
126
1800.00
1600.00
1400.00
GST Activity (nMoles/min/mg)
Fig. 1. This figure shows
hepatic
glutathione
Stransferase
in
nMoles
conjugated CDNB/minute/mg
protein versus time in hours.
Fudulus heteroclitis exposed
to 55mg/L of CdCl2 and
either 3.8EU/L of Microcystis
or 58.9EU/L of Lyngbya.
Samples were taken at 0.5, 1,
2, 4, 8, 16 and 24 hours.
1200.00
1000.00
800.00
600.00
Cd-Microcystis
Cd-Lyngbya
400.00
200.00
0.00
0
5
10
15
20
25
Time (hr)
When killifish were exposed to lipopolysaccharide preparations from either Microcystis aeruginosa
or Lyngbya sp. in addition to Cadmium, a change in glutathione S-transferase activity was documented
in the liver. These data show a trend of higher hepatic GST in the Fundulus exposed to Cadmium and
Lyngbya than in those exposed to Cadmium and Microcystis. Instead of the expected potentiation of
metal toxicity by cyanobacterial LPS, we recorded ameliorated toxicity values for CdCl2 in toxicity
tests that incorporated Lyngbya LPS. Toxicity tests that incorporated Microcystis LPS did not result in
any significant change in LC50 value from that of tests with CdCl2 as the sole variable. These data,
combined with the previous year’s work at MDIBL6 indicate that there may be a threshold level of
LPS for the amelioration of metal toxicity, and at lower levels LPS may in fact potentiate metal
toxicity. However, these data need to be further examined and additional experiments performed to
better understand interactions of a myriad of deleterious effects associated with both Cd and LPS
toxicity.
These preliminary studies warrant further investigation regarding mechanisms of potentiation and
amelioration of metal toxicity by lipopolysaccharides, as well as exploration of GST and other
detoxification processes involved in concurrent exposure with LPS and metals. This work was funded
in part by a New Investigator Award to G.D.M. from the Salisbury Cove Research Fund, Salisbury
Cove, ME.
1.
2.
3.
4.
5.
6.
Integrated Water Quality Assessment for Florida: 2004 305(b) Report and 303(d) List Update. Florida Department of
Environmental Protection, July 2004.
Best, J.H., Pflugmacher, S., Wiegand, C., Eddy, F.B., Metcalf, J.S., Codd, G.A. Effects of enteric bacterial and
cyanobacterial lipopolysaccharides, and of microcystin-LR, on glutathione S-transferase activities in zebra fish
(Danio rerio). Aq. Tox. 60: 223-231, 2002.
Basha, P.S., Rani, A.U. Cadmium-induced antioxidant defense mechanism in freshwater teleost Oreochromis
mossambicus (Tilapia). Ecotoxicology and Environmental Safety 56(2): 218-221, 2003.
Raziuddin, S., Siegleman, H.W., Tornabene, T.G. Lipopolysaccharides of the cyanobacterium Microcystis
aeruginosa. Eu. J. Biochem. 137: 333-336, 1983.
Habig, W.H., Pabst, M.J., Jakoby, W.B. Glutathione S-transferase: the first enzymatic step in mercapturic acid
formation. J. Biol. Chem. 249: 7130, 1974.
Mayer, G.D., Berry, J.P., Patenaude, C.A., Walsh, P.J. Effect of lipopolysaccharides from Microcystis and Lyngbya
on metal toxicity in Fundulus heteroclitus. Bull. Mt Desert Isl. Biol. Lab. 34:143-144, 2004.
127

INVITED REVIEW Mechanisms of Rectal Gland Secretion Franklin H

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