Inositol trisphosphate receptors in smooth muscle cells

Transcription

Am J Physiol Heart Circ Physiol 302: H2190 –H2210, 2012.
First published March 23, 2012; doi:10.1152/ajpheart.01146.2011.
Review
Inositol trisphosphate receptors in smooth muscle cells
Damodaran Narayanan, Adebowale Adebiyi, and Jonathan H. Jaggar
Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee
Submitted 8 December 2011; accepted in final form 22 March 2012
inositol 1,4,5-trisphosphate receptors; sarcoplasmic reticulum; calcium
(Ca2⫹) signals are produced by both
extracellular Ca2⫹ influx and intracellular Ca2⫹ release (22,
29). Ca2⫹ influx can occur through plasma membrane ion
channels and transporters, including voltage-dependent L-type
Ca2⫹ (CaV1.2) channels, nonselective cation channels, and the
Na⫹/Ca2⫹ exchanger (22, 29). Ca2⫹ can also be released from
intracellular stores, such as the sarcoplasmic reticulum (SR)/
endoplasmic reticulum (ER) and mitochondria (22, 29). Ca2⫹
signals in smooth muscle cells (SMCs) include Ca2⫹ flashes,
oscillations, puffs, ripples, sparklets, sparks, waves, and global
intracellular Ca2⫹ concentration ([Ca2⫹]i) (13, 26, 90, 144,
158, 254). These diverse Ca2⫹ signals regulate physiological
functions and are modified in diseases, leading to pathological
consequences.
Many plasma membrane receptors couple via heteromeric
Gq/11 proteins to PLC (22, 29). Upon agonist to receptor
binding, PLC-catalyzed hydrolysis of phosphatidylinositol 4,5bisphosphate generates diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3) (21, 57). IP3 binds to and activates
INTRACELLULAR CALCIUM
Address for reprint requests and other correspondence: J. H. Jaggar, Dept. of
Physiology, Univ. of Tennessee Health Science Center, Memphis, TN 38163
(e-mail: [email protected]).
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SR-localized IP3 receptors (IP3Rs) (21, 57). SMC IP3Rs were
first purified from aorta and, following reconstitution into
phospholipid vesicles, were identified as Ca2⫹ release channels
(34, 52, 134). IP3Rs regulate many cellular processes in SMCs,
including contractility, gene expression, migration, and proliferation (2, 60, 149, 156, 234, 237).
The focus of this review is to discuss IP3R expression,
cellular localization, channel activity, communication with ion
channels and organelles, Ca2⫹ signals generated, physiological
functions regulated, and pathological alterations in SMCs.
Excellent reviews have summarized knowledge gained from
studies of recombinant IP3R channels and those expressed in a
wide variety of different cell types (21, 57, 143, 171). Here,
IP3R signaling in non-SMC types will only be considered when
extrapolation to SMCs is appropriate or when discussion may
stimulate future research in SMCs.
IP3R Structure
IP3Rs are tetramers, with each subunit composed of three
principal regions: an NH2 terminus, a hydrophobic region
comprising six transmembrane domains, and a COOH-terminal
tail (Fig. 1) (139, 246). The cytosolic NH2-terminal region is
functionally divided into the IP3-binding domain, a suppressor
0363-6135/12 Copyright © 2012 the American Physiological Society
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Narayanan D, Adebiyi A, Jaggar JH. Inositol trisphosphate receptors in
smooth muscle cells. Am J Physiol Heart Circ Physiol 302: H2190 –H2210, 2012.
First published March 23, 2012; doi:10.1152/ajpheart.01146.2011.—Inositol 1,4,5trisphosphate receptors (IP3Rs) are a family of tetrameric intracellular calcium
(Ca2⫹) release channels that are located on the sarcoplasmic reticulum (SR) membrane of
virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we
have reviewed literature investigating IP3R expression, cellular localization, tissue
distribution, activity regulation, communication with ion channels and organelles,
generation of Ca2⫹ signals, modulation of physiological functions, and alterations
in pathologies in SMCs. Three IP3R isoforms have been identified, with relative
expression and cellular localization of each contributing to signaling differences in
diverse SMC types. Several endogenous ligands, kinases, proteins, and other
modulators control SMC IP3R channel activity. SMC IP3Rs communicate with
nearby ryanodine-sensitive Ca2⫹ channels and mitochondria to influence SR Ca2⫹
release and reactive oxygen species generation. IP3R-mediated Ca2⫹ release can
stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca2⫹ channels. SMC IP3Rs also signal to
other proteins via SR Ca2⫹ release-independent mechanisms through physical
coupling to TRP channels and local communication with large-conductance Ca2⫹activated potassium channels. IP3R-mediated Ca2⫹ release generates a wide variety
of intracellular Ca2⫹ signals, which vary with respect to frequency, amplitude,
spatial, and temporal properties. IP3R signaling controls multiple SMC functions,
including contraction, gene expression, migration, and proliferation. IP3R expression and cellular signaling are altered in several SMC diseases, notably asthma,
atherosclerosis, diabetes, and hypertension. In summary, IP3R-mediated pathways
control diverse SMC physiological functions, with pathological alterations in IP3R
signaling contributing to disease.
Review
IP3Rs IN SMOOTH MUSCLE CELLS
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domain that inhibits IP3 binding, and the regulatory domain
(Fig. 1, A and B) (57, 245). The regulatory domain contains
binding sites for ATP (Fig. 1A, red squares) and Ca2⫹ (Fig. 1A,
blue squares) and consensus sequences for phosphorylation
(Fig. 1A, black squares) (57, 139, 171). A coupling domain
through which IP3Rs physically interact with transient receptor
potential canonical (TRPC) channels is also located in the
regulatory domain (Fig. 1B) (2, 208). The transmembrane
domains anchor each IP3R subunit to the SR membrane (Fig. 1,
B and C) (139). The luminal loop between transmembrane
domains 5 and 6 forms the Ca2⫹-permeable pore (Fig. 1B)
(139). The COOH-terminal tail extends from transmembrane
domain 6 into the cytosol (Fig. 1, A and B) (139). The
transmembrane domains and COOH-terminal tail appear to be
essential for IP3R tetramerization (141, 188).
IP3R Isoforms and Distribution in SMCs of Different Tissues
To date, three IP3R isoforms (IP3R1–3) have been identified,
each of which is encoded by a different gene (57). Important
features of mammalian IP3R isoforms are summarized in
Table 1. SMCs express all three IP3R isoforms, with relative
levels of each determined by the tissue of origin and developmen-
tal stage. IP3R1 is the predominant isoform expressed in vascular
SMCs (64, 84, 147, 228, 254, 257). Quantitative PCR performed
on isolated cerebral artery SMCs indicated that IP3R1 mRNA was
the most abundant of the three isoforms, at 82% of total message,
with much of the residual IP3R message being IP3R3 (254).
Similarly, IP3R1 was the major isoform detected in basilar and
mesenteric arteries, whole thoracic aorta, cultured portal vein
SMCs, and A7r5 cells, an aortic SMC line (64, 84, 147, 211, 228,
257). Reports indicate that IP3R isoform expression in vascular
SMCs shifts during ontogeny and proliferation (210, 211). In
neonatal (2- to 4-day-old) rats, IP3R1 protein was lower and
IP3R3 protein was higher in SMCs of aorta and portal vein,
compared with those in juvenile (6 wk-old) rats (210). IP3R2 and
IP3R3 levels were higher in proliferating, cultured aortic SMCs
than in whole aorta homogenates (211). IP3R isoform expression
in nonvascular SMCs exhibits tissue variability. In isolated ureteric and myometrial SMCs, all three IP3R isoforms were detected
(26, 148). IP3R3 was the predominant isoform, with IP3R1 and
IP3R2 also present in tracheal SMCs (230). In contrast, cultured
ureteric and gastric SMCs expressed IP3R1 and IP3R3 but not
IP3R2 (147, 154). Tissue variability in IP3R isoform expression
may contribute to signaling differences in these SMC types.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.01146.2011 • www.ajpheart.org
Fig. 1. Inositol 1,4,5-trisphosphate receptor (IP3R) molecular structure. A: schematic representation of an IP3R subunit depicting important domains and regions.
Sites for ATP-binding (black squares), Ca2⫹-binding (blue squares), and phosphorylation (red squares) are indicated. B: single IP3R subunit illustrating important
domains, regions, and pore. C: tetrameric IP3R channel. SR, sarcoplasmic reticulum; N, NH2 terminus; C, COOH terminus.
Review
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Table 1. Important features of the three mammalian IP3R isoforms
IP3R1
IP3R2
–
Homology with human IP3R1
IP3R3
77% (242)
72% (123, 242)
Alternative splicing
Yes (163, 241)
None described
None described
Amino acid number (human)
2,695–2,743 (splice variation-dependent)
(69, 163, 241)
3p25–26 (241)
2,701 (242)
2,671 (123, 242)
12p11 (242)
6p21 (242)
Chromosomal localization
(human)
Cellular localization in SMCs
Central SR
Peripheral SR
Cultured aorta (199, 211)
Cultured aorta (199, 211), ureter (26)
Highest (0.10 ␮M)* (217)
Lowest [0.40 ␮M]* (217)
Yes [0.17 ␮M]* (216)
Yes [0.37 ␮M]* (216)
Decreases (33, 244)
Yes [0.15 ␮M]* (216)
Yes [0.16 ␮M]* (216)
None described
Yes [0.06 ␮M]* (216)
Yes [0.17 ␮M]* (216)
⬍500 nM increases, ⬎500 nM decreases
(33, 244)
3–4 (171)
High
[0.13 mM]* (217)
1 (171)
None (217)
2 (171)
Low
[2.0 mM]* (217)
IP3Rs, inositol 1,4,5-trisphosphate receptors; SMCs, smooth muscle cells; SR, sarcoplasmic reticulum. Numbers in parenthesis indicate reference numbers.
*EC50 for recombinant IP3Rs reconstituted into lipid bilayer.
Cellular Expression and Localization of SMC IP3Rs
Cellular levels of IP3Rs in SMCs are determined by an
equilibrium between gene transcription and protein degradation (3, 129, 171, 227). c-Myb, a transcription factor, binds to
the IP3R1 promoter and stimulates transcription in cultured
aortic SMCs (3). In A7r5 cells, retinoic acid inhibited IP3R1
gene transcription through a mechanism mediated by activator
protein-2, another transcription factor (45). In cultured aortic
SMCs, hydrogen peroxide (H2O2) stimulated proteasomal degradation of IP3R1 and IP3R3, whereas Jak2, a tyrosine kinase,
phosphorylated IP3R1 and prevented proteasomal degradation
(129, 227). Transforming growth factor-␤ (TGF-␤) reduced
IP3R1 and IP3R3 protein in renal arteriolar SMCs, although it
was not determined if this was due to protein degradation
(138). In A7r5 cells, vasopressin inhibited IP3R1 gene transcription and also stimulated protein degradation, suggesting
that a physiological agonist can modulate SMC IP3R levels
through both of these mechanisms (197). Therefore, regulation
of both gene transcription and protein degradation controls
IP3R cellular levels in SMCs.
In SMCs, IP3Rs locate to the SR in different cellular regions,
including central SR around the nucleus and peripheral SR
beneath the plasma membrane (Table 1) (2, 26, 63, 162, 199,
211, 224, 254). IP3R localization to central and/or peripheral
SR is isoform and tissue dependent. In cerebral artery SMCs,
IP3R1 distributes to both central and peripheral SR (Fig. 2A)
(2, 254). However, IP3R1 and IP3R3 located only to peripheral
SR in ileal and ureteric SMCs, respectively (26, 63). IP3Rs on
central SR around the nucleus may regulate Ca2⫹-dependent
gene expression through signals that do not impact global
[Ca2⫹]i. Distribution to peripheral SR may position IP3Rs in
close proximity to the plasma membrane, thereby permitting
local signaling to membrane proteins, including ion channels
(2, 255). Therefore, IP3R cellular localization in SMCs likely
contributes to distinct physiological functions regulated by
these proteins.
IP3R Channel Properties in Vascular SMCs
Single channel properties of vascular SMC IP3Rs have been
examined using purified aortic IP3Rs reconstituted into planar
lipid bilayers (134). Reconstituted IP3Rs were found to be
IP3-gated ion channels, most permeable to Ca2⫹, followed by
K⫹ and Cl⫺ (134). Bath application of 0.5 ␮M IP3 activated
⬃32-pS single channel currents after a delay of ⬃1 min in
standard buffer with 50 mM Ca2⫹ as the charge carrier (Fig.
2B) (134). The delayed response between bath application of
IP3 and stimulation of channel currents was suggested to be
related to the diffusion of IP3 in the bath (134). IP3-induced
channel currents were enhanced by ATP (Fig. 2C) and blocked
by heparin (134).
The kinetics of IP3R-mediated Ca2⫹ release in vascular
SMCs have been investigated using permeabilized portal vein
smooth muscle strips and flash photolysis of caged IP3 (198).
Binding of IP3 to a single IP3R subunit, as opposed to all four
subunits of the tetramer, was sufficient to induce channel
opening (198). IP3 photolysis elevated [Ca2⫹]i from ⬃100 to
175 nM, with a total IP3-releasable SR Ca2⫹ pool of ⬃126 ␮M
(198). The amount of IP3-induced Ca2⫹ release increased as a
function of IP3 concentration ([IP3]) with an estimated Kd of
⬃1 ␮M (198). The delay between IP3 photolysis and the
[Ca2⫹]i elevation was inversely proportional to [IP3], with
saturating [IP3] (⬎4 ␮M) and submaximal [IP3] (0.4 – 0.8 ␮M)
inducing Ca2⫹ release within 10 ms or after 420 ms, respectively (198). Collectively, these studies (134, 198) provided
important information regarding biophysical properties of
IP3Rs and the kinetics of IP3R-mediated Ca2⫹ release in
vascular SMCs.
IP3 affinity
Regulation by Ca2⫹
Activation
Inhibition
Effect of Ca2⫹ on IP3
binding
Regulation by ATP
Number of binding sites
Sensitivity
Cerebral artery (1, 2, 254), cultured and
noncultured aorta (162, 199, 211), A7r5
cells (224)
Cerebral artery (1, 2, 254), cultured aorta (199,
211), vas deferens (162), ileum (63)
Intermediate [0.27 ␮M]* (217)
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Cellular Regulators of SMC IP3R Activity
This section will describe ligands, protein kinases, regulatory
proteins, and other modulators that influence IP3R activity in
SMCs. The reader is referred to earlier reviews (57, 127, 172, 213)
where additional details of these and other IP3R regulators derived
from reports in non-SMC types are discussed.
Ligands. Ligands that regulate SMC IP3R activity include
IP3, cytosolic Ca2⫹, SR luminal Ca2⫹, and ATP, with IP3 and
cytosolic Ca2⫹ acting as principal modulators. Regulation of
IP3R channel activity by IP3, cytosolic Ca2⫹ concentration
([Ca2⫹]c), and ATP is summarized in Table 1.
IP3 is the major stimulus for inducing IP3R-mediated Ca2⫹
release in SMCs (34, 134, 198). [3H]IP3 binding assays indicated that IP3 bound to IP3Rs with a Kd of 2–5 nM in SMCs of
the aorta, colon, ileum, jejeunum, myometrium, and vas deferens (4, 24, 34, 150, 152, 182, 223, 251). Evidence from studies
in non-SMCs suggest that binding of IP3 induces a conformational change in the IP3-binding domain, which is transmitted
via the central regulatory domain to the transmembrane domains to elicit channel opening (141, 155, 222). SMC IP3R
activity is modulated by an intricate interplay between IP3 and
[Ca2⫹]c (77, 79, 134). SMC IP3R channel activation occurs at
[Ca2⫹]c ⬍300 nM, and released Ca2⫹ initially stimulates IP3Rs
via Ca2⫹-induced Ca2⫹ release (CICR) (77, 79). However, an
elevation in [Ca2⫹]c ⬎300 nM inhibits SMC IP3Rs (77, 79).
Studies (74, 140) in non-SMCs suggested that for Ca2⫹ to
inhibit IP3Rs, [Ca2⫹]c ⬎300 nM and CaM, a Ca2⫹-binding
protein, were both required. IP3R activity regulation by
[Ca2⫹]c is controlled by distinct stimulatory and inhibitory
Ca2⫹-binding sites (195, 196). One luminal and seven cytosolic Ca2⫹-binding sites (Fig. 1A, blue squares) have been identified on IP3Rs, suggesting that Ca2⫹ binds to and directly
influences IP3R activity (195, 196). A [Ca2⫹]c elevation can
also inhibit IP3 binding to SMC IP3Rs, thereby indirectly
regulating channel activity (16). Thus [Ca2⫹]c regulates IP3R
activity in SMCs via two simultaneously occurring mechanisms: 1) directly, by binding to stimulatory or inhibitory sites
on IP3Rs; and 2) indirectly, by modulating IP3 binding to
IP3Rs. Evidence also suggests that IP3 determines if Ca2⫹
stimulates or inhibits IP3Rs (124). IP3 binding to IP3Rs displaces Ca2⫹ from inhibitory sites, leading to a reduction in
Ca2⫹-dependent inhibition, and exposes stimulatory Ca2⫹binding sites (Fig. 1A, blue squares), thereby permitting Ca2⫹mediated activation (124). Therefore, IP3 and [Ca2⫹]c influence SMC IP3R activity directly, by binding to IP3Rs and
indirectly, by modulating effects of each other.
SMC IP3Rs are modulated not only by cytosolic Ca2⫹ but
also by SR luminal Ca2⫹. In A7r5 cells, an elevation in SR
Ca2⫹ concentration ([Ca2⫹]SR) enhanced IP3-induced Ca2⫹
release (30). Depletion of SR Ca2⫹ abolished IP3R-mediated
Ca2⫹ release in SMCs (81, 86, 117, 136, 151). Ca2⫹ binding to
a site located in the luminal loop between transmembrane
domains 5 and 6 of IP3Rs (Fig. 1A, blue square in the pore)
(195) stimulated cerebellar IP3Rs (23). [Ca2⫹]SR may also
regulate SR Ca2⫹ release in SMCs by determining the driving
force for Ca2⫹ efflux, which should modulate the amplitude of
IP3R-mediated Ca2⫹ signals. Therefore, [Ca2⫹]SR may influence SMC IP3Rs through more than one mechanism, although
experimental evidence for such regulation is limited.
ATP binds to three sites located in the IP3R regulatory
domain (Fig. 1A, black squares). ATP is not required for SMC
IP3R activation but enhances IP3 and Ca2⫹ sensitivity, thereby
augmenting channel activity (77, 80, 134). ATP alone did not
stimulate aortic IP3Rs reconstituted into planar lipid bilayers
but elevated IP3-induced currents (Fig. 2C) (134). Similarly, in
permeabilized portal vein SMCs, ATP dose-dependently increased IP3-induced Ca2⫹ release with a maximal effect at 0.5
mM ATP (80). ATP also elevates SMC IP3R Ca2⫹ sensitivity,
Fig. 2. Smooth muscle cell (SMC) IP3Rs.
A: immunofluorescence image illustrating
IP3R1 localization in an isolated cerebral
artery SMC. Scale bar ⫽ 10 ␮m. B and
C: single channel recordings of purified aortic IP3Rs reconstituted into lipid bilayers
demonstrating activation by IP3 (⫺50 mV
holding potential; B) and ATP-mediated enhancement of IP3-induced currents (C). B and
C: reproduced from Mayrleitner et al. (134) by
copyright permission (2011) from Elsevier.
Review
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and FK506 in colonic SMCs (15, 118). In contrast, in aorta,
FKBP12 did not coimmunoprecipitate with IP3Rs and IP3Rmediated Ca2⫹ release was unaffected by FK506 or rapamycin
(118, 119). Explanations for these tissue-specific differences in
FKBP12 regulation of SMC IP3Rs are unclear but may be due
to variable expression of FKBP12 and important regulatory
proteins, including calcineurin, FK506, and mTOR.
RACK1 is a scaffolding protein that shuttles PKC to its
substrates, thereby facilitating interaction (173). RACK1 coimmunoprecipitated with IP3R1 in PC12 cells, a non-SMC
line, and elevated [3H]IP3 binding to cerebellar IP3Rs, suggesting that RACK1 interacts with IP3Rs and increases IP3 affinity
(173). RACK1 enhanced agonist-induced IP3R-mediated Ca2⫹
release in A7r5 cells and cultured preglomerular microvascular
SMCs, suggesting that RACK1 also stimulates IP3R channel
activity in SMCs (36, 173). Although evidence suggests functional regulation of IP3R-mediated Ca2⫹ release by RACK1 in
SMCs, whether these effects are due to direct binding to IP3Rs
or by PKC-dependent pathways has not been resolved.
Other modulators. In addition to endogenous ligands, kinases,
and regulatory proteins, cellular pH and reactive oxygen species
(ROS) also modulate SMC IP3Rs. pH influences SMC IP3R
activity by controlling IP3 binding and IP3R Ca2⫹ sensitivity (38,
78, 152, 215, 251). Elevating pH activated recombinant IP3R1 and
IP3R3 channels, with IP3R1 being more sensitive at pH 6.8 and
IP3R3 at pH 7.5, suggesting that pH regulation is isoform dependent (44). IP3 binding to SR membranes from aortic, colonic, and
tracheal smooth muscle was weak at pH ⬍7 and maximal at pH
8 –9 (38, 152, 251). In portal vein and taenia caeci SMCs,
increasing pH from 6.7 to 7.0 –7.3 reduced the Ca2⫹ concentration
required for IP3R activation, thereby elevating IP3R-mediated
Ca2⫹ release (78, 215). Evidence suggests that H⫹ may compete
with IP3 and Ca2⫹ to inhibit their binding to SMC IP3Rs, resulting
in channel inhibition (215).
ROS regulate SMC IP3Rs through processes that include control of [IP3]i and modulation of IP3R IP3 affinity (31, 112, 183,
201). Superoxide reduced IP3 hydrolysis in SR vesicles of thoracic aorta, thereby enhancing [IP3]i and IP3-induced Ca2⫹ release
(201). Thimerosal, an oxidizing agent, enhanced the affinity of
IP3R1 for IP3 and potentiated IP3-induced Ca2⫹ release in A7r5
cells (31). Similarly, in SMCs of the pulmonary and systemic
vasculature, H2O2 stimulated IP3R-mediated Ca2⫹ release, suggesting that ROS enhance IP3R signaling in SMCs (112, 183). In
contrast, in coronary artery SMCs, H2O2, superoxide, and peroxynitrite inhibited the SR Ca2⫹-ATPase (SERCA), leading to a
reduction in [Ca2⫹]SR that attenuated IP3R-mediated Ca2⫹ release
(53, 65). In gallbladder and mesenteric artery SMCs, superoxide
and H2O2 did not alter IP3R activity (225, 238). Therefore, ROS
regulation of SMC IP3Rs is multimodal and may be dependent on
tissue of origin and ROS species involved.
In summary, IP3, [Ca2⫹]c, and ATP are major modulators of
IP3R activity in SMCs and protein kinases, pH, and ROS
regulate IP3Rs through phosphorylation, controlling [IP3]i, IP3
binding to IP3Rs, and Ca2⫹ sensitivity of IP3Rs.
IP3R Communication with Ion Channels and Mitochondria
in SMCs
SMC IP3Rs communicate with several SR- and plasma
membrane-localized ion channels and mitochondria to control
intracellular signals and influence physiological functions.
leading to an increase in Ca2⫹ activation, which can augment
CICR (77). Thus, in contrast to IP3, which activates IP3Rs by
reducing Ca2⫹-mediated inhibition, ATP stimulates IP3Rs by
enhancing Ca2⫹-dependent activation (57). In summary, IP3,
Ca2⫹, and ATP modulate multiple regulatory processes to
control and fine tune IP3R activity in SMCs.
Protein kinases. PKA and PKG inhibit SMC IP3Rs via
phosphorylation, through regulation of IP3 binding, and by
controlling IP3 generation. Consensus sequences for PKA- and
PKG-mediated IP3R phosphorylation are located in the regulatory domain of SMC IP3Rs (Fig. 1A, red squares) (43, 100).
PKG phosphorylated SMC IP3Rs at serine 1755 and inhibited
IP3-induced Ca2⫹ release in aortic and gastric SMCs (100,
154). PKG phosphorylated IP3Rs via IP3R-associated cGMP
kinase substrate (IRAG) in gastric and tracheal SMCs (153,
154, 189, 226). PKG/IRAG-dependent IP3R phosphorylation
and subsequent inhibition of IP3R-mediated Ca2⫹ release relaxed colonic and aortic SMCs (46, 58). These studies suggest
that PKG/IRAG regulation of IP3R phosphorylation and Ca2⫹
release regulates SMC contractility. PKA activation reduced
PLC-dependent IP3 generation and decreased the number of
binding sites for IP3 on IP3Rs, thereby attenuating IP3Rmediated Ca2⫹ release in tracheal SMCs (120, 191). Twodimensional phosphopeptide mapping revealed that PKA phosphorylated SMC IP3Rs at serine 1589 (43). In iris sphincter
SMCs, PKA inhibited IP3R-mediated Ca2⫹ release, resulting in
relaxation (48, 204). It has been proposed that PKG is required for
PKA-mediated phosphorylation of IP3Rs in SMCs. In gastric
SMCs and intact aorta, PKA phosphorylated IP3Rs at two sites:
serine 1589, a PKA phosphorylation site, and serine 1755, a
PKG-specific site, suggesting that PKA activates PKG to phosphorylate IP3Rs (101, 154). Therefore, PKA-PKG cross talk may
contribute to PKA-mediated phosphorylation of SMC IP3Rs.
PKC and tyrosine kinases enhance SMC IP3R signaling
through IP3R phosphorylation and by elevating intracellular
[IP3] ([IP3]i). PKC and tyrosine kinase phosphorylation sites on
SMC IP3Rs are yet to be identified. PKC-mediated IP3R
phosphorylation elevated [Ca2⫹]i and contracted gallbladder
SMCs and A10 cells, an aortic SMC line (240, 248). In cultured
aortic SMCs, tyrosine kinase inhibitors reduced ANG II-induced
PLC-␥1 phosphorylation and elevations in [IP3]i and [Ca2⫹]i,
suggesting that tyrosine kinases stimulate IP3 generation in SMCs
(126). Similarly, tyrosine kinase inhibitors attenuated agonistinduced elevations in [IP3]i and [Ca2⫹]i and contraction in cultured gluteal and pulmonary artery SMCs but not in cultured
aortic SMCs (55, 214). Collectively, these studies indicate that
protein kinases regulate SMC IP3R-mediated Ca2⫹ signaling and
contraction directly, through phosphorylation and indirectly, by
controlling IP3 generation and IP3 binding.
Regulatory proteins. Evidence suggests that cytosolic proteins, including the 12-kDa FK506 binding protein (FKBP12)
and receptor for activated PKC 1 (RACK1) influence IP3R
channel activity in SMCs (15, 36, 118, 173). FKBP12, an
intracellular ligand for immunosuppressants FK506 and rapamycin, interacts with and regulates SMC IP3Rs (15, 118).
FKBP12 coimmunoprecipitated with IP3R1 in colonic SMCs,
suggesting physical interaction (118). FKBP12 modulated colonic SMC IP3R activity via three effector proteins: calcineurin, FK506, and mammalian target of rapamycin (mTOR) (15,
118). FKBP12 potentiated IP3R-mediated Ca2⫹ release
through mTOR and inhibited IP3R activity through calcineurin
Review
activation (253). Cerebral artery SMC TRPC6 channels also
contain a CIRB domain but do not physically couple to IP3R1
due to spatial separation of these proteins (2). IP3R1-induced
TRPC3 channel activation induced a cation current (ICat),
resulting in membrane depolarization, CaV1.2 channel activation, and vasoconstriction (Fig. 3C) (237). This SR-Ca2⫹
release-independent mechanism is a major contributor to agonist-induced IP3R-mediated global [Ca2⫹]i elevation and vasoconstriction in cerebral arteries (2, 237).
SMC IP3Rs also activate TRP channels through SR Ca2⫹
release-dependent mechanisms (61, 106, 206). ET-1-induced
IP3R stimulation activated Ca2⫹ influx that was inhibited by
TRPC1 knockdown in cultured aortic SMCs (206). IP3Rmediated Ca2⫹ release also activated Ca2⫹-sensitive PKC
isoforms, leading to PKC-dependent TRPC1 activation in portal vein SMCs (106). These studies suggested that IP3R stimulation activates TRPC1 channels via SR Ca2⫹ release, although it was unclear if physical coupling was also involved.
In cerebral artery SMCs, SERCA and IP3R inhibitors reduced
TRPM4-mediated native ICat (61). TRPM4 is Ca2⫹ sensitive
and does not contain a CIRB domain (2), indicating that
SR-released Ca2⫹ and not direct interaction with IP3Rs is
likely to regulate TRPM4 activation.
A reduction in SR Ca2⫹ load following IP3R-mediated Ca2⫹
release may feedback to stimulate SOCE, which exists in some
but not all contractile and migratory/proliferative SMC types (5,
88, 97, 159, 185, 237, 239). In addition to Orai and STIM
proteins, TRP channels may be a molecular component of SOCE
channels (73, 180, 229). TRPC channels, including 1, 5, 6, and 7
contribute to SOCE in SMCs of coronary and mesenteric arteries
and portal vein (186, 207). IP3R-mediated SR Ca2⫹ depletion
activated TRPC-dependent SOCE in SMCs of inferior vena cava,
portal vein, and pulmonary arteries (108, 114, 160, 180).
SMC TRP channels are also activated by IP3R-independent
mechanisms. TRPC channels, including 1, 3, 4, 5, 6, and 7
were activated by pathways that required PLC, phosphatidylinositol 4,5-bisphosphate, and/or DAG, but not IP3 or IP3Rs, in
SMCs of coronary, ear, and mesenteric arteries, portal vein,
and gastric antrum (5, 6, 93, 109, 110, 174, 185). Therefore, in
SMCs, TRP channels are activated by PLC-mediated IP3Rdependent and -independent pathways. IP3R-dependent pathways include physical coupling with TRPC channels, Ca2⫹
release-mediated activation, and SR Ca2⫹ store depletioninduced stimulation.
BKCA CHANNELS. SMC IP3Rs communicate with nearby
plasma membrane BKCa channels via SR Ca2⫹ release-dependent and -independent mechanisms (255) (Fig. 3D). IP3Rmediated SR Ca2⫹ release activated BKCa channels in basilar
artery SMCs (95). IP3R1 is located in close spatial proximity
to, and coimmunoprecipitates with, plasma membrane BKCa
channels in cerebral artery SMCs (255). SMC IP3R1 activation
elevated BKCa channel Ca2⫹ sensitivity, thereby facilitating
channel activation at lower Ca2⫹ concentrations in cerebral
artery SMCs (255). The IP3R-mediated elevation in BKCa
channel Ca2⫹ sensitivity would enhance channel activation by
SR Ca2⫹ release by local IP3Rs (255). IP3R-induced BKCa
channel activation would oppose TRPC3 channel-mediated
membrane depolarization in SMCs and attenuate the IP3induced global [Ca2⫹]i elevation and vasoconstriction (255).
MACROMOLECULAR COMPLEXES CONTAINING IP3RS. IP3Rs, TRPC3,
and BKCa channels appear to be located in macromolecular
Ryanodine-sensitive Ca2⫹ release channels. Ryanodine-sensitive Ca2⫹ release channels (RyRs) are tetrameric proteins that
share a number of structural and functional characteristics with
IP3Rs, including substantial amino acid sequence homology of
the pore region and carboxy terminus (59, 142, 247). Analytical cell fractionation studies revealed that RyRs and IP3Rs are
both SR membrane localized in SMCs (28, 56, 91). There is
uncertainty as to whether IP3Rs and RyRs release Ca2⫹ from
the same or distinct pools in SMCs. IP3Rs and RyRs have been
suggested to share the same Ca2⫹ pool in SMCs of mesenteric,
small pulmonary and renal arteries, portal vein, and small
intestine (86, 91, 168, 219, 256). In colonic SMCs, two SR
populations were identified: one expressing only RyRs and
another with both RyRs and IP3Rs (56). Reports have also
suggested that RyRs and IP3Rs do not share the same Ca2⫹
pool and that each channel can generate distinct Ca2⫹ signals
in SMCs of the ureter and mesenteric and large pulmonary
arteries (26, 32, 91, 219). Some of this discrepancy may have
arisen due to different experimental conditions used to obtain
data. Whether IP3Rs or RyRs release Ca2⫹ from the same or
distinct pools may also depend on anatomical origin and
physiological functions of the SMC types studied. Additional
data, including that obtained by using identical experimental
approaches in different SMC types, will be required to investigate these possibilities.
Ca2⫹ released from an IP3R or RyR activates the other
channel type via CICR (12, 25, 28, 62, 91, 232). IP3R activation stimulated RyR-mediated Ca2⫹ release in SMCs of gallbladder, gastric antrum, portal vein, and renal artery (12, 25,
28, 62, 91, 232). RyR inhibitors also attenuated norepinephrine- and phenylephrine-induced IP3R-dependent Ca2⫹ release in
portal vein and renal artery SMCs (28, 91). Therefore, IP3Rmediated Ca2⫹ release activates RyRs in SMCs and, in turn, RyRs
also feedback to regulate IP3R activity. However, ryanodine, a
RyR blocker, and anti-RyR channel antibodies did not alter
agonist-induced IP3R-mediated Ca2⫹ release in pulmonary artery
and ureteric SMCs (39, 91), suggesting that IP3R-RyR cross talk
may not exist in all SMC-containing tissues.
Plasma membrane-localized ion channels. Research over
the last decade has demonstrated that IP3Rs can regulate the
activity of plasma membrane TRPC and large-conductance
Ca2⫹-activated K⫹ (BKCa) channels through both SR Ca2⫹
release-dependent and -independent mechanisms in SMCs.
Evidence suggests that IP3R communication with these ion
channels is enabled by caveolae, which are plasma membrane
microdomains located between the SR and plasma membranes
in many cell types, including SMCs (Fig. 3, A and B).
TRPC CHANNELS. IP3R activation stimulates TRPC channels
directly, through physical coupling and indirectly, through
Ca2⫹-dependent activation and induction of store-operated
Ca2⫹ entry (SOCE). Endothelin (ET)-1 and uridine 5=-triphosphate, PLC-coupled receptor agonists, and IP3 stimulated physical coupling between the IP3R1 NH2-terminal TRPC coupling
domain and the TRPC3 channel COOH-terminal CaM and
IP3R binding (CIRB) domain in cerebral artery SMCs (Fig.
3C) (1, 2, 253). In mammals, the TRPC coupling domain
sequence (L⫺⫺⫺⫺⫺⫺EEVWL⫺W⫺D) and the CIRB domain sequence (Y⫺⫺⫺MK⫺LV⫺RYV) are conserved
among the three IP3R isoforms and seven TRPC channels,
respectively (208). The IP3R coupling domain displaces inhibitory CaM from the TRPC CIRB domain, leading to channel
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complexes that span from the SR membrane to plasma membrane
caveolae in SMCs. Caveolae disruption, knockdown of caveolin-1 (cav-1), a caveolae scaffolding protein, and a cav-1
scaffolding domain peptide spatially separated IP3R1 and
TRPC3 channels and attenuated IP3-induced ICat activation in
cerebral artery SMCs (1, 47, 83). Coimmunoprecipitation and
immunofluorescence studies indicated that BKCa channels associate with cav-1 in aortic and cerebral artery SMCs (7, 255).
Thus cav-1 likely maintains close spatial proximity of SR
IP3R1 channels with both TRPC3 and BKCa channels in SMCs
(Fig. 3, C and D) (1, 7, 255). Caveolae may permit localized
coupling of IP3R1 to both TRPC3 and BKCa channels to
modulate SMC membrane potential and arterial contractility.
Fig. 3. IP3Rs communicate with plasma membrane ion channels in SMCs. A: transmission
electron microscopic image of a portal anterior
mesenteric vein section illustrating close spatial
proximity between junctional SR and plasma
membrane. Electron-opaque material (small arrows) traverses the gap of ⬃20 –25 nm between
the 2 opposing membranes. B: caveolae, termed
“surface vesicles” (SV; small arrows) in the
original publication, can be found where the SR
and plasma membrane (arrowheads) are in
close spatial proximity. A mitochondrion (M) is
also located nearby both the SR and plasma
membranes (arrowheads). Scale bar ⫽ 100 nm.
A and B: reproduced from Devine et al. (47) by
copyright permission (2011) of the Rockefeller
University Press. C: an elevation in intracellular
IP3 concentration ([IP3]i) elevation induces
physical coupling of IP3R1 to transient receptor
potential canonical 3 (TRPC3) channels in arterial SMCs, leading to vasoconstriction.
D: IP3R1 activation elevates the Ca2⫹ sensitivity of nearby large-conductance Ca2⫹-activated
K⫹ (BKCa) channels, thereby amplifying stimulation by released Ca2⫹ to oppose IP3-induced
SMC contraction. DAG, diacylglycerol.
Therefore, IP3Rs, TRPC3, and BKCa channels appear to be
located within the same cav-1-containing macromolecular
complex that bridges the SR and plasma membrane in arterial
SMCs (1, 255). RyRs generate Ca2⫹ sparks that activate
nearby plasma membrane BKCa channels in SMCs, inducing
relaxation (90, 159). Given that SMC RyR channels can be
located immediately (⬃20 nm) beneath the plasma membrane
(90), IP3Rs and RyRs may also be contained within the same
macromolecular complexes and communicate locally to each
other and nearby plasma membrane ion channels. Conceivably,
SMC RyRs may communicate with TRP and BKCa channels,
thereby regulating membrane potential and contractility. Future research should be directed at exploring cellular localiza-
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H2197
Ca2⫹ (175, 187). In SMCs, physiological global [Ca2⫹]i, which
is ⬃100 –300 nM, should not modify [Ca2⫹]mito (175, 187), a
conclusion supported by published data (156). Local high
[Ca2⫹]i elevations generated by nearby Ca2⫹ channels are
necessary to elevate SMC [Ca2⫹]mito (35, 175). Receptor
agonists that elevate IP3 and activate IP3Rs increase [Ca2⫹]mito
to micromolar concentrations in permeabilized A10 cells and
in cultured preglomerular afferent arteriole, cultured and noncultured aortic, pulmonary artery, and colonic SMCs (50, 66,
137, 146, 169, 176). SMC [Ca2⫹]mito measurements in these
studies were obtained primarily by using inorganic fluorescent
indicators. Recent evidence (156) obtained using a genetically
encoded mitochondria-targeting Ca2⫹ indicator (2mt8CG2)
indicated that IP3R-mediated Ca2⫹ waves, but not global
[Ca2⫹]i, elevated [Ca2⫹]mito and stimulated mitochondria-derived ROS generation in cerebral artery SMCs (Fig. 4B). This
study provided evidence that in arterial SMCs, Ca2⫹ waves
may be the IP3R-mediated Ca2⫹ signal that communicates with
Fig. 4. IP3Rs interact locally with mitochondria. A: electron microscopy image illustrating
multiple mitochondria ensheathed by the SR
network in a tracheal SMC. Scale bar ⫽ 200
nm. Reproduced from Dai et al. (41) by copyright permission (2011) from Elsevier. B: signaling pathways by which IP3Rs regulate mitochondrial Ca2⫹ concentration ([Ca2⫹]mito),
mitochondrial reactive oxygen species (ROS)
generation, NF-␬B activity, and CaV1.2 channel gene expression in arterial SMCs. IP3Rmediated SR Ca2⫹ release activates NF-␬B
both by elevating mitochondrial ROS and via a
secondary mitochondria-independent pathway,
leading to CaV1.2 gene expression. ETC, mitochondrial electron transport chain.
tion of RyRs and local signaling to IP3Rs and plasma membrane ion channels in SMCs.
Mitochondria. Spatial localization of mitochondria nearby
the SR permits local signaling between these two organelles.
Given their local proximity, SR Ca2⫹ release can alter local
mitochondrial activity and mitochondria can feedback to regulate nearby SR Ca2⫹ channels.
IP3R REGULATION OF MITOCHONDRIAL CA2⫹ CONCENTRATION. In
airway and vascular SMCs, mitochondria are located near the
SR membrane and IP3R activation elevates mitochondrial
Ca2⫹ concentration ([Ca2⫹]mito) (Fig. 3B and 4A) (41, 50, 66,
137, 146, 162, 169, 176, 203). In tracheal SMCs, ⬃99% of
mitochondria were within 30 nm of the SR membrane and
⬃82% of mitochondria were ensheathed by the SR (Fig. 4A)
(41). Such close spatial proximity between mitochondria and
SR enables local Ca2⫹ signaling between these two organelles
in SMCs (41). The mitochondrial Ca2⫹ uniporter, the major
mitochondrial Ca2⫹ uptake pathway, is sensitive to micromolar
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H2198
nearby mitochondria to control [Ca2⫹]mito and mitochondrial
ROS generation. In A10 cells and cultured aortic SMCs,
agonist-induced [Ca2⫹]mito elevations measured using targeted
aequorin closely matched the kinetics of [Ca2⫹]i transients
(175, 203). In contrast, in noncultured aortic and pulmonary
artery SMCs, agonist- and IP3-induced [Ca2⫹]mito elevations
outlasted [Ca2⫹]i transients by minutes (50, 66). These data
suggest that changes in [Ca2⫹]i and [Ca2⫹]mito are not always
synchronous and that mitochondria may retain Ca2⫹ in SMCs.
The variability in results may also reflect experimental differences in the mitochondrial Ca2⫹ indicator used, protocol, or
effects of cell culture.
MITOCHONDRIAL REGULATION OF IP3R-MEDIATED CA2⫹ RELEASE.
IP3Rs and Intracellular Ca2⫹ Signals in SMCs
IP3R activation generates a wide variety of Ca2⫹ signals in
SMCs, including flashes, puffs, oscillations, ripples, sparks,
waves, and global [Ca2⫹]i (13, 26, 90, 144, 254). Other Ca2⫹
channels, including nonselective cation, RyR, TRP, SOCE, and
voltage-dependent Ca2⫹ channels, also contribute to these events
in certain SMC types. The following section will discuss IP3Rdependent Ca2⫹ signals and the modulation of these Ca2⫹ signals
by other Ca2⫹ channels in SMCs. Properties, cell types, contributing Ca2⫹ channels, and physiological functions of IP3R-regulated Ca2⫹ signals in SMCs are summarized in Table 2.
Ca2⫹ puffs. Ca2⫹ puffs arise from the synchronous opening
of ⬃30 IP3R channels distributed within a ⬃400-nm diameter
cluster in Xenopus oocytes (170, 194). Ca2⫹ puffs correspond
to a Ca2⫹ current of 11–23 pA, with a Ca2⫹ current of ⬃0.4 pA
per IP3R (170, 194). In SMCs, Ca2⫹ puffs occur due to IP3Rmediated SR Ca2⫹ release, with a minor contribution from
Mitochondria regulate SMC IP3Rs through multiple mechanisms, including buffering local and global [Ca2⫹]i, controlling
ATP synthesis and ROS signaling, and by modulating other ion
channels that control membrane potential and [Ca2⫹]i (31, 35,
57, 105, 134, 137, 164, 175, 176, 201–203). Inhibition of
mitochondrial Ca2⫹ uptake and oxidative metabolism attenuated IP3R-mediated Ca2⫹ transients in colonic, gallbladder,
cultured aortic, and tail artery SMCs (11, 137, 164, 202, 203).
These data suggested that mitochondria directly modulate IP3R
activity and, in turn, influence generation and propagation of
Ca2⫹ signals in SMCs (137, 203). It has been proposed that in
SMCs, mitochondrial Ca2⫹ uptake may decrease [Ca2⫹]i
nearby IP3Rs, resulting in a reduction in Ca2⫹-dependent
inhibition, thereby stimulating further Ca2⫹ release (137, 203).
ATP enhances IP3R IP3 and Ca2⫹ sensitivity and is required
for SERCA activity in SMCs (57, 105, 134). Thus mitochondria may control SMC IP3R activity and Ca2⫹ release indirectly through ATP generation. Mitochondrial ROS and redox
potential may also modulate SMC IP3R expression and activity, thereby influencing IP3R-mediated Ca2⫹ release (31, 201).
In vascular SMCs, mitochondria regulate the activity of several
plasma membrane ion channels, including voltage-dependent
Ca2⫹, Ca2⫹-activated K⫹ (KCa), Ca2⫹-activated Cl⫺ (ClCa),
and SOCE channels (175). Mitochondrial modulation of these
channels will influence membrane potential and also feedback
to alter local and global Ca2⫹ signals, thereby indirectly
influencing SMC IP3R channel activity. Therefore, mitochondrial regulation of IP3Rs in SMCs involves several integrated
signaling pathways.
RyRs. In colonic SMCs, purinergic receptor stimulation elevated Ca2⫹ puff frequency and amplitude, which was attenuated by both xestospongin C (XeC), an IP3R blocker, and
ryanodine, indicating involvement of both IP3Rs and RyRs
(14). However, ryanodine and an anti-RyR antibody did not
alter spontaneous and ACh-induced Ca2⫹ puffs in ureteric
SMCs, suggesting that IP3R activation alone can also generate
these Ca2⫹ transients (26). Similarly, in guinea pig colonic
SMCs, localized photolysis of IP3 generated Ca2⫹ puffs, which
were abolished by 2-aminoethoxydiphenyl borate, an IP3R
inhibitor (164). Thus Ca2⫹ puffs are stimulated by IP3R activation, with RyRs contributing in certain SMC types. Clustering of IP3Rs on the SR membrane has been proposed to be
essential for Ca2⫹ puff generation in SMCs (26, 62, 91, 164).
Consistent with this conclusion, IP3R clustering and Ca2⫹
puffs were observed in colonic and ureteric SMCs but neither
were detected in portal vein and pulmonary artery SMCs (26,
62, 91, 164). The physiological functions of Ca2⫹ puffs in
SMCs have not been identified.
Ca2⫹ flashes. Spontaneous, rapid [Ca2⫹]i events termed
“Ca2⫹ flashes” were observed during rhythmic phasic contractions of unstimulated gallbladder SMCs (13). Ca2⫹ flash frequency was reduced by inhibiting voltage-dependent Ca2⫹
channels, IP3Rs, and RyRs, suggesting that all these channels
contribute to these Ca2⫹ signals in SMCs (13). In contrast, in
mesenteric artery and urinary bladder SMCs, Ca2⫹ flashes
induced by electrical field stimulation were unaltered by depleting SR Ca2⫹, indicating that IP3Rs and RyRs do not
generate these signals in certain SMC types (104, 157). Ca2⫹
flashes also occurred in ⬃2% of resting tail artery SMCs but
such low occurrence prevented detailed study of the contribution of IP3Rs to these events (9).
Ca2⫹ oscillations. Ca2⫹ oscillations are repetitive, nonpropagating global [Ca2⫹]i elevations generated by periodic,
pulsatile release of SR Ca2⫹ in SMCs (10, 18, 22, 29, 111).
Ca2⫹ oscillations occur due to cyclical positive and negative
feedback of [Ca2⫹]i on IP3R channel activity with contributions from RyRs and TRPC channels (10, 18, 29, 111, 190). In
isolated retinal arteriole SMCs, ET-1 increased Ca2⫹ oscillation frequency and this elevation was inhibited by blockers of
IP3Rs and RyRs, indicating that IP3R-RyR cross talk stimulates
Ca2⫹ oscillations (190, 218). Ca2⫹ oscillations in A7r5 cells
required both IP3R1-mediated SR Ca2⫹ release and TRPC6 as
a receptor-operated Ca2⫹ influx pathway (111). In airway
SMCs, IP3R inhibition reduced Ca2⫹ oscillation frequency,
leading to relaxation (10, 18). Similarly, in basilar artery
SMCs, IP3R-mediated Ca2⫹ oscillations activated ClCa channels, leading to depolarization, Ca2⫹ influx, and vasoconstriction (67). These studies suggested that IP3R-dependent Ca2⫹
oscillations induce SMC contraction. In contrast, agonist-induced IP3R-mediated Ca2⫹ oscillations in mesenteric artery
SMCs did not contribute Ca2⫹ for vasoconstriction (144). Thus
the physiological functions of Ca2⫹ oscillations in some SMC
types remain poorly understood.
Ca2⫹ ripples. Ca2⫹ ripples are spontaneous, low amplitude,
propagating, IP3R-mediated Ca2⫹ signals that have been observed in unstimulated aortic, mesenteric, and tail artery SMCs
(9, 249). Ca2⫹ ripples occur due to PLC activation and SR
Ca2⫹ release that may be due to IP3Rs in tail artery SMCs (9).
Future studies would need to be performed using alternative
approaches, including selective IP3R antagonists and IP3R
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H2199
Table 2. SMC Ca2⫹ signals regulated directly or indirectly by IP3Rs
Ca2⫹ Signal
2⫹
Properties
SMC Type
Additional Ca2⫹ Channels
Involved
Physiological Function
0.02–0.04 Hz/cell
1.13–2.02
57–160 ms
107–250 ms
1.89–2.51 ␮m
Colon (14, 164), renal artery (91),
ureter (26)
RyR (14, 91, 164)
Unclear
Ca2⫹ flash
Frequency:
Amplitude*:
Rise time:
t1/2 decay:
0.2–0.5 Hz/cell
1.5–2.6
0.2–0.8 s
0.5–1.5 s
Gallbladder (13)
RyR, voltage-dependent
Ca2⫹ (13)
Contraction (13)
Ca2⫹ oscillation
Frequency:
Amplitude*:
Rise time:
t1/2 decay:
0.07–0.4 Hz/cell
1.1–6.5
2–6 s
3–18 s
A7r5 cells (111), airway (10, 18), basilar
artery (67), mesenteric artery (144),
retinal arteriole (190, 218)
RyR (218), TRPC6 (111)
Contraction (10, 18, 67)
Ca2⫹ ripple
Frequency:
Amplitude*:
Rise time:
t1/2 decay:
Velocity†:
0.05–0.19 Hz/cell
1.05-1.8
1–2 s
4–6 s
⬃18 ␮m/s
Aorta (9), mesenteric artery (249), tail
artery (9)
Ca2⫹ spark
Frequency:
Amplitude*:
Rise time:
t1/2 decay:
Spatial spread:
0.07–0.59 Hz/cell
1.34–2.05
10–65 ms
30–150 ms
0.52–4.7 ␮m
Choroidal and retinal arterioles (190,
218), pulmonary artery (252), trachea
(116), vas deferens (233)
RyR (116, 190, 218, 233,
252)
Contraction (190, 218)
Ca2⫹ wave
Frequency:
Amplitude*:
Rise time:
t1/2 decay:
Spatial spread:
Velocity†:
0.04–1.41 Hz/cell
1.4–6.9
0.5–2 s
2–10 s
8.8–50 ␮m
7–126 ␮m/s
Cerebral artery (60, 61, 88, 89, 151, 156,
234, 254), choroidal and retinal
arteriole (190), cremaster artery and
arteriole (177, 231), mesenteric artery
(86, 104, 133, 249), pulmonary artery
(76, 219), tail artery (49, 81), airway
(17), cecum (70, 82), colon (15, 70,
117, 135, 136), duodenum (28),
gallbladder (12), ileum (167), inferior
vena cava (42, 107, 108, 184), portal
vein (25, 28, 62, 145), trachea (94,
103, 179), ureter (26), urinary bladder
(157)
Na⫹/Ca2⫹ exchanger
(107, 108),
nonselective cation
(42), RyR (12, 17, 25,
28, 62, 70, 81, 88, 94,
145, 151, 179, 184,
231), SOCE (107,
108), voltagedependent Ca2⫹ (12,
42, 60, 70, 88, 107,
108, 179)
Contraction (17, 28, 42,
49, 70, 76, 103, 104,
107, 108, 133, 151,
184, 231, 249), gene
expression (60),
mitochondrial ROS
generation (156),
proliferation (234),
relaxation (15)
Global [Ca2⫹]i
Amplitude:
⬃30–685 nM
Aorta (206, 220), cerebral artery (2, 60,
61, 67, 71, 88, 89, 151, 156, 237),
choroidal arteriole (190), cremaster
artery and arteriole (177, 231),
femoral artery (99), mesenteric artery
(86, 104, 133, 249, 250), pulmonary
artery (27, 76, 87, 91, 98, 160, 180,
219, 235), renal artery (91), retinal
arteriole (190, 218), tail artery (9, 49,
81), airway (17, 115, 128), cecum (70,
82), colon (15, 70, 102, 117, 135, 136,
164), ileum (167), iliac lymph vessels
(121), inferior vena cava (42, 107,
108, 184), portal vein (25, 28, 62,
114, 145), prostate (54), small
intestine (96, 122), trachea (94, 103),
ureter (26), vas deferens (233)
Nonselective cation (15,
42), RyR (17, 27, 28,
54, 67, 70, 71, 81,
86–88, 91, 94, 96,
115, 145, 151, 184,
190, 218, 219, 231,
235), SOCE (98, 107,
108, 114, 160, 177,
180, 220, 250),
TRPC1 (206), TRPC3
(2, 237), TRPM4 (61),
voltage-dependent
Ca2⫹ (2, 42, 60, 61,
67, 70, 71, 76, 88, 96,
107, 108, 237)
Contraction (2, 9, 15,
17, 27, 28, 42, 49,
54, 67, 70, 71, 76,
86, 87, 96, 99, 102104, 107, 108, 115,
121, 122, 128, 133,
151, 160, 167, 177,
180, 184, 190, 218,
231, 235, 237, 249,
250, 254), gene
expression (60)
puff
Contraction (9, 249)
ROS, reactive oxygen species; RyR, ryanodine-sensitive Ca2⫹ release channels; SOCE, store-operated Ca2⫹ entry; TRPC, transient receptor potential
canonical. *F/F0. †Propagation velocity.
knockdown, to confirm that Ca2⫹ ripples occur due to IP3Rmediated Ca2⫹ release in SMCs. In SMCs of pressurized
mesenteric arteries, Ca2⫹ ripples were observed only after the
development of myogenic tone (249). Similarly, ANG II receptor antagonists abolished Ca2⫹ ripples and reduced myogenic tone in tail arteries (9). It was concluded that ANG II
produced locally within the arterial wall stimulates Ca2⫹ rip-
ples, which regulate myogenic tone (9). Therefore, IP3Rmediated Ca2⫹ ripples may be a Ca2⫹ signal by which the local
renin-angiotensin system in the arterial wall contributes to
myogenic constriction (9, 249).
Ca2⫹ sparks. Ca2⫹ sparks are localized, intracellular Ca2⫹
transients that occur due to the concerted opening of several
spatially localized RyRs (37, 90, 159). RyR-mediated Ca2⫹
Frequency:
Amplitude*:
Rise time:
t1/2 decay:
Spatial spread:
Ca
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and arterioles, suggesting that IP3Rs contribute to these signals
in these SMC types (12, 15, 62, 70, 219, 231, 249). Basal
release of receptor ligands by endothelial cells and circulating
receptor agonists may also stimulate Gq/PLC-mediated IP3
generation in SMCs, leading to spontaneous IP3R-mediated
Ca2⫹ waves (61, 156). However, in cerebral artery SMCs,
neither 50% knockdown of IP3R1 or XeC altered baseline
Ca2⫹ wave frequency, suggesting that IP3Rs do not contribute
to the generation of spontaneous Ca2⫹ waves in all SMC types
(254). Thus the contribution of IP3Rs to spontaneous Ca2⫹
waves that occur in the complete absence of agonists in
vascular SMCs is unclear. Ligands that bind to PLC-coupled
receptors enhanced Ca2⫹ wave generation in several SMC
types, including cerebral, mesenteric, pulmonary, and tail arteries, cecum, colon, duodenum, gallbladder, ileum, trachea,
inferior vena cava, portal vein, and ureter (12, 26, 28, 42, 49,
76, 82, 103, 104, 135, 167, 254). IP3R inhibitors, anti-IP3R
antibodies, and IP3R knockdown attenuated agonist-induced
Ca2⫹ waves, indicating that IP3R activation is essential for
generation of these Ca2⫹ signals (17, 25, 26, 28, 42, 60, 62, 70,
76, 94, 104, 156, 157, 254). Thapsigargin, ryanodine, and
anti-RyR antibodies also inhibited agonist-induced Ca2⫹ waves in
certain SMCs, suggesting that RyRs may also contribute to
these events (17, 25, 27, 28, 62, 70, 81, 94, 104, 151, 179, 184).
Therefore, IP3Rs and RyRs can both influence the generation
of spontaneous and agonist-induced Ca2⫹ waves, with relative
involvement of each appearing to vary in different SMC types.
The contribution of IP3Rs and RyRs to the propagation of
Ca2⫹ waves has also been examined in SMCs (28, 62, 135,
184, 231). In colonic SMCs, carbachol and IP3 photolysis
stimulated local SR Ca2⫹ release, which transformed into a
propagating Ca2⫹ wave only in the presence of cytosolic Ca2⫹
Fig. 5. IP3R-mediated Ca2⫹ waves in SMCs of different tissues. Images illustrate Ca2⫹ waves induced by endothelin-1 (ET-1) in cerebral artery SMCs (A),
spontaneously in tail artery SMCs (B), by IP3 photolysis in a colonic SMC (C), and spontaneously in gallbladder SMCs (D). Traces corresponding to changes
in [Ca2⫹]i over time as determined in regions highlighted by boxes or regions in confocal images are also shown. White boxes in A indicate regions where Ca2⫹
sparks occurred (not shown). Reproduced with copyright permission (2012): Narayanan et al. (156) from Wolters Kluwer Health (A), Dreja et al. (49) from John
Wiley and Sons (B), McCarron et al. (136) from the American Society for Biochemistry and Molecular Biology (C), and Balemba et al. (12) from the American
Physiological Society (D).
sparks have been described in SMCs of a wide variety of
tissues, including arteries, arterioles, veins, gallbladder, gastric
antrum, intestine, trachea, ureter, and urinary bladder (72, 90,
178, 181, 230). IP3Rs have been reported to contribute to Ca2⫹
sparks, albeit in a small number of SMC types (116, 190, 218,
252). RyR- and IP3R-mediated SR Ca2⫹ release both stimulated Ca2⫹ sparks in SMCs of choroidal and retinal arterioles,
pulmonary artery, and trachea (116, 190, 218, 252). In contrast,
IP3R inhibition elevated Ca2⫹ spark frequency in vas deferens
SMCs, although this may have occurred due to an [Ca2⫹]SR
elevation (233). Reports (190, 218) in retinal and choroidal
arteriole SMCs suggested that IP3R-mediated Ca2⫹ sparks may
contribute to global [Ca2⫹]i and contractility. However, whether
localized Ca2⫹ sparks directly influence SMC global [Ca2⫹]i is
uncertain.
Ca2⫹ waves. Ca2⫹ waves are propagating elevations in
[Ca2⫹]i that occur due to SR Ca2⫹ release in vascular and
nonvascular SMCs (12, 17, 28, 62, 70, 76, 89, 104, 135, 156,
167). The activation of IP3Rs, RyRs, or both channels can
control both the generation and propagation of spontaneous
and agonist-induced Ca2⫹ waves in SMCs. Figure 5 illustrates
IP3R-mediated Ca2⫹ waves imaged in SMCs of vascular and
nonvascular tissues.
Spontaneous Ca2⫹ wave generation was inhibited by ryanodine in SMCs of cecum, colon, gallbladder, cerebral, cremaster and pulmonary arteries, and portal vein, suggesting that
RyRs contributed to these events (12, 62, 70, 88, 151, 219,
231). Caffeine stimulated Ca2⫹ waves, supporting the view
that RyR activation alone can generate Ca2⫹ waves in SMCs
(70, 71, 145, 184). In contrast, IP3R blockers reduced spontaneous Ca2⫹ waves in SMCs of cecum, colon, gallbladder,
portal vein, pulmonary, mesenteric, and cremaster feed arteries
Review
have also suggested that IP3Rs do not regulate global [Ca2⫹]i
in certain SMC types (88, 144, 157, 254). In mesenteric,
cerebral artery, and urinary bladder SMCs, IP3R-mediated
Ca2⫹ waves and oscillations did not elevate global [Ca2⫹]i.
Tissue-specific variability in IP3R modulation of global
[Ca2⫹]i could be due to multiple factors, including differences
in the amplitude of Ca2⫹ signals generated, activation and
contribution of other Ca2⫹ channels, and regulation of IP3R
activity by tissue-specific proteins. In summary, IP3R activation, sometimes with the involvement of other Ca2⫹ channels,
produces a wide variety of intracellular Ca2⫹ signals, which
control several physiological functions in SMCs.
IP3R Regulation of SMC Physiological Functions
IP3Rs regulate SMC physiological functions, including contractility, gene expression, migration, and proliferation (Table
2). IP3R control of these functions can occur via the modulation of intracellular Ca2⫹ signals and via local communication
with ion channels and organelles.
Contractility. IP3R-mediated SR Ca2⫹ release stimulates
contraction in both vascular and nonvascular SMCs. In gallbladder SMCs, IP3R activation contributed to Ca2⫹ flashes and
action potentials that preceded rhythmic contractions of individual smooth muscle bundles (13). IP3R-mediated Ca2⫹ oscillations induced airway and basilar artery contraction (10, 18,
67). Ca2⫹ sparks generated by IP3R and RyR activation contributed to vasoconstriction in retinal and choroidal arterioles
(190, 218). Reports in mesenteric, pulmonary, and tail arteries,
inferior vena cava, and portal vein proposed that IP3R-mediated Ca2⫹ waves directly induce vasoconstriction (28, 42, 49,
76, 86, 104, 107, 108, 133, 184, 249). XeC inhibited agonistinduced contraction in iliac lymph vessels, pulmonary arteries,
and small femoral artery branches, indicating that agonists
induce vasoconstriction through IP3R activation (87, 99, 121,
128, 235). Similarly, in nonvascular SMCs of ileum, prostate,
small intestine, and trachea, IP3R blockers inhibited spontaneous and agonist-induced contraction (54, 103, 122, 167). In
airway and intestinal SMCs, membrane depolarization activated IP3Rs and RyRs, which contributed to contraction (17,
70, 96, 115). Collectively, IP3R-mediated Ca2⫹ release regulates spontaneous and agonist-induced contraction of vascular
and nonvascular SMCs. IP3R activation and the resulting SR
Ca2⫹ store depletion also stimulated SOCE channel-dependent
Ca2⫹ influx, which induced contraction of SMCs of aorta,
inferior vena cava, cremaster, and pulmonary arteries (107,
108, 160, 177, 220, 250). IP3R activation has also been reported to induce relaxation of certain SMC types (15, 102). In
colonic SMCs, IP3R-mediated Ca2⫹ release stimulated KCa
channels, resulting in spontaneous transient outward currents,
hyperpolarization, and relaxation (15, 102). However, in urinary bladder SMCs, electrical field stimulation enhanced Ca2⫹
waves, which did not induce contraction (157), suggesting that
IP3Rs do not control contractility in certain SMC types.
IP3R regulation of pressure-induced myogenic vasoconstriction has been examined in several vascular beds. U-73122, a
PLC inhibitor, dilated pressurized cerebral and ophthalmic
arteries, suggesting that DAG/PKC and/or IP3/IP3Rs contribute
to myogenic tone (85, 92, 166, 237). Inhibitors of PLC or
IP3Rs attenuated Ca2⫹ waves and dilated pressurized cremaster
muscle feed arteries and arterioles, suggesting that IP3R-de-
and a steady [IP3]i (135). This study indicated that IP3R
activation alone is sufficient for propagation of these Ca2⫹
signals in SMCs. An alternative concept of Ca2⫹ wave progression suggests that IP3R-mediated Ca2⫹ release initiates a
Ca2⫹ wave in SMCs, which then propagates exclusively due to
RyR-dependent CICR (28, 62, 184). RyR contribution to Ca2⫹
wave propagation has been examined primarily by using ryanodine to inhibit these channels (28, 62, 184). However, ryanodine may inhibit Ca2⫹ wave propagation by depleting SR
Ca2⫹ and not solely by blocking RyRs (81, 86, 117, 136, 151).
In contrast, tetracaine, which blocks RyRs without depleting
SR Ca2⫹, attenuated spontaneous Ca2⫹ waves in SMCs of
cremaster feed arteries, suggesting that RyRs can regulate
Ca2⫹ wave propagation in certain SMC types (231). Studies
have proposed that Ca2⫹ influx via membrane proteins, including the Na⫹/Ca2⫹ exchanger, nonselective cation channels,
SOCE channels, and voltage-dependent Ca2⫹ channels, also
contributes to Ca2⫹ wave propagation and maintenance in
certain SMC types (12, 42, 60, 70, 88, 107, 108, 179).
In summary, IP3Rs contribute to Ca2⫹ wave generation and
propagation in SMCs. Evidence, obtained in many cases by
using nonspecific pharmacological tools, also suggests that
RyRs and other Ca2⫹ channels regulate Ca2⫹ wave propagation in some SMC types.
Global [Ca2⫹]i. Global [Ca2⫹]i is spatially averaged cytosolic [Ca2⫹]i. An elevation in global [Ca2⫹]i induces contraction, whereas a reduction in global [Ca2⫹]i results in relaxation
(22, 29, 90). IP3R activation elevates SMC global [Ca2⫹]i
directly, through SR Ca2⫹ release and indirectly, by stimulating Ca2⫹ influx via plasma membrane-localized ion channels.
IP3R-mediated SR Ca2⫹ release elevates global [Ca2⫹]i in a
wide variety of different SMC types (Table 2). IP3R activation
stimulated plasma membrane-localized TRPC1, TRPM4, and
voltage-dependent Ca2⫹ channels, leading to Ca2⫹ influx and
global [Ca2⫹]i elevation in basilar, cerebral artery, and aortic
SMCs (61, 67, 206). In cerebral artery SMCs, physical coupling of IP3R1 to TRPC3 channels results in membrane depolarization and an indirect elevation in global [Ca2⫹]i via voltage-dependent Ca2⫹ channel activation (2). In SMCs of aorta,
mesenteric, and pulmonary arteries, cremaster arterioles, inferior vena cava, and portal vein, IP3R-mediated SR Ca2⫹
depletion activated SOCE, leading to a global [Ca2⫹]i elevation (98, 107, 108, 114, 160, 177, 180, 220, 250). Collectively,
these studies indicate that IP3R activation can elevate global
[Ca2⫹]i directly, through SR Ca2⫹ release and indirectly,
through stimulation of plasma membrane ion channel-dependent Ca2⫹ influx in SMCs.
The relative contribution of IP3R-mediated SR Ca2⫹ releasedependent and -independent mechanisms to global [Ca2⫹]i
elevations was examined in cerebral artery SMCs (237). SR
Ca2⫹ depletion reduced IP3-induced global [Ca2⫹]i elevation
by ⬃25%, suggesting that the contribution of IP3R-mediated
SR Ca2⫹ release to global [Ca2⫹]i is minor (237). TRPC3
knockdown reduced IP3-induced global [Ca2⫹]i elevation by
⬃70%, indicating that IP3R physical coupling to TRPC3 channels and the resulting plasma membrane Ca2⫹ influx are
responsible for the majority of the global [Ca2⫹]i elevation
(237). Therefore, IP3R-mediated Ca2⫹ release can make a minor
direct contribution to global [Ca2⫹]i, with IP3R control of membrane potential and Ca2⫹ influx being prominent indirect
mechanisms by which IP3Rs influence global [Ca2⫹]i. Reports
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Review
H2202
and upregulation of all three IP3R isoforms in mesenteric artery
SMCs (3, 20). Consistent with these observations, Ca2⫹ wave
frequency was elevated in proliferating cerebral artery SMCs
(234). PLC and IP3R antagonists attenuated Ca2⫹ waves and
proliferation in cerebral artery SMCs, indicating that IP3Rmediated Ca2⫹ release stimulates proliferation (234). Similarly, in cultured aortic SMCs, IP3R-dependent intercellular
Ca2⫹ waves promoted proliferation and migration (149). An
elevation in IP3R-mediated Ca2⫹ release also stimulated proliferation of cultured preglomerular microvascular SMCs (36). XeC
inhibited pulsatile pressure-induced aortic SMC migration, and
IP3R1 knockdown reduced A7r5 cell proliferation (205, 228).
Taken together, these findings provide strong evidence that IP3R
activation stimulates SMC migration and proliferation.
IP3Rs and SMC Pathologies
Vascular SMC diseases. Vascular SMC IP3R expression,
[IP3]i, and Ca2⫹ signaling are altered in several diseases,
including hypertension, atherosclerosis, and diabetes-related
vascular complications (19, 125, 130, 138, 192, 193, 236).
Genetic hypertension in rats is associated with elevations in both
[IP3]i and IP3R IP3-binding affinity in SMCs. Basal and phenylephrine-induced [IP3]i were both higher in cultured aortic SMCs
of spontaneously hypertensive rats than Wistar-Kyoto controls
(236). A [3H]IP3 binding assay indicated that the IP3-binding
capacity of IP3Rs was significantly higher in aortic SMCs of
spontaneously hypertensive rats compared with Wistar-Kyoto rats
(19). Although myogenic tone and global [Ca2⫹]i are higher in
vascular SMCs of hypertensive rats, compared with normotensive
controls (92), altered vascular SMC IP3R expression and Ca2⫹
signaling have not been reported.
During the pathogenesis of atherosclerosis, vascular SMCs
are exposed to oxidized LDL, which stimulates plaque formation (130). In aortic SMCs exposed to oxidized LDL and in
atherosclerotic aorta, IP3R1 protein and IP3R-dependent Ca2⫹
release were reduced (125, 130). SERCA2b expression was
also downregulated in atherosclerotic aorta, suggesting that an
[Ca2⫹]SR reduction may also contribute to the impaired IP3Rmediated Ca2⫹ release in atherosclerosis (125, 130).
Diabetes-related vascular complications are associated with
a reduction in IP3R expression and Ca2⫹ release in SMCs of
aorta and glomerular arterioles (138, 192, 193). In renal glomerular arteriolar SMCs of diabetic rats, IP3R protein was
lower than in nondiabetic controls (138). Protein expression of
all three IP3R isoforms and IP3R-mediated [Ca2⫹]i signals
were attenuated in aortic SMCs of genetic and inducible animal
models of diabetes (192). High glucose reduced IP3R protein in
A7r5 cells, suggesting that a diabetes-induced reduction in
vascular SMC IP3R levels may be due to direct effects of
hyperglycemia (192). IP3R1 protein and IP3R-mediated [Ca2⫹]i elevations were reduced, and TGF-␤2 levels were elevated in
diabetic rat aorta (193). Intraperitoneal administration of an
anti-TGF-␤ antibody partly restored IP3R1 expression and
IP3R-mediated [Ca2⫹]i elevations in aortic and glomerular
arteriolar SMCs and prevented the development of glomerular
hypertrophy (138, 193). These reports suggested that TGF-␤induced suppression of IP3R expression underlies vascular
dysfunction, leading to diabetic glomerular hypertrophy. Collectively, these studies indicate that an alteration in IP3R
pendent Ca2⫹ waves regulate myogenic tone in these vessels
(231). Similarly, in pressurized mesenteric and tail arteries,
IP3R-mediated Ca2⫹ ripples were proposed to stimulate myogenic constriction (9, 249). SR-dependent Ca2⫹ waves contributed to myogenic tone at low intravascular pressure in cerebral
artery SMCs, although the contribution of IP3Rs was not
determined (151). These studies suggest that IP3R-mediated
Ca2⫹ release regulates the myogenic response. In contrast, in
pressurized mesenteric and cerebral arteries, IP3R-dependent
Ca2⫹ oscillations and waves did not contribute significantly to
myogenic vasoconstriction (88, 144, 254). Explanations for
these different observations include that the frequency and
amplitude of IP3R-mediated Ca2⫹ signals are likely to be
important factors that determine functional impact on contractility. In addition, the activation status of other signaling
pathways that may enhance the sensitivity of the contractile
apparatus to IP3R-induced Ca2⫹ release is also likely to modify
functional responses.
IP3Rs also control arterial SMC contractility via an SR Ca2⫹
release-independent mechanism that involves physical coupling to plasma membrane TRPC3 channels (Fig. 3C) (1, 2,
237, 254). In cerebral arteries at physiological intravascular
pressure, the majority (⬃60%) of IP3-induced vasoconstriction
occurred through IP3R1-TRPC3 physical coupling, with a
minor contribution from IP3R-mediated SR Ca2⫹ release
(237). At low intravascular pressure, where [Ca2⫹]SR is low
and the driving force for cation influx is high, IP3R1-TRPC3
physical coupling fully accounted for IP3-induced vasoconstriction (237). Thus, in cerebral artery SMCs, IP3Rs induce
contraction primarily via an SR Ca2⫹ release-independent
mechanism that involves TRPC3 channel activation. In summary, SMC contractility regulation by IP3Rs appears to be
tissue and stimulus dependent. Contrary to what was previously a prevailing view, IP3R-mediated Ca2⫹ release induces
contraction in some, but not all, SMC types, with IP3R activation also stimulating contraction via SR Ca2⫹ release-independent mechanisms.
Gene expression. IP3R-mediated Ca2⫹ release can activate
transcription factors, thereby regulating gene expression in
SMCs (40, 51, 60, 156). In cerebral artery SMCs, IP3Rmediated SR Ca2⫹ release activated NF-␬B both directly and
indirectly, by stimulating mitochondrial ROS production (Fig.
4B) (156). IP3R-dependent NF-␬B activation stimulated
CaV1.2 channel ␣1C subunit gene expression, leading to vasoconstriction (156). NF-␬B p105/p50 subunit expression was
also upregulated by IP3R-mediated SR Ca2⫹ release via a
mitochondrial ROS- and NF-␬B-independent pathway (156).
In cerebral artery and cultured aortic SMCs, IP3R-dependent
SR Ca2⫹ release stimulated nuclear factor of activated T-cell c3,
a transcription factor that can downregulate KCa channel ␤1subunit and Kv2.1 channel expression (8, 60, 113, 161). IP3Rmediated Ca2⫹ release also activated cAMP response elementbinding protein, a Ca2⫹-dependent transcription factor, in SMCs
of cerebral arteries and the portal vein (40, 51). Therefore, Ca2⫹
released by IP3Rs modulates multiple transcription factors that
can control ion channel gene expression in SMCs.
Migration and proliferation. Vascular SMCs proliferate and
migrate during vasculogenesis and in response to injury and are
associated with elevated IP3R expression and Ca2⫹ release (3,
20, 149, 205, 228, 234). Proliferation correlated with an elevation in IP3R1 expression in aortic and carotid artery SMCs
Review
insulin resistance, and diet-induced diabetes (243). A report
(200) that examined SMC function in IP3R1⫺/⫺ mice determined that gastric SMCs exhibited irregular bursts of spike
potentials, resulting in reduced contractility.
Population analysis studies have identified human diseases
caused by IP3R gene mutations. Similar to observations in knockout mice, there is a strong correlation between IP3R gene mutations and neurological disease. A heterozygous deletion and a
mutation in the IP3R1 gene have been identified in patients with
spinocerebellar ataxia and tremors (68, 221). SMC pathologies
have not been reported that are associated with deletions or
mutations in IP3R genes. Recent studies (75, 165) reported that
single nucleotide polymorphisms in genes encoding IP3R3 and
IP3 3-kinase C, which phosphorylates IP3 to IP4, predisposed
children with Kawasaki disease, a pediatric systemic vascular
autoimmune disease, to coronary artery aneurysms.
Summary
Since the discovery of IP3Rs more than 20 years ago,
research has identified these proteins as SR-located ion channels that are expressed in virtually all cell types. In SMCs, IP3R
Fig. 6. IP3R signaling in SMCs. Illustrated are the following: pathways controlling IP3R cellular expression (A), regulators of IP3R channel activity (B), ion
channels and organelles that communicate with IP3Rs (C), Ca2⫹ signals generated by IP3R-mediated Ca2⫹ release (D), and physiological functions modulated
by IP3Rs (E). AP-2, activator protein 2; RyR, ryanodine-sensitive Ca2⫹ release channels; CREB, cAMP response element-binding protein; NFATc3, nuclear
factor of activated T-cell c3; RACK, receptor for activated PKC 1; FKBP12, 12-kDa FK506 binding protein.
expression and IP3R-mediated Ca2⫹ signaling in vascular
SMCs contributes to the pathogenesis of vascular diseases.
Asthma. Asthma is associated with enhanced SMC [IP3]i,
IP3R signaling, and contractility, leading to airway hyperresponsiveness and remodeling (132, 209). In Fisher rats, an
asthmatic animal model, IP3-5-phosphatase expression and
activity were both reduced, leading to elevations in [IP3]i and
IP3R-mediated Ca2⫹ release in airway SMCs (209). This
enhanced IP3-induced Ca2⫹ release may underlie airway SMC
hyperresponsiveness in asthma (209). 2-Aminoethoxydiphenyl
borate inhibited acidic pH-induced remodeling of airway
SMCs, suggesting that IP3Rs may also regulate extracellular
matrix formation and airway remodeling in asthma (132).
Pathologies associated with IP3R gene deletion and mutation.
Studies performed in IP3R knockout (IP3R⫺/⫺) mice have provided valuable information regarding pathologies associated
with these proteins, although limited information is available
regarding SMC dysfunction. IP3R1⫺/⫺ mice are rarely born
alive, indicating that IP3R1 is crucial for embryonic development (131). Animals that survive exhibit severe neurological
pathologies, including ataxia and epilepsy (131). IP3R1⫹/⫺
mice displayed increased susceptibility to glucose intolerance,
H2203
Review
H2204
Future Directions
This review has summarized studies investigating IP3R expression, cellular localization, activity, Ca2⫹ signals generated,
physiological functions regulated, and associated pathologies
in SMCs. There are still major questions that remain regarding
IP3R functionality in SMCs. Studies are necessary to better
determine mechanisms that regulate IP3R gene transcription
and protein expression in SMCs. IP3R signaling in non-SMCs
is modulated by a wide variety of proteins, including calmodulin, Ca2⫹-binding protein 1, Ca2⫹- and integrin-binding protein 1, chromogranins, ERp44, IP3R-binding protein released
with IP3, and G␤␥ (57, 212). Modulation of IP3R signaling by
these proteins should be examined in SMCs. Studies should
also be aimed at determining other cellular organelles and ion
channels that interact with IP3Rs in SMCs. A better understanding of the contribution of RyRs and other Ca2⫹ channels
to IP3R-mediated Ca2⫹ signals in SMCs is also needed. Research is required to elucidate mechanisms that underlie tissuespecific variability in IP3R signaling in SMCs. Studies would
benefit from the availability of novel, selective, high-affinity,
membrane-permeant IP3R antagonists to investigate IP3R signaling, physiological functions, and pathological alterations.
SMC-specific IP3R animal models, including those that induce
knockout, express IP3R mutations associated with human diseases, or alter known IP3R coupling domains, would also
provide the capability to investigate the contribution of these
proteins to physiological functions and pathological alterations
both in vivo and in vitro. SMC dysfunctions in IP3R knockout
mice also need to be examined. Screening of humans to
identify potential IP3R mutations associated with SMC diseases is also necessary.
ACKNOWLEDGMENTS
We thank Drs. John Bannister, Sarah Burris, and Charles Leffler for critical
reading of the manuscript.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-67061, HL-110347, and HL-094378 (to J. H. Jaggar) and K01-HL096411 (to A. Adebiyi).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: D.N. prepared figures; D.N., A.A., and J.H.J. drafted
manuscript; D.N., A.A., and J.H.J. edited and revised manuscript; D.N., A.A.,
and J.H.J. approved final version of manuscript.
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Transcription

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