© 2001 American Heart Association, Inc.
Cellular Biology |
Crucial Role of Type 1, but Not Type 3, Inositol 1,4,5-Trisphosphate (IP3) Receptors in IP3-Induced Ca2+ Release, Capacitative Ca2+ Entry, and Proliferation of A7r5 Vascular Smooth Muscle Cells
From the Human Genome Center (Yuepeng Wang, H.H., Y.S.), the Department of Molecular Neurobiology (K.M.), Institute of Medical Science, the Second Department of Internal Medicine (J.C., Yue Wang, Y.H., M.O.), the Health Service Center (T.T.), University of Tokyo, Tokyo, Japan; Department of Pharmacology (C.W.T.), University of Cambridge, Cambridge, UK.
Correspondence to Yuepeng Wang, Laboratory of Functional Genomics, the Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail srwang-tky{at}umin.ac.jp
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Abstract—Stimulation of G protein– or tyrosine kinase–coupled receptors regulates cell proliferation through intracellular Ca2+ ([Ca2+]i) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP3) mediates vasopressin (VP)-evoked Ca2+ release from intracellular stores and showed that types 1 (IP3R1) and 3 (IP3R3) IP3 receptors were expressed. Using antisera selective for IP3R1 or IP3R3 and another that interacted equally well with both subtypes, together with membranes from Sf9 cells expressing only single IP3R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP3R1 and 19% IP3R3. To elucidate the contributions of IP3R1 and IP3R3 to Ca2+ signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP3R subtypes were selected. Mild inhibition of IP3R1 (71±8% of control level) slightly attenuated the IP3-evoked Ca2+ release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca2+ entry (CCE) and proliferation. Moderate inhibition (34±6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP3R3 expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP3R1 and IP3R3. A high dose of heparin had a similar effect to complete inhibition of IP3R1 expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP3R1, but not IP3R3, is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.
Key Words: inositol 1,4,5-trisphosphate receptors • IP3-induced Ca2+ release • capacitative Ca2+ entry • proliferation • vascular smooth muscle cell
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Introduction |
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In most cell types, hormones, neurotransmitters, or growth factors stimulate membrane receptors and activate phospholipase C, which catalyzes the production of inositol 1,4,5-trisphosphate (IP3).1 IP3 is a well-known second messenger mediating IP3-induced Ca2+ release (IICR) from the intracellular Ca2+ ([Ca2+]i) stores by binding to IP3 receptors (IP3Rs). IICR produces an initial [Ca2+]i spike and induces the subsequent capacitative Ca2+ entry (CCE).1 2 The [Ca2+]i signaling mediated by IICR is versatile, almost ubiquitous, and essential in regulating fertilization,3 development,4 differentiation,5 cell proliferation,5 6 7 and cell death.8 9 In mammalian cells, at least three IP3R genes (IP3R1, IP3R2, and IP3R3) have been identified encoding proteins with up to 70% amino acid similarity.2 Almost all tissues and cell lines express all three IP3R subtypes to varying degrees, but IP3R1 is most ubiquitously expressed.2 10 11 Most IP3Rs are found in the sarcoplasmic/endoplasmic reticulum, but they may also reside in the plasma membrane and nucleus.1 2
Regarding function, IP3R1 has been most elucidated in the central nervous system where IP3R1 is the predominant subtype.2 The majority of homozygous knockout mice lacking the IP3R1 died in utero; those that survived had severe behavioral abnormalities in the form of ataxia and epileptic seizures.12 IP3R1 is essential for the development of nerve growth cones in cultured neurons and for the proliferation of neuroblasts and differentiation of imaginal progenitor cells in Drosophila.5 7 The function of IP3R2 has not been clearly delineated, despite its predominance in liver and heart,11 although in pancreatic islets it may contribute to insulin excretion.13 In bronchial mucosa, IP3R3 may be part of a mechanism coping with oxidative stress.14
In the vascular system, we detected only IP3R1 in endothelial cells and both IP3R1 and IP3R3 in vascular smooth muscle cells (VSMCs).15 16 In endothelial cells, IP3R1 is essential for [Ca2+]i signaling, because ATP- or bradykinin-induced IICR and the subsequent CCE can be inhibited by microinjection of antibody or expression of antisense against IP3R1.15 16 In VSMCs, the contributions of IP3R1 and IP3R3 to Ca2+ signaling and proliferation are unclear, although in B lymphocytes IP3Rs in the plasma membrane have been implicated in control of apoptosis.8 17 Abnormal proliferation of VSMCs is a pivotal step in the pathogenesis of arteriosclerotic lesions and the formation of restenosis after angioplasty.18 19 In the present study, we quantified the relative levels of expression of IP3R1 and IP3R3 in A7r5 cells and examined the effects on Ca2+ signaling and cell proliferation of attenuating the expression or function of IP3R1 and/or IP3R3 expression using antisense and heparin. Our results suggest that IP3R1 is more important than IP3R3 for IICR, CCE, and proliferation of VSMCs. CCE is more susceptible than IICR to mild inhibition of IP3R1 and may be responsible for the significantly retarded proliferation of the cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
A7r5 cells were grown and passaged in DMEM, as described previously.20
Western Blot
Total cell lysates were separated by 6% SDS-PAGE and
immunoblotted with antibodies against each type of
IP3R (R1Ab,
R2Ab, and
R3Ab).16
Quantification of Relative Expression of
IP3R1 and
IP3R3
Full-length rat IP3R1 and
IP3R3 cDNAs were
expressed in Sf9 cells, and
membrane fractions
(Sf9/IP3R1
and
Sf9/IP3R3,
respectively) were
prepared.21 Three antisera
(Ab1, Ab3, and AbC) were raised in rabbits using peptides corresponding
to the C-terminal of rat
IP3R1
([C]LLGHPPHMNVNPQQPA) and
IP3R3 (LGFVDVQNCMSR) and
to the N-terminal residues that are similar in all
IP3Rs
(PMNRYSAQKQFWKA[C]).21 22
Four identical blots with lanes containing
Sf9/IP3R1,
Sf9/IP3R3,
and A7r5 cells were probed with the antisera. Using the ratios for
Sf9/IP3R1
(Ab1:AbC) and
Sf9/IP3R3
(Ab3:AbC), densitometric measurements of
IP3R1 and
IP3R3 staining (detected
with Ab1 and Ab3) from A7r5 cells were converted into units of AbC
staining and quantified.
Construction of Vectors
A 99-bp fragment (nucleotide position -90 to +9)
that shares no homology between
IP3R1 and
IP3R3 was designed. Total
RNA was extracted from A7r5 cells, and reverse transcription polymerase
chain reaction (RT-PCR) was performed with primers
(IP3R1:
5'-CAAGGAGCTGACTACA-3' and 5'-GTCAGACATGTCCTTG-3';
IP3R3:
5'-TTTCCGCCCAGCGCGC-3' and 5'-TTCATTCATG- GCTTTG-3'). The RT-PCR
products were inserted into
pEGFPC1 (Clontech), and
antisense
(pG.R1AS
and
pG.R3AS)
and sense
(pG.R1S
and
pG.R3S)
orientation of the insert was confirmed by sequencing. The RT-PCR
products were also inserted into
pIND (Invitrogen) in antisense
(pI.R1AS
and
pI.R3AS)
and sense
(pI.R1S
and
pI.R3S)
orientation.
Transient and Stable Transfection
Transient transfection of
pG.R1AS
and
pG.R3AS
was carried out as described
previously.16 For stable
transfection, cells were cotransfected using a mixture of 3 µg of
pVgRXR and 3 µg of
pI.R1AS
or
pI.R3AS
and 15 µL of lipofectin per 10-cm-diameter dish. One day after
transfection, the medium was changed to DMEM. Two days later, the cells
were subcultured in DMEM with 400 µg/mL G418 and 700 µg/mL zeocin.
For each construct, 12 to 24 G418/zeocin-resistant clones were isolated
and passaged. Cells were used for experiments 24 to 72 hours after
induction with 5 µg/mL ponasterone A
(Invitrogen).
Microinjection
The amount of heparin (MW=5000) microinjected into
cells was controlled by varying heparin concentration and injecting
pressure.16
Measurement of
[Ca2+]i and
Mn2+ Influx
The
[Ca2+]i responses
of individual transfected and untransfected cells, as well as
heparin-injected and -uninjected cells, in the same observation field,
were separately analyzed after stimulation with vasopressin (VP) or
thapsigargin (TG). For measurement of the
Mn2+ influx rate,
Mn2+-induced quenching of fura-2
fluorescence was recorded at an excitation wavelength of 360
nm.16 20
Immunocytostaining
Cells grown on a CELLocate coverslip (Eppendorf) were
fixed, permeabilized, and immunostained with
R1Ab or
R3Ab.16
RNase Protection Assay
A 493-bp DNA fragment (nucleotide position +90 to
+403) of rat IP3R1, a
421-bp DNA fragment (+980 to +1400) of rat
IP3R2, and a 150-bp DNA
fragment (-129 to +21) of rat
IP3R3 were ligated to
pOPRSVI/MCS (Stratagene). The
antisense RNA probes, transcribed from plasmids with T7 RNA polymerase,
were labeled with biotin. Twenty micrograms of total RNA, isolated from
each clone after adding ponasterone A (5 µg/mL) for 72 hours, was
hybridized with the probes, followed by detection using an Ambion
Biodetect kit.
Cell Count
At 5% confluence, photomicrographs of cells were
taken in a fixed observation field at intervals of 24 hours after
ponasterone A (5 µg/mL) treatment; this was continued for 8 to 9
days.
Statistics
Results are expressed as mean±SEM. Statistical
significance was determined by paired
t test or by ANOVA.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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VP-Induced [Ca2+]i Release and Quantification of Relative Expression Levels of IP3R1 and IP3R3 in A7r5 Cells
In Ca2+-free medium, caffeine (3 to 60 mmol/L) did not evoke a [Ca2+]i signal in A7r5 cells, but the cells did respond strongly to VP (3 µmol/L) (Figure 1A
). Ryanodine (10 to 60 µmol/L) neither directly
evoked a [Ca2+]i
signal nor did it affect the very small response evoked by caffeine
(Figure 1B). Immunoblotting showed that
R1Ab and R3Ab each
reacted with a single 260-kDa band in A7r5 cells
(Figure 1C). These results suggest that the release of
Ca2+ from intracellular stores evoked by VP
is likely to be mediated by
IP3R1 and
IP3R3 but not by
ryanodine receptors. Previous studies have similarly concluded that
ryanodine receptors do not contribute to
Ca2+ signaling in A7r5
cells23 and that
IP3 receptors are required for VP-evoked
Ca2+
mobilization.24
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Next, the relative expression levels of
IP3R1 and
IP3R3 protein were
quantified. As shown in
Figure 1D
, four parallel blots loaded with a range of
concentrations of membranes from
Sf9/IP3R1,
Sf9/IP3R3,
and A7r5 cells, were immunoblotted with Ab1, Ab3, or AbC. There was a
linear relationship between the quantity of membranes loaded and the
densitometric measurements of IP3R bands
obtained with each antiserum. The ratio of the immunostaining obtained
using the subtype-selective and common antisera was 1.41±0.20 for
Sf9/IP3R1
(Ab1:AbC) and 1.56±0.21 for
Sf9/IP3R3
(Ab3:AbC). These ratios together with the parallel measurements of
immunostaining from A7r5 cells indicated that A7r5 cells express
80.1±2.6% IP3R1 and
19.2±2.3% IP3R3,
consistent with previous estimates of relative mRNA
levels.11
IICR and Ca2+ Entry
After Inhibition of
IP3R1 or
IP3R3
In the cells (
10%) successfully transiently
transfected with
pG.R1AS,
16% showed substantial inhibition of
IP3R1 expression by
immunocytostaining
(Figure 2A). The average
[Ca2+]i in
unstimulated cells was 148±8 nmol/L. In medium without extracellular
Ca2+
([Ca2+]o), the peak
[Ca2+]i signal
evoked by VP in untransfected cells was 587±18 nmol/L, but it was
consistently almost abolished in the cells where transfection with
pG.R1AS
had substantially inhibited
IP3R1 expression (180±6
nmol/L, n=7;
Figure 2F). The sustained
[Ca2+]i response in
the presence of
[Ca2+]o was also
inhibited (data not shown). These results are similar to those obtained
from endothelial cells.16 In
contrast, although 13% of the cells successfully transfected with
pG.R3AS
showed marked inhibition of
IP3R3 expression
(Figure 2D), the responses of these cells to VP in
[Ca2+]o-free medium
were only slightly reduced (523±16 nmol/L, n=9;
Figure 2H) and there was no detectable inhibition of the
sustained [Ca2+]i
response (data not shown). Normal staining for
IP3 receptors and normal IICR responses occurred
in all transfectants with
pEGFPC1 (584±19 nmol/L, n=10;
Figures 2C and 2G),
pG.R1S
(590±20 nmol/L, n=14;
Figures 2B and 2G), and
pG.R3S
(601±23 nmol/L, n=11;
Figures 2E and 2I). These results suggest that
IP3R1 may be more
important than IP3R3 for
IICR and Ca2+ entry in A7r5
cells.
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By using promoter-inducible antisense in stable clones, the
inhibition of expression of IP3R subtypes could
be both graded and more complete. The extent to which
IP3R protein levels were reduced could be
classified, according to the results of densitometric measurements of
Western blots, into mild (50% to 90% remaining), moderate (10% to
49%), and complete (<10%). The amount of
IP3R1 protein was mildly
decreased to 71±8% in four clones, moderately decreased to 34±6% in
four clones, and abolished (0.8±0.4%) in three of the 24 clones of
pI.R1AS,
compared with the amount of
IP3R1 in 12 clones of
pI.R1S
(Figure 3). In the
pI.R3AS
clones, the amount of
IP3R3 protein was mildly
decreased to 74±7% in three clones, moderately decreased to 30±5%
in three clones, and abolished (2.7±2.7%) in three of the 18 clones,
compared with the amount of
IP3R3 in 12 clones of
pI.R3S.
For both IP3R1 and
IP3R3, the results from
Western blotting were consistent with those from measurements of mRNA
(Figure 3). No compensatory increases in mRNA or the amounts
of IP3R1 or
IP3R3 protein were found
in clones of
pI.R3AS
or
pI.R1AS,
nor was novel transcription or expression of
IP3R2
detected.
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To evaluate the effects of graded inhibitions of
IP3R expression on IICR, responses to VP were
measured in
[Ca2+]o-free medium
(Figure 4). Clones with mild inhibition of
IP3R1 expression
exhibited a slight attenuation of IICR (510±7 nmol/L, n=4) compared
with clones of
pI.R1S
(549±10 nmol/L, n=12). After moderate inhibition of
IP3R1 expression, IICR
was markedly decreased (323±7 nmol/L, n=4), and it was almost
abolished (196±5 nmol/L, n=3) in the clones with nearly complete
inhibition of IP3R1
expression. In contrast, three clones with moderately inhibited
IP3R3 expression
demonstrated normal IICR (564±6 nmol/L), and even after expression was
completely inhibited, there was only a slight decrease in IICR (526±7
nmol/L, n=3), compared with clones of
pI.R3S
(559±11 nmol/L, n=12;
P>0.05). Microinjection of a
low concentration of heparin (pipette concentration 50 mg/mL, injection
pressure 20 mm H20), to inhibit
IP3 binding to all IP3R
subtypes, reduced IICR (400±31 nmol/L, n=6) relative to
vehicle-injected cells (591±24 nmol/L, n=6)
(Figure 5). This response was similar to that in clones with
a mild or moderate reduction of
IP3R1 expression
(Figure 4). A high concentration of heparin (200 mg/mL, 80
mm H2O) completely inhibited IICR (154±4
nmol/L, n=7;
Figure 5), an effect that was indistinguishable from that
observed in transient transfectants with substantial loss of
IP3R1 expression
(Figure 2) or stable clones with complete loss of
IP3R1 expression
(Figure 4).
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To evaluate the effects of graded reductions of
IP3R expression on
Ca2+ entry across the plasma membrane,
VP-evoked Ca2+ signals were measured in the
presence of [Ca2+]o
(Figure 6). Four clones with mildly inhibited
IP3R1 expression showed
only slight decreases in the amplitude of the initial
[Ca2+]i spike
(540±17 nmol/L), compared with clones of
pI.R1S
(576±20 nmol/L, n=12;
P>0.05). Surprisingly, there
was a significant decrease in the sustained
[Ca2+]i signal in
these cells (228±4 nmol/L at 210 seconds after stimulation) compared
with clones of
pI.R1S
(345±11 nmol/L, P<0.05).
Previous studies of A7r5 cells established that VP stimulated both CCE
and a second Ca2+ entry pathway that was
activated by arachidonic acid; activation of only the former required
depletion of the IP3-sensitive stores, and only
it was permeable to
Mn2+.24
The decreased Ca2+ entry in cells with fewer
IP3 receptors suggests the involvement of CCE;
this was examined directly by measuring VP-evoked
Mn2+ quench of fura-2 fluorescence. The
results demonstrate that fluorescence quenching in the cells with
mildly inhibited IP3R1
expression was significantly reduced (16±3% at 210 seconds after
stimulation), compared with clones of
pI.R1S
(43±3%, P<0.05). Four clones
with moderate loss of
IP3R1 expression revealed
an additional decrease in the sustained phase of the VP-evoked
[Ca2+]i signal
(218±4 nmol/L) and Mn2+ quenching
(13±2%), and in three clones with complete loss of expression, there
was an almost complete disappearance of the sustained
[Ca2+]i phase
(207±4 nmol/L) and Mn2+ entry
(10±3%).
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In contrast, three clones with moderately inhibited IP3R3 expression demonstrated no changes in the VP-evoked sustained [Ca2+]i phase (360±6 nmol/L) or Mn2+ entry (39±3%), and in three clones with complete loss of IP3R3 expression, there were insignificant decreases in the sustained [Ca2+]i phase (358±8 nmol/L) and Mn2+ entry (41±2%), compared with clones of pI.R3S (364±10 nmol/L, 41±3%, n=12). Cells injected with a low dose of heparin exhibited no marked change in the initial VP-evoked [Ca2+]i spike, but a significant decrease in the sustained [Ca2+]i phase (199±7 nmol/L) and Mn2+ entry (11±3%, n=16), compared with vehicle-injected cells (336±8 nmol/L, 38±3%, n=14; P<0.05). These findings were similar to those from clones with mild inhibition of IP3R1. A high dose of heparin further inhibited the sustained [Ca2+]i phase (161±3 nmol/L) and Mn2+ entry (6±1%, n=17), results that were indistinguishable from those in the clones with complete inhibition of IP3R1.
TG-induced CCE was also measured
(Figure 7). In clones with mild, moderate, or complete
inhibition of IP3R1
expression, there was only very modest attenuation of both the
sustained phase of the TG-evoked
[Ca2+]i signal
(343±4, 330±5, and 320±4 nmol/L, respectively, at 15 minutes after
stimulation; n=3 to 4) and Mn2+ quenching
(28±2%, 28±3%, and 24±2%, respectively, at 15 minutes after
stimulation), compared with clones of
pG.R1S
(386±8 nmol/L, 31±5%, n=11;
P<0.05). In three clones with
complete inhibition of
IP3R3 expression, the
sustained [Ca2+]i
signal (409±8 nmol/L) and Mn2+ quenching
(34±3%) were similar to those in
pI.R3S
clones (423±10 nmol/L, 33±3%, n=12). A low dose of heparin
attenuated the sustained
[Ca2+]i signal
(386±7 nmol/L) and Mn2+ quenching (28±3%,
n=11), compared with vehicle-injected cells (435±6 nmol/L, 35±3%,
n=9; P<0.05). A high dose of
heparin further reduced the sustained
[Ca2+]i signal
(341±4 nmol/L) and Mn2+ quenching (24±3%,
n=21). These results suggest that in A7r5 cells,
IP3R1 is more important
than IP3R3 for IICR, that
there is a graded IICR response based on the extent of
IP3R1 inhibition, and
that VP-evoked CCE is more susceptible than IICR to mild inhibition of
IP3R1.
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Cell Proliferation After Inhibition of
IP3R1 or
IP3R3 Expression
For each clone, cell proliferation was monitored by
serial photomicrographs
(Figure 8). In four clones with mildly reduced
IP3R1 expression,
proliferation was retarded (369±23 cells per observation field on day
8), compared with
pI.R1S
clones (825±25 cells per field, n=12,
P<0.05). Four clones with
moderate inhibition revealed markedly decreased proliferation (157±17
cells per field; P<0.05 versus
pI.R1S clones). In three clones with complete
loss of IP3R1 expression,
proliferation was arrested (46±3 cells per field). In contrast, clones
with moderate or complete loss of
IP3R3 expression showed,
respectively, proliferation rates of 698±39 (n=3) and 687±33 (n=3)
cells per field on day 8, which are not significantly different from
that of
pI.R3S
clones (723±37 cells per field, n=12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mammalian cells, where all three IP3R subtypes are often expressed and then assembled into homotetrameric and heterotetrameric Ca2+ channels, it has proved difficult to disentangle the contributions of each IP3R subtype to the different patterns of [Ca2+]i signaling.2 22 25 Several studies reported that blocking expression of one IP3R subtype did not significantly change the expression of the remaining subtypes.12 26 27 After inhibition of one or two IP3R subtypes, the remaining subtype(s), at least if expressed at a sufficient level, are often able to functionally compensate in mediating IICR and proliferation, suggesting a degree of functional redundancy.5 6 7 12 25 26 27 In Jurkat T cells, which express largely IP3R1 with only small amounts of IP3R2 and IP3R3,8 elimination of IP3R1 by antisense abolished IICR and arrested proliferation.6 8 In contrast, mouse T cells express similar amounts of all three IP3R subtypes, and T cells isolated from IP3R1–/– mice demonstrated normal IICR and proliferation.26 In avian B cells, IP3R1 and IP3R2 predominate,25 but abolishing expression of one or two subtypes was accompanied by an increased expression of the remaining subtype(s). Thus, IICR appeared normal after deleting only one IP3R subtype, was attenuated markedly after deleting both IP3R1 and IP3R2, and was abolished when all three IP3R subtypes were deleted.27 Chromophore-assisted laser inactivation of IP3R1 resulted in growth arrest in neurons, where almost all IP3R are type 1.7 In vivo, neonatal IP3R1–/– mice at a low birthrate showed a 50% decrease in body weight and died at weaning.12 In Drosophila, which has only one IP3R subtype similar to IP3R1, knockout of the gene caused fatal damage or extraordinary retardation of growth.5
In the present study, there were no compensatory increases in the expression of the remaining types of IP3R in the stable clones lacking either IP3R1 or IP3R3. In some clones, there was no detectable IP3R1 or IP3R3 mRNA, suggesting that it was degraded, presumably by ribozyme activity in the complex of endogenous IP3R1 or IP3R3 mRNA with antisense mRNA. Cells completely lacking IP3R1 showed no IICR, nor did they proliferate. In cells with moderately inhibited IP3R1 expression, the retarded proliferation was similar to that reported in Jurkat T cells.6 Even complete inhibition of IP3R3 expression only slightly attenuated IICR and had no effect on proliferation. These results suggest that the 19% of IP3R that are type 3 could not alone trigger IICR and proliferation. However, the 81% of IP3R that are type 1 could compensate for the lack of IP3R3 and trigger most of the proliferation and the IICR evoked by maximal stimulation with VP. These results are similar to those from B cells.27
In addition to their levels of expression, the relative affinities of the IP3R subtypes for IP3 will also affect their contributions to [Ca2+]i signaling. IP3R1 and IP3R3 have been reported to differ in their relative affinities, with most,10 although not all,21 studies suggesting that IP3R1 has greater affinity for IP3. IP3Rs are also regulated by [Ca2+]i, which contributes to the oscillatory patterns of [Ca2+]i release21 25 required for maintaining vascular tone after sympathetic stimulation.28 Although the issue remains contentious,29 it seems likely that IP3R1 and IP3R3 are each biphasically regulated by [Ca2+]i, however with different [Ca2+]i sensitivities and possibly different mechanisms that may also contribute to the relative importance of the IP3R subtypes in mediating physiological responses.21 22
Ca2+ influx mediated by various Ca2+ channels including CCE channels is crucial for proliferation of VSMCs.30 31 We and others have demonstrated that blocking Ca2+ influx through L-type Ca2+ channels by verapamil or nifedipine inhibited proliferation of A7r5 cells by 22% to 61%.31 32 Blocking Ca2+ influx through the growth factor–regulated Ca2+ channel33 by inhibiting channel expression blocked proliferation by 40% (personal communication, I. Kojima, 1999). In cells completely lacking IP3R1, VP could no longer empty the Ca2+ stores and was therefore unable to stimulate CCE. However, in cells with mild inhibition of IP3R1, VP-induced IICR was almost normal, yet CCE was significantly inhibited. These results indicating that CCE is more susceptible to inhibition of IP3R1 expression than IICR are consistent with recent work in rat basophilic leukemia cells in which CCE was activated only after substantial depletion of the intracellular stores.34 35 In both A7r5 cells with mild inhibition of IP3R and rat basophilic leukemia cells stimulated with low levels of IP3, substantial IICR was induced without any activation of CCE,35 because the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase may be able to effectively counteract the IP3-evoked [Ca2+]i leak and so prevent the stores from emptying to a level at which they activate CCE.34
In contrast, VP-induced CCE was not decreased in cells lacking IP3R3: IICR was only slightly inhibited, and CCE was barely affected. Our results therefore exclude an essential role for IP3R3 in regulating CCE8 and establish that IP3R1 are more important than IP3R3 in controlling IICR, CCE, and proliferation in VSMCs. VP still evoked a very small [Ca2+]i rise (192 to 206 nmol/L) after complete inhibition of IP3R1 and/or IP3R3. This [Ca2+]i rise may result from Ca2+ influx through the noncapacitative pathway,24 which may help maintain cell viability, without being sufficient for proliferation.
Compared with the substantial inhibition of VP-evoked
Ca2+ entry in cells with mild loss of
IP3R1, by either
IP3R1AS or
heparin36
(Figure 6), the TG-evoked
[Ca2+]i rise and
Mn2+ entry were attenuated only slightly,
even in cells with complete loss of
IP3R1. These results
therefore suggest that although we cannot wholly eliminate a role for
IP3R in coupling depletion of
[Ca2+]i stores to
CCE,37 38 the
major effect of IP3 receptor inhibition in A7r5
cells is to prevent IP3 from emptying the stores
and consequently inhibits CCE.
VSMCs also coexpress IP3R1 and IP3R3 in vivo and the relative amounts, as shown by the amounts of mRNA, may change with age.39 In rat, IP3R1 is the minor subtype in neonates, but the major subtype in adults,19 whereas the reverse is true for IP3R3.39 40 Our group observed that expression of IP3R1, but not IP3R3, increased in VSMCs of adult human arteriosclerotic plaques and in the rat carotid artery neointima after balloon injury (authors’ unpublished data). Increased IP3R1 expression may therefore be involved in hypertension and arteriosclerosis. Because complete inhibition of IP3R1 expression is likely to be detrimental,7 12 but mild inhibition is effective in blocking VSMC proliferation, our results are of potential application in blocking VSMC proliferation, especially in restenosis after coronary artery angioplasty,18 by mild inhibition of IP3R1 expression.
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Acknowledgments |
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This study was supported by the Research Foundation of Japan Society for the Promotion of Science, the Honjo International Scholarship Foundation, the Japanese-Chinese Medical Research Collaboration Foundation, and the Wellcome Trust.
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Footnotes |
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Original received July 13, 2000; revision received December 18, 2000; accepted December 19, 2000.
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References |
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