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(Circulation Research. 2001;88:202.)
© 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

Yuepeng Wang, Jie Chen, Yue Wang, Colin W. Taylor, Yasunobu Hirata, Hisashi Hagiwara, Katsuhiko Mikoshiba, Teruhiko Toyo-oka, Masao Omata, Yoshiyuki Sakaki

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

	Abstract

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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

	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.

	Materials and Methods

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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.

	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

View larger version (29K): [in this window] [in a new window]   Figure 1. IICR and quantitative analysis of the expression levels of IP3R1 and IP3R3 in A7r5 cells. A, [Ca2+]i responses to 30 mmol/L caffeine in [Ca2+]o-free medium, with or without subsequent addition of VP (3 µmol/L). B, In Ca2+-free medium, neither caffeine (60 mmol/L), nor ryanodine (10 µmol/L), nor caffeine after ryanodine caused a significant increase in [Ca2+]i. The results (A and B) are typical of those from 3 experiments. C, Total lysates (15 µg) of cultured A7r5 cells (1, 3, and 5), bovine aortic endothelial cells (2), Chinese hamster ovary cells (4), and COS cells (6) were electrophoresed on 6% SDS-PAGE. The pairs of lanes were then immunoblotted with R1Ab (1 and 2) R2Ab (3 and 4), and R3Ab (5 and 6). D, Lanes were loaded with 400 µg of membranes prepared from A7r5 cells (7 and 8) or 1 µg (9), 2 µg (10), or 4 µg (11) of Sf9/IP3R1 (a and b) or Sf9/IP3R3 (c and d) membranes. Lane 12 was loaded with 2 µg of Sf9/IP3R3 (a and b) or 1 µg of Sf9/IP3R1 (c and d). The blots were probed with the indicated antisera (Ab1, Ab3, and AbC). The results shown are typical of 4 experiments.

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.

View larger version (42K): [in this window] [in a new window]   Figure 2. 2D images of [Ca2+]i responses, IP3R1 or IP3R3 expression, and [Ca2+]i dynamics induced by VP (3 µmol/L, arrows) in transient transfectants of pG.R1AS (A and F), pG.R1S (B and G), pEGFPC1 (C and G), pG.R3AS (D and H), or pG.R3S (E and I) in [Ca2+]o-free medium. A through E, Transfectants expressing reporter EGFP (a), fura-2–loaded cells (b), peak [Ca2+]i response (c), and the immunocytostaining (d) of IP3R1 (A through C) or IP3R3 (D and E). F through I, [Ca2+]i dynamics in typical single cells.

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.

View larger version (89K): [in this window] [in a new window]   Figure 3. RNase protection assay (left) and Western blot (right) in clones transfected with pI.R1AS, pI.R1S, pI.R3AS, and pI.R3S. Lanes 1 through 9, Levels of expression of IP3R1, IP3R3, and IP3R2 in the pI.R1AS-transfected clones R1AS1, R1AS4, R1AS6, R1AS8, R1AS11, R1AS16, R1AS12, R1AS15, and R1AS17, respectively. Lanes 10 through 18, Changes in IP3R1 in pI.R1S–transfected clones R1S1, R1S3, R1S6, R1S7, R1S9, R1S12, R1S4, R1S10, and R1S11, respectively. Lanes 19 through 24, Levels of expression of IP3R1, IP3R3, and IP3R2 in the pI.R3AS–transfected clones R3AS3, R3AS4, R3AS5, R3AS7, R3AS10, and R3AS14, respectively. Lanes 25 through 30, Changes in IP3R3 in pI.R1AS–transfected clones R3S1, R3S2, R3S3, R3S4, R3S6, and R3S9, respectively. The density values for the protein bands in the right panel are as follows: lanes 1 through 9, IP3R1 bands: 8.8, 15.1, 11.6, 25.3, 0.0, 34.7, 6.0, 17.7, and 0.3, respectively; for the IP3R3 bands, the average from all 9 lanes was 8.1±0.6. Lanes 10 through 18, Average value (IP3R1) was 29.1±1.9; lanes 19 through 24, IP3R3 bands: 0.7, 8.8, 0.0, 5.2, 0.0, and 1.8, respectively; for the IP3R1 bands, the average from all 6 lanes was 33.5±1.6; and lanes 25 through 30, average value (IP3R3) was 8.7±0.7.

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).

View larger version (21K): [in this window] [in a new window]   Figure 4. [Ca2+]i responses evoked by VP (3 µmol/L, arrows) in [Ca2+]o-free medium in clones of pI.R1AS (A and B) and pI.R3AS (C). Results are typical of 3 experiments.

View larger version (84K): [in this window] [in a new window]   Figure 5. 2D images of [Ca2+]i responses and [Ca2+]i dynamics evoked by VP (3 µmol/L, arrows) in cells injected with low (A and D) or high (B and E) doses of heparin or vehicle (C and F) in [Ca2+]o-free medium. A through C, Positions of the injected cells (* in a), fura-2–loaded cells (b), and peak [Ca2+]i responses (c). D through F, Time courses of the [Ca2+]i signals.

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%).

View larger version (21K): [in this window] [in a new window]   Figure 6. Sustained [Ca2+]i signals (A through C) and Mn2+ entry (0.5 mmol/L [D through F]) evoked by VP (3 µmol/L, arrows) in clones of pI.R1AS (A, B, E, and F) and pI.R3AS (C and G) and heparin-injected cells (D and H). The responses were recorded in the presence of 1 mmol/L [Ca2+]o and are typical of those recorded from 3 experiments.

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.

View larger version (18K): [in this window] [in a new window]   Figure 7. Sustained [Ca2+]i signals (A through C) and Mn2+ entry (0.28 mmol/L [D through F]) evoked by TG (1 µmol/L, arrows) in clones of pI.R1AS (A and D) and pI.R3AS (B and E) and heparin-injected cells (C and F). The responses were recorded in the presence of 1 mmol/L [Ca2+]o and are typical of those recorded from 3 experiments.

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).

View larger version (29K): [in this window] [in a new window]   Figure 8. Cell proliferation in clones of pI.R1AS (A) and pI.R3AS (B).

	Discussion

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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 release^{21 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.

	Acknowledgments

 
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.

	Footnotes

 
Original received July 13, 2000; revision received December 18, 2000; accepted December 19, 2000.

	References

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