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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1225.)
© 2000 American Heart Association, Inc.

Vascular Biology

Intracellular Ca²⁺ Handling in Vascular Smooth Muscle Cells Is Affected by Proliferation

Olivier Vallot; Laurent Combettes; Philippe Jourdon; Jocelyn Inamo; Isabelle Marty; Michel Claret; Anne-Marie Lompré

From CNRS EP 1088 (O.V., P.J., J.I., A.-M.L.) and INSERM U 442 (L.C., M.C.), IFR-FR 46 Signalisation cellulaire, Université Paris-sud, Orsay, France, and DBMS/BMC (I.M.), CEA, Grenoble, France.

Correspondence to Dr A.-M. Lompré, Biochime, Faculté de Pharmacie, IFR-75, Université Paris-sud, F 92296-Chatenay-Malabry, France. E-mail anne-marie.lompre{at}egm.u-psud.fr

	Abstract

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Abstract—Despite intensive interest in the dedifferentiation process of vascular smooth muscle cells, very little data are available on intracellular Ca²⁺ signaling. The present study was designed to investigate the evolution of the intracellular Ca²⁺ pools when rat aortic smooth muscle cells (RASMCs) proliferate and to define the mechanisms involved in the functional alterations. RASMCs were cultured in different conditions, and [Ca²⁺]_i was measured by use of fura 2. Expression of the sarco(endo)plasmic reticulum Ca²⁺ pumps (SERCA2a and SERCA2b), Ca²⁺ channels, the ryanodine receptor (RyR), and the inositol trisphosphate receptor (IP3R) was studied by reverse transcription–polymerase chain reaction and immunofluorescence. Antibodies specific for myosin heavy chain isoforms were used as indicators of the differentiation state of the cell, whereas an anti–proliferating cell nuclear antigen antibody was a marker of proliferation. SERCA2a, SERCA2b, RyR3, and IP3R-1 mainly were present in the aorta in situ and in freshly isolated RASMCs. These cells used the 2 types of Ca²⁺ channels to release Ca²⁺ from a common thapsigargin-sensitive store. Proliferation of RASMCs, induced by serum or by platelet-derived growth factor-BB, resulted in the disappearance of RyR and SERCA2a mRNAs and proteins and in the loss of the caffeine- and ryanodine-sensitive pool. The differentiated nonproliferative phenotype was maintained in low serum or in cells cultured at high density. In these conditions, RyR and SERCA2a were also present in RASMCs. Thus, expression of RyR and SERCA2a is repressed by cell proliferation, inducing loss of the corresponding Ca²⁺ pool. In arterial smooth muscle, Ca²⁺ release through RyRs is involved in vasodilation, and suppression of the ryanodine-sensitive pool might thus alter the control of vascular tone.

Key Words: vascular smooth muscle • cell proliferation • Ca²⁺ pumps • ryanodine receptors • inositol trisphosphate receptors

	Introduction

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Vascular smooth muscle cells, present in normal blood vessels, exhibit a quiescent differentiated phenotype characterized by a unique repertoire of contractile proteins, ion channels, and receptors involved in the contractile function of the cell. They undergo a transition from a contractile phenotype to a proliferative or synthetic phenotype when they proliferate,¹ a situation that is observed in chronic vascular disease and, in particular, in atherosclerosis or restenosis. This is accompanied by changes in the phenotype of many contractile proteins (see Reference ² for review). Voltage-gated Ca²⁺ currents also vary.^{3 4 5} Freshly dissociated rat aortic smooth muscle cells (RASMCs) have functional L- and T-type Ca²⁺ channels, but when they proliferate, the proportion of the L-type channels decreases, whereas that of the T-type channels increases. The changes are dependent on the cell cycle.⁶ Furthermore, alteration in expression of the ₁, ß₂, and ß₃ subunits of the L-type Ca²⁺ channel was recently reported in culture.⁵

Changes in the response to caffeine, ryanodine, and angiotensin II have been reported during vascular smooth muscle cell culture.^{7 8 9} This suggests modifications in the intracellular Ca²⁺ stores, but the molecular events underlying these pharmacological properties are unknown. We postulated that alteration in the expression of sarco(endo)plasmic reticulum Ca²⁺ pumps and channels may account, at least in part, for the functional alterations. Ca²⁺ is released from the intracellular stores by 2 types of receptors, the inositol trisphosphate (IP3) receptor and the ryanodine receptor (IP3R and RyR, respectively), the expression of which may vary during proliferation. The type 1 isoform is the major IP3R in adult porcine¹⁰ and rat aorta,¹¹ whereas type 3 predominates in neonatal aorta.¹¹ The RyR isoform that is present in vascular smooth muscle cells has not been definitively identified.^{12 13} Refilling of the Ca²⁺ stores depends on the sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCAs). The 2 spliced variants of the SERCA2 gene, 2a and 2b, are present in the smooth muscle cells, with SERCA2b being the most abundant isoform (see Reference ¹⁴ for review). Maturation of the rat aorta is associated with an increase in the level of SERCA2a mRNA, with the amounts of SERCA2b mRNA being identical in young and adult animals.¹⁵

The goal of the present study was (1) to characterize the intracellular Ca²⁺ pools in isolated RASMCs and to precisely identify the sarco(endo)plasmic reticulum Ca²⁺ pumps and Ca²⁺ channels in this cell type, (2) to study the changes in functional Ca²⁺ pools and expression of genes encoding the sarco(endo)plasmic reticulum Ca²⁺ pumps and Ca²⁺ channels during RASMC proliferation in culture, and (3) to gain new insight into the mechanisms involved in the alteration of the phenotype of sarcoplasmic reticulum (SR) proteins during proliferation.

	Methods

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Cell Culture
RASMCs were isolated from the medial layer of thoracic aorta (aortic cross excluded) of 180- to 200-g male Wistar rats by enzymatic digestion with collagenase (CLS2, 50 U/mL, Worthington) and pancreatic elastase (0.25 mg/mL, Sigma) for 2 hours at 37°C. After periods of 20 minutes, the suspension was centrifuged at 1500 rpm for 5 minutes, and the cells were collected and placed in DMEM+20% FCS. The cells obtained in the first three 20-minute periods were discarded because they had a nonmuscle phenotype.¹⁶ Those obtained in the other cycles were pooled, centrifuged, suspended in DMEM containing 10% FCS, plated onto glass coverslips coated with collagen I (rat tail, Sigma) at a density of 1.3x10⁴ cells per square centimeter, and incubated at 37°C in 5% CO₂/95% air. The medium was changed every 2 days with DMEM containing 10% FCS.

Experiments were also performed at high density (10⁵ cells per square centimeter) in the presence of 10% FCS. For some experiments, the cells were plated at normal density and cultured for 2 days in the presence of 10% FCS. The serum was then withdrawn, and the cells were cultured in 0.1% FCS for 1 day and then in either 0.1% FCS alone or 0.1% FCS plus 50 ng/mL platelet-derived growth factor (PDGF)-BB (rat recombinant, Sigma) for 3 more days.

Loading With Fura 2 and [Ca²⁺]_i Measurement
RASMCs were loaded with 3 µmol/L fura 2-AM for 40 minutes at 37°C in 5% CO₂/95% air. The coverslips were washed twice with saline solution (10 mmol/L HEPES, 116 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl₂, 0.8 mmol/L MgCl₂, 0.96 mmol/L NaH₂PO₄, 5 mmol/L NaHCO₃, and 2 g/L glucose, pH 7.4). For experiments in extracellular Ca²⁺-free medium ([Ca²⁺]_e=0), CaCl₂ was replaced by 0.1 mmol/L EGTA. Observations were made at 34°C with a Zeiss Axiovert 35 microscope set up for epifluorescence microscopy. The ratio was calculated from 340- and 380-nm frame pairs (1 ratio per 3 seconds) to measure a true image subjected to ratio calculation for up to 30 minutes. After background correction, Ca²⁺ values were calculated on whole RASMCs, which are elongated cells (70x10 µm in size) corresponding to an area of 100x15 pixels with the x40 objective used.

Calibration of fura 2 fluorescence in terms of [Ca²⁺]_i was performed in individual cells and calculated from the ratio of 340-nm/380-nm fluorescence values by using a K_d (Ca²⁺ dye) of 250 nmol/L. Maximum and minimum fluorescence ratios were determined as described previously.¹⁷ The resting [Ca²⁺]_i was determined by averaging Ca²⁺ values obtained for 30 seconds on 5 cells from 3 different coverslips and 3 cell preparations (n=45). The cytosolic Ca²⁺ concentration elevation induced by an agonist ([Ca²⁺]_i) was determined by averaging the values obtained for 36 cells from 3 different cultures.

Cells were continuously superfused at a rate of 1.5 to 2 mL/min with control or test solutions by 6 inlet tubes converging on the coverslip chamber. The perfusion chamber volume was 0.2 mL. Caffeine and ATP were dissolved in perfusion medium, and stock solutions of thapsigargin (TG) were prepared in dimethyl sulfoxide.

Immunofluorescence Study
The SERCA2a and SERCA2b antibodies (a gift from F. Wuytack, University of Leuven, Leuven, Belgium) have been described elsewhere.¹⁸ The IP3R type I antibody was from Affinity Bioreagents. The polyclonal anti-RyR antibody was against the RyR purified from pig skeletal muscle. It reacted with skeletal muscle¹⁹ and cardiac tissue.²⁰ Immunolabeling with antibodies specific for the smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, was used as a marker of the contractile phenotype, whereas the nonmuscle MHC-B (NMB-MHC) antibody was a marker of the undifferentiated phenotype. These antibodies were from Yamasa Corp and have been previously described.²¹ An anti–proliferating cell nuclear antigen antibody (a-PCNA, clone PC10, Dako) was used as marker of proliferation.

The cells were fixed in paraformaldehyde (4%) for 20 minutes at room temperature, washed in PBS and in PBS containing 50 mmol/L NH₄Cl, and permeabilized with 0.1% Triton X-100 (6 minutes). They were washed 3 times and incubated 2 hours at room temperature with diluted antibodies (PBS containing 3% BSA): anti (a)-SERCA2a (1/250), a-SERCA2b (1/250), a-IP₃R (1/120), a-RyR (1/150) or a-SM2-MHC (1/1000), a-SM1-MHC (1/1000), a-NMB-MHC (1/2000), and a-PCNA (1/50). They were incubated 1 hour with biotinylated anti-mouse or anti-rabbit Ig (1/200, Amersham) and 45 minutes with streptavidin–Texas red (1/200) at room temperature. For double immunofluorescence studies, cells were fixed in -20°C methanol (8 minutes) and washed in PBS. PCNA was detected with Texas red–conjugated anti-mouse IgG (Amersham), and the antibodies specific for Ca²⁺ pumps or Ca²⁺ release channels, with biotinylated anti-rabbit immunoglobulin and fluorescein streptavidin (Amersham). Coverslips were mounted in glycerol/PBS medium (FluoroGuard antifade Reagent, Bio-Rad).

Polymerase Chain Reaction
Total RNA from rat brain, aorta, heart, skeletal muscles, cardiomyocytes, hepatocytes, Caco-2 (human colon carcinoma cells), and aortic smooth muscle cells in culture was extracted by the RNA-InstaPure procedure (Eurogentec). Total RNA (1 µg) was used for reverse transcription (RT) by using 2.5 µmol/L random hexamers (Boehringer-Mannheim) or oligo-dT primers (Promega) according to the polymerase chain reaction (PCR) II protocol (Perkin-Elmer). Two PCR reactions were set up from each first-strand reaction. SERCA2b was used as a standard for each reaction. A control without reverse transcriptase was included in each experiment. The reactions were performed in a final volume of 100 µL in a Perkin-Elmer GeneAmp PCR system according to the Perkin-Elmer protocol with 40 cycles of 15 seconds at 95°C, 30 seconds at the specific annealing temperature for the primers, and 30 seconds at 72°C.

Primers for SERCA2a and SERCA2b amplified nucleotides 2615 to 3122 and 3121 to 3630 of published sequences,^{22 23} respectively. RyR1 primers generated a 435-bp fragment from the mouse sequence.¹³ RyR2 primers were from the rabbit cardiac RyR2 cDNA²⁴ and amplified from nucleotides 8603 to 9144. RyR3 primers were as described.²⁵ Sizes of amplified DNA fragments were 614 bp for RyR3-II and 273 bp for RyR3-I. Primers common to all 3 IP3R subtypes were as in Reference ²⁶ . Twenty to 24 nucleotide primers were annealed at 60°C, 50°C, 62°C, 52°C, and 60°C for SERCA2a and SERCA2b, RyR1, RyR2, RyR3, and IP3R, respectively.

The PCR products were subjected to electrophoresis on 8% polyacrylamide gels, stained by using Vistra-green (Amersham), and scanned with a PhosphorfluorImager (Storm, Molecular Dynamics). For RyR3 and IP3R, the DNA was transferred to Hybond N+ membranes and detected by probing with specific oligonucleotides labeled with [-³²P]ATP (NEN) by using T4 kinase (Eurogentec). The RyR3 oligonucleotide corresponds to a region upstream from the splice site.²⁵ The IP3R isotype–specific oligonucleotides used in Southern blotting were as described.²⁶ Hybridization was performed in 6x standard saline citrate (1x SSC contains 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate), 0.1% Ficoll, 0.1% BSA, 0.1% polyvinylpyrrolidone, 0.5% SDS, and 100 µg/mL salmon sperm DNA at 60°C. The membranes were washed with 6x SSC and 2x SSC at room temperature and then with 0.5x SSC at 60°C for RyR3, 50°C for IP3R1 and IP3R2, and 40°C for IP3R3. The membranes were then exposed to the PhosphorImager screen, and signals were recorded by use of Storm.

Statistical Analysis
Sensitivity to caffeine, ATP, or caffeine+ATP was estimated by counting the responsive cells on each coverslip. Results were expressed as a percentage of reactive cells. The resting Ca²⁺ level was determined for each cell population by averaging Ca²⁺ values during 30 seconds for 45 cells (from 3 cultures at each time point and 3 coverslips each). The values of Ca²⁺ peaks, [Ca²⁺]_i, after caffeine or ATP stimulation were estimated on 36 cells (6 coverslips in 3 cultures at each time point). The results were expressed as mean±SEM. For each culture, normality was assessed by using the Kolmogorov-Smirnov test. One-way ANOVA or Kruskal-Wallis ANOVA on ranks, followed by the Student-Newman-Keuls test, was used for the statistical comparison of multiple groups. An unpaired t test was used for comparison of the [Ca²⁺]_i values obtained with caffeine and ATP. Data were analyzed by using the SigmaStat Statistical Software Package, Version 1.0 (Jandel Scientific).

	Results

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RASMCs Contain Connected RyR-Sensitive and IP3-Sensitive Ca²⁺ Pools
Experiments were performed in the absence of external Ca²⁺ and in the presence of EGTA to eliminate any possible effect of Ca²⁺ influx. Caffeine induced transient increases in [Ca²⁺]_i, but little or no increase was observed after subsequent ATP application (Figure 1A). After incubation in normal Ca²⁺ medium, an increase in [Ca²⁺]_i was obtained when ATP was added, but subsequent application of caffeine had no effect (Figure 1A). Thus, both agonists released Ca²⁺ from the same store. Incubation of the same cell with TG (500 nmol/L), a specific inhibitor of the SERCAs, in Ca²⁺-free medium resulted in a slow increase in the intracellular Ca²⁺ level, causing depletion of the Ca²⁺ pools (Figure 1B). Neither caffeine nor ATP induced an increase in [Ca²⁺]_i in the presence of TG. Thus, the caffeine-sensitive and IP3-sensitive pools are both present in freshly dissociated RASMCs, and they are both TG sensitive.

View larger version (19K): [in this window] [in a new window]   Figure 1. Caffeine (Caf)-sensitive and IP3-sensitive stores are present in differentiated RASMCs and are TG sensitive. Representative tracings (of 321 cells from 27 coverslips and 7 cultures) of an experiment performed in the absence of external Ca2+ on RASMCs in culture for 1 day are shown. A, It represents a sequential application of 10 mmol/L Caf and 20 µmol/L ATP. Intracellular Ca2+ stores were then refilled in normal Ca2+ medium, and the order of application of ATP and Caf was reversed. When the Ca2+ store was depleted by one of the agonists, the other did not release Ca2+. B, The stores were refilled by incubation in normal Ca2+ medium for 5 minutes, and then 500 nmol/L TG was added and incubated for 5 minutes in Ca2+-free medium. Subsequent application of 20 µmol/L ATP and 10 mmol/L Caf had no effect on [Ca2+]i.

RASMCs Lose the Caffeine-Sensitive Ca²⁺ Pool in Culture
To determine whether the Ca²⁺ pools were stable or were altered during culture, the percentage of cells responsive to caffeine, ATP, and both agonists and the percentage of nonresponsive cells were determined on days 1, 3, 5, and 12 of culture (Figure 2). The values of resting Ca²⁺ concentration and of the peaks of Ca²⁺ released by each agonist were also determined (Table). On day 1, a total of 321 cells (7 cultures and 3 to 7 coverslips per culture) were analyzed. No statistical difference in sensitivity to caffeine (P=0.214), ATP (P=0.06), or caffeine+ATP (P=0.229) was observed between the 7 different cultures tested. In our cell population, 77% of cells were sensitive to caffeine and ATP, 3% were sensitive to caffeine only, and 8% were sensitive to ATP only, whereas 12% were insensitive to both drugs (Figure 2). The resting Ca²⁺ concentration was 169±6 nmol/L (n=45), and the increases in cytosolic Ca²⁺ concentration induced by caffeine and ATP were not different (736±25 nmol/L and 817±36 nmol/L, respectively; P>0.05; Table).

View larger version (37K): [in this window] [in a new window]   Figure 2. Changes in RASMC sensitivity to caffeine and ATP in primary culture. The percentages of caffeine-responsive, ATP-responsive, caffeine- and ATP- responsive, and nonsensitive cells were determined on days 1, 3, 5, and 12. The fraction of responding cells in each group is expressed as a percentage of the total number of cells. There was a significant difference in ATP sensitivity on days 1, 3, 5, and 12 (P<0.001) and in caffeine+ATP sensitivity on days 1, 3, and 12 (P<0.001). All cells were tested according to the protocol shown in Figure 1A. Caffeine (10 mmol/L) was used for cells in culture for 1 day, and 40 mmol/L caffeine was used for cells in culture at the other stages.

View this table: [in this window] [in a new window]   Table 1. Resting [Ca2+]i and Maximum Peak Values in [Ca2+]i After Caffeine or ATP Application During Culture of RASMCs

When RASMCs were actively proliferating (day 5), caffeine did not induce Ca²⁺ release even at concentrations as high as 40 mmol/L, whereas a transient increase in [Ca²⁺]_i (882±54 nmol/L) was observed after application of ATP (Table). All cells had lost their functional caffeine-sensitive pool, 80% were sensitive to ATP only, and 20% were insensitive to both agonists (Figure 2). This disappearance was progressive, because on day 3, 17% of the cells (16 of 92 cells from 11 coverslips and 3 cultures) were still sensitive to caffeine, whereas none of the cells tested (0 of 190 cells from 3 cultures and 14 coverslips) were sensitive to caffeine on day 5 (Figure 2). In our conditions, the cells were subconfluent and had stopped proliferating after 12 days. By day 12, 54% of the cells (240 of 442 cells from 4 cultures and 19 coverslips) had reacquired sensitivity to caffeine (Figure 2). The cells did not respond to 5 or 10 mmol/L caffeine, and 40 mmol/L caffeine was required to induce Ca²⁺ release. There was no significant difference between the different cultures on day 12 in ATP sensitivity (P=0.444) or ATP+caffeine sensitivity (P=0.186). However, when comparing days 1, 3, 5, and 12, there was a significant difference in ATP+caffeine sensitivity (P<0.001) or in ATP sensitivity alone (P<0.001).

During the time of culture, the resting Ca²⁺ concentration did not vary significantly (P=0.092), and the size of the ATP-sensitive Ca²⁺ pool was also similar (P=0.063, Table). Furthermore, the amounts of Ca²⁺ released by caffeine and ATP were not different on day 1 (P=0.069) or on day 12 (P=0.095). Thus, the difference in SR Ca²⁺ load is not responsible for the absence of sensitivity to caffeine.

Each Freshly Dissociated RASMC Expresses 2 SERCA Isoforms and 2 Ca²⁺ Release Channels
To characterize the phenotype of the isolated RASMCs, the pattern of expression of the genes encoding the SR Ca²⁺ pumps and Ca²⁺ channels was determined, at the protein and RNA levels, on cells exposed to the culture medium for 1 day. Expression of the MHC isoforms and the PCNA antigen were used as markers.

All cells in culture for 1 day in the presence of serum expressed SM1 and SM2 MHCs; NMB-MHC was present only at a low level. This indicates that the cells had a differentiated contractile phenotype. The PCNA antigen was not detected, indicating that the RASMCs were quiescent (Figure 3A).

View larger version (114K): [in this window] [in a new window]   Figure 3. A, RASMCs in culture for 1 day are still differentiated and quiescent. Immunolabeling of RASMCs in culture for 1 day with antibodies specific for the smooth muscle MHCs SM1 (a) and SM2 (b), for NMB-MHC (c), and for PCNA (d) is shown. Bar=10 µm. B, Two Ca2+ pumps and 2 Ca2+ release channels are present in each differentiated RASMC. RASMCs in culture for 1 day were labeled with a-SERCA2a (a), a-SERCA2b (c), a-RyR (e), and a-IP3R (g). In panel B, a, c, e, and g are immunolabelings, and b, d, f, and h represent interference contrast micrographs. Bar=10 µm.

Two SERCA2 mRNA isoforms are present in rat aorta.^{15 27} We used immunofluorescence and interference contrast images to show that SERCA2a and SERCA2b were both expressed in each individual smooth muscle cell. Similarly, RyR and IP3R were present in each cell (Figure 3B). The immunolabeling with a-IP3R was mainly but not exclusively perinuclear, but confocal images revealed no labeling inside the nucleus (not shown). The a-RyR antibody was not isoform specific and reacted with cardiac as well as skeletal microsomes.^{19 20} No reaction was obtained with a monoclonal antibody specific for RyR2, which also reacts weakly with RyR1 (MA3-916, Affinity Bioreagents; not shown).

We checked for the specificity of RyR in smooth muscle by performing RT-PCR with oligonucleotides specific for RyR1, RyR2, and RyR3 (Figure 4). RyR1 mRNA was present in skeletal muscle and brain but not in cardiac muscle. It was also present in aortic preparations containing endothelium but not in those free of endothelium. RyR2 mRNA was present in cardiac muscle, in brain, and in aortic preparations containing endothelium but only at low levels in skeletal muscle or aortic preparations free of endothelium. Two RyR3 mRNA isoforms were detected: the upper band is compatible with RyR3-II (614 bp), and the lower band is compatible with RyR3-I (273 bp).²⁵ RyR3-I was constitutively expressed, whereas RyR3-II was present in brain, total aorta, and aorta without endothelium but not in skeletal muscle or hepatocytes.

View larger version (40K): [in this window] [in a new window]   Figure 4. Detection of RyR1 (A), RyR2 (B), and RyR3 (C) mRNA isoforms. A, RT-PCR was performed with RyR1-specific primers and total RNA from total aorta, aorta free of endothelium (aorta w/o endoth), brain, skeletal muscle, and heart. Skeletal muscle RNA in the absence of reverse transcriptase, skeletal muscle (-), served as a negative control. B, Amplification of RyR2 mRNA by RT-PCR on RNA from total aorta, aorta w/o endoth, brain, skeletal muscle, and heart is shown. RNA from heart in the absence of reverse transcriptase, heart (-), served as a negative control. C, Detection of RyR3 mRNA by RT-PCR in the brain, total aorta, aorta w/o endoth, skeletal muscle, and hepatocytes is shown. RNA from brain and no reverse transcriptase, brain (-), served as a negative control. Amplified cDNA fragments were stained with Vistra-green and detected with a FluorImager (A and B). For DNA size ladder, HaeIII-digested X-174-RF DNA (marker IV, Pharmacia) was used. In panel C, fragments were detected by Southern blotting by using a labeled oligonucleotide (see Methods). Two fragments of sizes consistent with RyR3-I (273 bp) and RyR3-II (614 bp) were amplified and present in aorta.

RASMCs Lose SERCA2a and RyR but Keep SERCA2b and IP3R in Culture
In agreement with previous results,²⁸ the RASMCs had lost the SM2-MHC in culture for 5 and 12 days, but the SM1-MHC was still expressed in each cell, indicating their smooth muscle phenotype. The NMB-MHC was highly expressed, especially at days 5 and 12 (Figure 5A).

View larger version (61K): [in this window] [in a new window]   Figure 5. A, Immunodetection of SM1, SM2, and NMB-MHCs in RASMCs in culture for 3, 5, or 12 days. B, Double-immunolabeling of RASMCs in culture for 1, 3, 5, and 12 days with a-SERCA2a, a-SERCA2b, a-RyR, and a-IP3R (green) and with a-PCNA (red). Bar=10 µm.

To determine whether change in the SR Ca²⁺-transporting enzyme phenotype could account for the loss of caffeine sensitivity that we observed in culture, we first analyzed the protein pattern by immunofluorescence with the use of specific antibodies. The cells were simultaneously labeled with a-PCNA to determine whether proliferation was associated with change in phenotype (Figure 5B). On day 1, all cells were labeled with the antibodies for SERCA2a, SERCA2b, RyR, and IP3R (green), but none were labeled with a-PCNA (red). On day 3, the cells were labeled with a-PCNA, indicating that they entered the cell cycle. SERCA2b and IP3R were expressed at high levels, whereas SERCA2a and RyR were present only at low levels. On day 5, all cells were labeled with a-PCNA. They expressed SERCA2b and IP3R but not SERCA2a and RyR. When cells were subconfluent (day 12), they stopped dividing and did not express the PCNA antigen, and SERCA2a and RyR were detectable again. These data indicate that the expression of SERCA2 and RyR is regulated during culture.

To determine whether regulation of gene expression is translational or pretranslational, we analyzed the Ca²⁺ pumps and Ca²⁺ channel mRNA levels by RT-PCR (Figure 6). SERCA2 (Figure 6A), IP3R (Figure 6B), and RyR (Figure 6C) mRNA isotypes were amplified from total RNA from the aorta, from RASMCs cultured for 1, 3, 5, and 12 days, and from control RNAs. SERCA2b was used as a control in the PCR reaction. SERCA2a was present in RASMCs on day 1 but was absent on days 3 and 5 and was detected again on day 12 (Figure 6A). SERCA2a mRNA was not detected on day 3, whereas the corresponding protein was still present (Figure 5B). This can be explained by differences in the half-lives of mRNA and protein. SERCA2a mRNA was detected again on day 12, together with the corresponding protein

View larger version (27K): [in this window] [in a new window]   Figure 6. Detection of SERCA2 (A), IP3R (B), and RyR (C) mRNA isoforms by RT-PCR in total aorta and RASMCs in culture. Total RNA from skeletal muscle (Sk.muscle), heart, cardiomyocytes, and Caco cells were used as controls. In panel A, PCR products were detected by using Vistra-green. In panel B, IP3R mRNA was amplified by using primers common to all isoforms. Total PCR products were stained by Vistra-green with SERCA2b used as control (2 upper lanes). Each IP3R mRNA isoform was then detected by Southern blotting with specific oligomers (3 lower lanes). Southern blot hybridized with type 1 was exposed for 4 hours, whereas the membranes hybridized with types 2 and 3 were exposed for 16 hours. In panel C, RyR isoforms were detected by using SERCA2b as a control. The cDNAs corresponding to RyR1 and RyR2 were stained by using Vistra green and detected with a FluorImager, whereas the 2 RyR3 isoforms were visualized by Southern blotting. Two alternatively spliced RyR3 mRNAs, RyR 3-I (273 bp) and RyR 3-II (614 bp), were present; RyR3-II disappeared progressively during culture.

IP3R mRNA was detected in each mRNA preparation tested, whereas SERCA2b was below the detection level in isolated cardiomyocytes (Figure 6B). IP3R-1 was the major isoform in the aorta and RASMCs, and its level was rather constant during culture, whereas IP3R-2 was present at low but constant levels in all RASMC preparations. In cardiomyocytes, type 2 was present at high levels as demonstrated,²⁶ but some IP3R-1 was also detected in our experiments. IP3R-3 was not detected in RASMCs at any stage of culture but was present at very high levels in Caco cells, as previously demonstrated (J. Mesonero, personal communication, 1999). Thus, 2 IP3R isotypes were detected in RASMCs, but their mRNA levels did not change during culture.

RyR3-I and RyR3-II mRNAs were present in total aorta and in cultured cells (Figure 6C). RyR3-I but not RyR3-II was also present in skeletal and cardiac muscle. RyR3-I mRNA was produced at all stages of proliferation, whereas RyR3-II disappeared progressively on days 3 and 5 and was not present on day 12. This suggests that splicing of the RyR3 gene is regulated during proliferation as for the SERCA2 gene. Surprisingly, large amounts of RyR3-I mRNA were detected on day 5, whereas there was no RyR protein (Figures 5B and 7B) and no caffeine sensitivity (Figure 2). This suggests that RyR3-I mRNA is not translated or that the protein is unstable. We tested whether another RyR isoform was induced during proliferation and performed RT-PCR with oligonucleotides specific for RyR1 and RyR2 mRNA. RyR2, the major isoform in heart, was not detectable in the aorta or in RASMCs at any stage of culture. However, RyR1 mRNA, the major isoform in skeletal muscle, which was not detected in the aorta or in cells in culture for 1, 3, or 5 days, was present on day 12 (Figure 6C).

View larger version (50K): [in this window] [in a new window]   Figure 7. A, RASMCs in culture for 6 days in different conditions were immunolabeled with antibodies specific for SM2- or NMB-MHCs. Culture conditions were as follows: 6 days in 10% FCS (a and b), 2 days in 10% FCS and 4 days in 0.1% FCS (c and d), 2 days in 10% FCS, 1 day in 0.1% FCS, and 3 days in 0.1% FCS+50 ng/mL PDGF-BB (e and f), and high-density cell culture (105 cells/cm2) with 10% FCS for 6 days (g and h). Bar=10 µm. B, Double immunolabelings of RASMCs cultured as in panel A, with a-SERCA2a, a-SERCA2b, a-RyR, and a-IP3R (in green) and with a-PCNA (in red). Bar=10 µm.

Loss of SERCA2a and RyR Is Associated With the Proliferative State of RASMCs
To gain better insight into the mechanisms of alteration of SR Ca²⁺ pump and channel phenotypes, RASMCs were cultured in different conditions of medium and density, and the phenotype was analyzed by immunofluorescence and RT-PCR.

Expression of SM2- and NMB-MHC was used as a marker of the differentiated and undifferentiated phenotype, respectively (Figure 7A). In 10% FCS, SM2-MHC was absent, and NMB-MHC was present at high levels. In low serum (0.1%), the differentiated phenotype and expression of SM2-MHC were maintained. Addition of PDGF-BB to low serum reversed the phenotype of MHC toward the undifferentiated NMB-MHC. When grown at high density in presence of 10% FCS, the cells had a differentiated phenotype, as shown by the presence of the SM2-MHC.

Double immunolabeling with a-PCNA (in red) and antibodies specific for the SR Ca²⁺ pumps and channels (in green) were performed on the same cell cultures (Figure 7B). In 10% FCS, RASMCs were labeled with a-PCNA and had lost SERCA2a and RyR. In low serum, the cells did not proliferate and were not labeled with a-PCNA. In these conditions, they still expressed SERCA2a and RyR. In the presence of PDGF-BB, the PCNA antigen was detected, indicating that the cells had entered the cell cycle, and SERCA2a and RyR were absent. Plating at high density in the presence of 10% FCS prevented proliferation (no PCNA antigen detected) and loss of SERCA2a and RyR.

To confirm the immunofluorescence data and to determine whether regulation of SERCA2 and RyR gene expression was translational or pretranslational, SERCA2a, SERCA2b, and RyR mRNA levels were measured on the same cultures by RT-PCR (Figure 8). SERCA2a mRNA was present in RASMCs cultured in low serum or at high density, whereas it was absent from cells cultured in 10% FCS or PDGF-BB. RyR3-I was ubiquitously expressed. RyR3-II was absent from cells cultured in high serum or PDGF-BB but was present in cells cultured in low serum or at high density.

View larger version (40K): [in this window] [in a new window]   Figure 8. Detection of SERCA2a, SERCA2b, and RyR3 mRNA isoforms by RT-PCR in total aorta and in RASMCs in the various culture conditions described in legend to Figure 7. PCR products were detected on a FluorImager by using Vistra-green for SERCA2a and SERCA2b. The cDNAs corresponding to RyR3-I and RyR3-II isoforms were detected by Southern blotting with a labeled oligonucleotide. Membrane was exposed for 4 hours. The ladder is the SmartLadder SF (Eurogentec).

These 2 sets of data demonstrate that expression of SERCA2 and RyR3 is regulated at a pretranslational level when RASMCs proliferate.

	Discussion

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Our data demonstrate the following: (1) In freshly isolated RASMCs, Ca²⁺ is released from internal stores by 2 mechanisms, a caffeine- and ryanodine-sensitive mechanism and an IP3-sensitive mechanism, both of which are sensitive to TG. This is supported by the presence in each individual cell of 2 Ca²⁺ release channels, RyR3 and IP3R, and 2 Ca²⁺ pumps, SERCA2a and SERCA2b. (2) Reorganization of the intracellular Ca²⁺ compartments is part of the dedifferentiation process that occurs during culture: the RASMCs lose their caffeine- and ryanodine-sensitive store because of pretranslational changes in the expression of the RyR3 and SERCA2 genes. (3) Expression of RyR3 and SERCA2a is repressed at a pretranslational level when cells acquire a proliferative phenotype.

We have demonstrated that the caffeine sensitivity is lost in proliferating cells but reappears when cells are subconfluent. This observation is consistent with previous results showing that the ryanodine-sensitive Ca²⁺ store was absent from proliferating cultures of RASMCs but was present in subconfluent cultures.⁹ Furthermore, it has been shown that 80% of quiescent RASMCs in the early phase of culture responded to high K⁺, angiotensin II, and caffeine by an increase in [Ca²⁺]_i but that most of the cells had lost their [Ca²⁺]_i responsiveness to the 3 agents in the proliferating phase.^{7 29}

Several explanations may be found for changes in reactivity to pharmacological agents affecting Ca²⁺ release from internal stores. Our data indicate that changes in the caffeine sensitivity cannot be attributed to changes in the SR Ca²⁺ load because the SR Ca²⁺ content, estimated by the amount of Ca²⁺ released by high concentrations of caffeine or ATP, was similar throughout the culture period. The basal cytosolic Ca²⁺ concentration was also unchanged. We show that alteration in caffeine sensitivity is associated with changes in the phenotype of SR Ca²⁺ pumps and channels. Caffeine-insensitive cells do not express RyR but do express IP3R. Similarly, SERCA2a is absent, but the ubiquitous SERCA2b isoform is still present. Subconfluent cells regain sensitivity to high doses of caffeine, but RyR1 rather than RyR3 is present. The low sensitivity to caffeine cannot be explained by the presence of a different RyR isoform because both are sensitive to caffeine.³⁰ Other mechanisms that control the activity of the Ca²⁺ channels, such as the level of intrareticular proteins that regulate Ca²⁺ release and the Ca²⁺ sparks, are also regulated during proliferation. Alternatively, the frequency of the Ca²⁺ sparks, the physiological stimuli of the RyRs, may vary independently of the fact that the RyRs are functional and Ca²⁺ is released by caffeine. Such a situation has been described recently during ontogeny of the cerebral arteries.³¹ All these changes will participate to alter the sensitivity of the vessel to pharmacological vasoconstricting drugs.

Very little is known about the mechanisms of control of the expression of the SR Ca²⁺ pumps and Ca²⁺ channels in smooth muscle. The 2a isoform of the SERCA2 gene is the muscle-specific variant, and splicing of the SERCA2 gene is regulated by myogenic factors during differentiation of a muscle cell line.³² A similar phenomenon may occur in smooth muscle cells, and myogenic factors that are induced when smooth muscle cells differentiate may favor splicing of the SERCA2 gene toward SERCA2a. We have previously shown that the level of SERCA2a mRNA increases in the rat aorta during maturation of the vessel, whereas the level of SERCA2b mRNA remains stable.¹⁵ These 2 sets of results are consistent, and in both cases, the differentiated smooth muscle phenotype was associated with the presence of SERCA2a. We demonstrate that SERCA2a is downregulated by PDGF-BB in RASMCs, whereas upregulation of SERCA2a was observed in porcine aortic cells after 16 to 34 hours in the presence of PDGF-BB.^{33 34} Differences in the initial phenotype of the cells and in the level of action of PDGF-BB may account for some of the discrepancies. Indeed, porcine aortic cells were passaged, and the effect of PDGF-BB was mainly translational,³³ whereas we used primary cultures, and our results indicate that regulation was pretranslational. Differentiation of muscle cell lines was also associated with changes in the caffeine sensitivity and expression of the ryanodine receptor gene,^{35 36} but at that time, the various RyR isoforms were not yet described. Transforming growth factor-ß was shown to induce the expression of RyR3 together with the release of Ca²⁺ in response to ryanodine in mink lung epithelial cells.³⁷

The process of dedifferentiation of vascular smooth muscle cells has been subject to intensive interest because of its importance in vascular pathology. It is clearly demonstrated that when vascular smooth muscle cells reenter the cell cycle, they undergo a transition from a contractile to a synthetic phenotype,¹ and this is associated with major changes in the phenotype of contractile proteins.² They lose the differentiated MHC isoform, SM2, whereas they gain the undifferentiated isoform, NMB-MHC, as shown in the present study. Changes in the expression of the voltage-dependent Ca²⁺ channels were also recently reported.^{5 6} We demonstrate that alteration in the expression of the proteins involved in intracellular Ca²⁺ handling is also part of this process: proliferation is associated with loss of SERCA2a and RyR3. Only SERCA2b and IP3R are present, at RNA and protein levels, in proliferating cells. In our experiments, RyR3 is the major isoform in the aorta, as also shown by Ledbetter et al,¹² whereas RyR2 seems to be predominant in the cerebral arteries.³¹ Type 1 IP3R is the major isoform in the aorta, as previously described^{10 11} but a low amount of type 2 was also detected. No shift in the relative expression of the IP3R isoforms could be observed, and especially type 3, which is predominant in the neonate,¹¹ did not reappear in culture. In nonproliferating RASMCs, SERCA2a and RyR3 are present together with the SM2-MHC isoform. However, in subconfluent cells, the cells do not divide (no PCNA antigen) but express only a very low amount of SM2-MHC; this may be due to the long half-life of the MHC.

Thus, the proliferating RASMCs lose the Ca²⁺-induced Ca²⁺ release mechanism by pretranslational modification in the expression of the RyR3 and SERCA2 genes. In arterial smooth muscles, Ca²⁺ release through RyRs activates Ca²⁺-sensitive K⁺ channels to oppose vasoconstriction.³⁸ Thus, in the absence of this mechanism, the vessel may not be able to counteract the effects of vasoconstricting agents and will become hypersensitive to these drugs. Hypersensitivity to vasoactive stimuli is a common finding in atherosclerotic arteries, which are characterized by the presence of undifferentiated smooth muscle cells. It remains to be demonstrated whether the RyRs are absent from proliferating smooth muscle cells in vivo as they are from cells in culture. If loss of RyR occurs in vivo, this may play an important role in the pathology of arterial and coronary vasospasm and may be an important factor in myocardial ischemia and infarction.

Many factors may turn on and off the smooth muscle program, such as growth factors, cell-cell interaction, extracellular matrix,² and mechanical stresses.³⁹ The extracellular matrix cannot be involved in the modifications described in the present study because the same cell substrate, collagen-type 1, was used in all experiments. Several soluble factors present in the serum may also control gene expression. However, in the presence of 10% FCS, the cells keep the differentiated phenotype when grown at high density or at subconfluence, whereas in the proliferative phase, they are dedifferentiated. Thus, the medium by itself is not sufficient to explain the changes in phenotype that we observed. Cell-cell interaction is certainly an important factor because redifferentiation occurs when cells are confluent and are kept in high-density cultures. The absence of pressure and flow³⁹ and the absence of the endothelial cell lining may also be important factors in the dedifferentiation process. Additional experiments are now needed to define the factors involved in the regulation of SERCA2, RyR, and IP3R gene expression in vascular smooth muscle cells.

	Acknowledgments

 
This study was supported by the Ministère de l’Education nationale, de l’Enseignement Supérieur, de la Recherche, et de l’Insertion professionnelle (ACC-SV9 No. 9509012), Recherche et Partage, and Fondation pour la Recherche Médicale. The authors are grateful to Dr F. Wuytack (University of Leuven, Belgium) for providing the a-SERCA 2. They thank José Mesonero (INSERM U442, Orsay) for sharing unpublished data and providing the Caco cell total RNA.

Received September 6, 1999; accepted November 18, 1999.

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