Ca2 handling is altered when arterial myocytes progress ... - CiteSeerX

Am J Physiol Cell Physiol 295: C779 –C790, 2008. First published July 2, 2008; doi:10.1152/ajpcell.00173.2008.

Ca2⫹ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture Roberto Berra-Romani, Amparo Mazzocco-Spezzia, Maria V. Pulina, and Vera A. Golovina Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland Submitted 25 March 2008; accepted in final form 25 June 2008

Berra-Romani R, Mazzocco-Spezzia A, Pulina MV, Golovina VA. Ca2⫹ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture. Am J Physiol Cell Physiol 295: C779 –C790, 2008. First published July 2, 2008; doi:10.1152/ajpcell.00173.2008.—Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca2⫹ handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP3)- and ryanodine (Ry)-sensitive Ca2⫹ stores, store-operated Ca2⫹ entry (SOCE), and receptor-operated Ca2⫹ entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca2⫹ levels and increased IP3-sensitive Ca2⫹ store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA) inhibitor]-induced Ca2⫹ transients in Ca2⫹-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca2⫹ release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP3 receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP3/CPAsensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP3 receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca2⫹ handling that occurs during vascular growth and remodeling.

smooth muscle cell (ASMC) functions requires different cell phenotypes. In contrast to skeletal and cardiac muscle cells, which differentiate terminally, ASMCs retain a high degree of plasticity and are able to modulate from a contractile phenotype to a proliferative, secretory phenotype. The latter is responsible for vascular growth and remodeling and is an essential element in the adaptation of arteries to physiological and pathological stimuli associated with injury, atherosclerosis, and hypertension (31, 61). Similar phenotypic modulation occurs when vascular myocytes are grown in primary culture in the presence of serum (13, 29, 70).

Phenotype modulation can alter expression of ion channels, transporters, receptors, and contractile proteins. This leads to alterations in Ca2⫹ handling and to the loss of contractility, a critical feature of the differentiated cells (13, 26, 30, 71). The aim of this report is to elucidate mechanisms of Ca2⫹ handling alterations that occur during progression of myocytes from a contractile to a proliferative state in culture. Therefore, in this study we used freshly dissociated and primary cultured ASMCs. Alterations in Ca2⫹ handling associated with cell proliferation have been extensively investigated in heterologous expression systems (63, 73) and in primary cultured vascular myocytes grown in the presence of serum (5–10%) and then in serum-free or low-serum (0.1%) medium (22, 26, 71). Cultured ASMCs stop proliferating in low-serum medium (26, 71); they retain a flat morphology and differ from elongated (spindle shaped), freshly dissociated contractile myocytes. There are also a few reports on modulation of Ca2⫹ signaling during serum-free organ culture of intact vascular tissue (6, 18, 19, 74). Serum-free organ culture of intact vessels preserves contractility but does not stimulate cell proliferation (48). The Ca2⫹ handling alterations may involve changes in the organization of the sarcoplasmic reticulum (SR) Ca2⫹ stores and expression of SR receptors and transporters, as well as changes in Ca2⫹ entry pathways including store-operated channels (SOCs), receptor-operated channels (ROCs), and voltagegated Ca2⫹ channels. Cell proliferation is regulated by intracellular Ca2⫹; maintenance of a sufficient ionized Ca2⫹ concentration in the cytosol ([Ca2⫹]cyt) and within the SR ([Ca2⫹]SR) or endoplasmic reticulum (ER) is required for cell growth and proliferation (8, 26, 63). The contribution of L-type voltage-gated Ca2⫹ channels, critical in regulating smooth muscle contraction, is markedly decreased in dedifferentiated, proliferating ASMCs (41, 59). Data from cell culture studies indicate that intracellular Ca2⫹, required for the cell’s proliferative machinery, can be provided by store-operated Ca2⫹ entry (SOCE) (26). SOCE refills the Ca2⫹ stores and maintains adequate Ca2⫹ levels in the cytosol and nucleus (5, 9, 44, 58). The role of ROCs, activated by agonists acting on G proteincoupled receptors (45), in phenotype-dependent alterations of Ca2⫹ handling is not defined. Evidence indicates that mammalian SOCs and ROCs are homo- or heterotetramers formed by members of a family of seven proteins (TRPC1–TRPC7) that are homologous to the Drosophila transient receptor potential (TRP) channel involved in phototransduction (5, 65). In particular, TRPC1, TRPC4, and TRPC5 may form or be part of the endogenous SOCs

Address for reprint requests and other correspondence: V. A. Golovina, Dept. of Physiology, Univ. of Maryland School of Medicine, 685 W. Baltimore St. HSF1, Rm. 565, Baltimore, MD 21201 (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

store-operated calcium entry; receptor-operated calcium entry; canonical transient receptor potential proteins; stromal interaction molecule 1 and Orai proteins THE DIVERSITY OF ARTERIAL

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activated by SR Ca2⫹ store depletion (5, 55, 77, 78), whereas TRPC3 and TRPC6 can be activated by inositol 1,4,5-trisphosphate (IP3) and/or diacylglycerol and may not be store dependent (38, 45). Recently, two families of transmembrane proteins, Orai and stromal interacting molecule 1 (STIM1) were shown to be essential for the activation of SOCs mainly in nonexcitable cells (36, 79). The role of Orai1 in SOCE was also confirmed in human airway smooth muscle cells (57). Orai1 may form the Ca2⫹ selectivity filter of the Ca2⫹ releaseactivated Ca2⫹ (CRAC) channel (79). There are two other potential homologs of Orai1 in the mammalian genome, Orai2 and Orai3 (20). Orai2 may also constitute or contribute to SOCs (52), but not in all tested cells (27, 28, 57). The role of Orai3 in SOCE is less confirmed (28). There is no evidence to date, however, that Orai proteins play a role in SOCE in vascular smooth muscle. STIM1, the putative Ca2⫹ sensor in the ER, regulates SOC and CRAC channels but not ROCs (36, 56, 60, 67). STIM1 and Orai1 may interact with TRPC proteins (36, 53); the dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in SOC activation in human salivary glands (53). TRPC channels have important functions in vascular smooth muscle (4, 5, 16). We (26) suggested that TRPC1 may be involved in ASMC growth. Antisense DNA targeted to TRPC1 or TRPC6 mRNA inhibits the proliferation of pulmonary artery smooth muscle cells in culture (66, 80). There is one report on the plasticity of TRPC channels during adaptation of intact vessels cultured in serum-free medium (6). Also, TRPC1 is increased in the hyperplasia that follows arterial injury in vivo (42). Expression of TRPCs in these studies, however, is detected mostly by RT-PCR, real-time PCR, and immunocytochemistry (6, 26, 42). Information is lacking about expression of Orai and STIM1 proteins in native vascular smooth muscle and how its expression changes during ASMC phenotype modulation. In this study, we examined whether SOCE and receptoroperated Ca2⫹ entry (ROCE) correlate with expression of various TRPC, Orai, and STIM1 proteins during modulation of arterial myocytes from a contractile to a proliferative state in culture. Furthermore, we investigated the contribution of IP3and ryanodine (Ry)-sensitive Ca2⫹ stores to Ca2⫹ signaling during the ASMC phenotype modulation. Finally, the expression of all three isoforms of IP3 receptors (IP3Rs), Ry receptors (RyRs), and SR Ca2⫹-ATPase (SERCA) 2a and 2b was examined in parallel in freshly dissociated and primary cultured arterial myocytes. Using fura-2 imaging and Western blot analysis, we have shown that modulation of ASMCs from a contractile to a proliferative phenotype in culture is associated with altered characteristics of SR Ca2⫹ stores, increased SOCE and ROCE, and augmented expression of TRPC, Orai, and STIM1 proteins. MATERIALS AND METHODS

Freshly dissociated ASMCs. Protocols involving the use of experimental animals for all experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Arterial myocytes were isolated from rat mesenteric arteries as described previously (7). The superior mesenteric artery from a euthanized male 9- to 10-wk-old SpragueDawley rat was rapidly removed and placed in ice-cold low-Ca2⫹ physiological salt solution 1 (PSS1) with the following composition AJP-Cell Physiol • VOL

(in mM): 140 NaCl, 5.36 KCl, 0.34 Na2HPO4, 0.44 K2HPO4, 10 HEPES, 1.2 MgCl2, 0.05 CaCl2, and 10 D-glucose, pH 7.2. The artery was cleaned of fat and connective tissue and then digested in lowCa2⫹ PSS1 containing 2 mg/ml collagenase type XI, 0.16 mg/ml elastase type IV, and 2 mg/ml bovine serum album (BSA; fat free) for 35 min at 37°C. After digestion, the tissue was washed three times with low-Ca2⫹ PSS1 at 4°C. A suspension of single cells was obtained by gently triturating the tissue with a fire-polished Pasteur pipette in low-Ca2⫹ PSS1. Smooth muscle cells were differentiated by their characteristic elongated morphology. Approximately 200 cells were collected immediately through applied suction by aspiration into a wide-bore patch-clamp pipette and stored at ⫺80°C until use for Western blot analysis. Dispersed cells were also directly deposited on glass coverslips for use in fluorescent microscopy experiments. ASMCs on coverslips were stored at 4°C and used within 4 h. Cells were allowed to settle on the coverslips for 20 –30 min before being loaded with fura-2. Freshly dissociated cells that were markedly contracted under resting conditions (⬍5%) were excluded from evaluation. At the conclusion of Ca2⫹ imaging experiments, the same cells were labeled for smooth muscle ␣-actin to characterize the ASMCs (24). In these experiments, nuclei also were identified by labeling for 5 min with a 50 ␮M solution of 4⬘,6⬘-diamidino-2-phenylindole (DAPI) (22). Primary cultured ASMCs. The methods used for isolation and culture of rat ASMCs have been published previously (24). Briefly, the superior mesenteric artery was isolated in sterile conditions from an euthanized male 9- to 10-wk-old Sprague-Dawley rat, as described above. The artery was incubated for ⬃45 min at 37°C in Ca2⫹- and Mg2⫹-free Hanks’ balanced salt solution (HBSS) containing 1 mg/ml collagenase type 2. After the incubation, the adventitia was carefully stripped and the endothelium was removed (24). ASMCs in the remaining smooth muscle were dissociated by digestion for 35– 40 min at 37°C in HBSS containing 1 mg/ml collagenase and 0.5 mg/ml elastase type IV. The dissociated cells were resuspended and plated on either 25-mm coverslips for use in fluorescent microscopy experiments or 10-cm culture dishes for Western blot analysis. The plated cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere of 5% CO2-95% air at 37°C. The medium was changed on days 4 and 7. Experiments were performed on subconfluent cultures on days 7 and 8 in vitro if not indicated otherwise. The purity of ASMC cultures was verified by positive staining with smooth muscle-specific ␣-actin (22, 24). Most of the cells (⬎99.5%) were ␣-actin positive. The cells also did not cross-react with fibroblast (CD90/Thy-1)- and endothelial (factor VIII, von Willebrand)-specific antigens (24). Proliferative phenotype of cultured ASMCs was confirmed by positive staining with proliferating cell nuclear antigen (a-PCNA) (71). Cultured arterial myocytes lose myosin and become noncontractile, as confirmed by the absence of expression of a specific marker of the contractile phenotype (SM2) smooth muscle myosin heavy chain isoform (24, 71). Primary human aortic myocytes were purchased from Lonza Walkersville (Walkersville, MD). Cells were cultured in smooth muscle basal medium (SmBM) containing 5% FBS at 37°C in a humidified atmosphere of 5% CO2. The subconfluent cells from passages 2– 4 were used for the experiments. Calcium imaging. Cytosolic Ca2⫹ concentration was measured with fura-2 by using digital imaging. Details of fluorescence imaging and analysis techniques were published previously (11). Freshly dissociated or primary cultured ASMCs were loaded with fura-2 by incubation for 35 min in PSS1 or culture medium, respectively, containing 3.3 ␮M fura-2 AM (20 –22°C, 5% CO2-95% O2). After dye loading, the coverslips were transferred to a tissue chamber mounted on a microscope stage, where cells were superfused for 15–20 min (35–36°C) with PSS2 to wash away extracellular dye. The PSS2 contained (in mM) 140 NaCl, 5.9 KCl, 1.2 NaH2PO4, 5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES, pH

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7.4. Cells were studied for 40 – 60 min during continuous superfusion with PSS2 (35°C). The imaging system included a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY). Fluorescent images were recorded using a Gen III ultrablue intensified charge-coupled device camera (Stanford Photonics, Palo Alto, CA). To improve the signal-to-noise ratio, four to eight consecutive video frames were usually averaged at a video frame rate of 30 frames/s. Images were acquired at a rate of one average image every 1–5 s when [Ca2⫹]cyt was changing and every 30 – 60 s when [Ca2⫹]cyt was stable. [Ca2⫹]cyt was calculated by determining the ratio of fura-2 fluorescence excited at 380 and 360 nm as described previously (22, 23). Intracellular fura-2 was calibrated in situ separately in freshly dissociated and primary cultured ASMCs (22). Intracellular Ba2⫹ measurements are shown as fura-2 340/380 excitation ratio with fluorescent emission at 510 nm (11). Immunocytochemistry. ASMCs were fixed and cross-reacted with monoclonal anti-actin, ␣-smooth muscle-FITC antibody (Sigma-Aldrich, St. Louis, MO), monoclonal anti-SM2 antibody (Abcam, Cambridge, MA), or monoclonal anti-proliferating cell nuclear antigen antibody (Dako, Carpinteria, CA). This enabled us to visualize the distribution of specific labels with a fluorescence microscope (Zeiss Axiovert 100; Carl Zeiss). Details were published previously (22, 24). Western immunoblot analysis. Membrane proteins were solubilized in sodium dodecyl sulfate (SDS) buffer containing 5% 2-mercaptoethanol and were separated by polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (11). The following antibodies were used: rabbit polyclonal anti-TRPC1, antiTRPC3, anti-TRPC4, anti-TRPC5, and anti-TRPC6 (Allomone Laboratories, Jerusalem, Israel); rabbit polyclonal anti-RyR-I, antiRyR-II, and anti-RyR-III (gifts from Dr. V. Sorrentino, University of Siena, Siena, Italy); rabbit polyclonal anti-IP3R-I, anti-IP3R-II, and anti-IP3R-III (gifts from Dr. R. J. H. Wojcikiewicz, State University of New York Upstate Medical University, Syracuse, NY); rabbit polyclonal anti-SERCA2a and anti-SERCA2b (gifts from Dr. F. Wuytak, Katholieke University, Leuven, Belgium); rabbit polyclonal anti-Orai1 and anti-Orai2 (Allomone Laboratories); rabbit polyclonal anti-Orai3 (ProSci, Poway, CA); and rabbit polyclonal anti-STIM1 (a gift from Dr. J. Roos, Torrey Pines Therapeutics, La Jolla, CA). Gel loading was controlled with polyclonal or monoclonal anti-␤-actin antibodies (Sigma-Aldrich) or monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Abcam). Neither GAPDH nor ␤-actin expression was changed during induction of proliferation. Materials. FBS was obtained from Atlanta Biologicals (Lawrenceville, GA). SmBM was purchased from Lonza Walkersville. All other tissue culture reagents were obtained from GIBCO-BRL (Grand Island, NY). Human aorta protein medley (homogenate) was purchased from Clontech (Mountain View, CA). Jurkat whole cell lysate was obtained from Abcam (Cambridge, MA). Fura-2 AM and DAPI were obtained from Molecular Probes, Invitrogen Detection Technologies (Eugene, OR). 1-Oleoyl-2-acetyl-sn-glycerol (OAG) and ryanodine were purchased from Calbiochem (San Diego, CA). Collagenase (type 2) was obtained from Worthington Biochemical (Freehold, NJ). Cyclopiazonic acid (CPA), dimethyl sulfoxide, ␤-actin, smooth muscle ␣-actin, collagenase (type XI), elastase (type IV), BSA, caffeine (Caf), ATP, nifedipine, ionomycin, penicillin G, and streptomycin were purchased from Sigma-Aldrich. All other reagents were analytic grade or the highest purity available. Statistical analysis. The numerical data presented in RESULTS are means ⫾ SE from n single cells (1 value per cell). The number of animals is also presented, where appropriate. Data from 6 –18 rats were obtained for most protocols. Statistical significance was determined using Student’s t-test and ANOVA. AJP-Cell Physiol • VOL

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Functionally distinct SR Ca2⫹ stores in freshly dissociated ASMCs. Primary cultured mesenteric artery smooth muscle cells possess two spatially and functionally different types of SR Ca2⫹ stores: release from one (presumably IP3 sensitive) store is abolished by a specific SERCA inhibitor, CPA, whereas release from the Caf/Ry-sensitive store is unaffected (25). The question is, are the functionally distinct Ca2⫹ stores seen in cultured ASMCs also observed in native vascular smooth muscle, i.e., in freshly dissociated myocytes from mesenteric artery? As shown in Fig. 1, mobilization of Ca2⫹ from the Caf/Ry-sensitive Ca2⫹ store did not appear to be essential for the CPA-evoked responses in freshly dissociated ASMCs. Ry, at low concentrations, locks Ca2⫹-induced Ca2⫹ release (CICR) channels in an open, low-conductance state (64); this promotes depletion of the Caf/Ry-sensitive store by Caf. Even increasing the Caf concentration from 10 to 20 mM failed to induce any further Ca2⫹ response in the presence of Ry (not shown). Nevertheless, the Ca2⫹ response induced by CPA (Fig. 1, A and B) was not significantly altered after the Caf/Ry-sensitive stores were depleted by repeated application of Caf in the presence of 1 ␮M Ry (142 ⫾ 7 vs. 145 ⫾ 8 nM in control, untreated cells; n ⫽ 83). Restoration of extracellular Ca2⫹ in the presence of Caf, Ry, and CPA (to prevent both stores from refilling) increased [Ca2⫹]cyt due to SOCE. This [Ca2⫹]cyt rise, after depleting both stores, was significantly larger (318 ⫾ 14 nM; n ⫽ 28) than the SOCE-activated rise induced after depletion of only the CPA-sensitive (134 ⫾ 7 nM; n ⫽ 26) or only the Caf-sensitive store (242 ⫾ 14 nM; n ⫽ 74). To verify that the Ca2⫹ transients were induced in arterial myocytes, immediately after Ca2⫹ measurements, the cells were stained with anti-smooth muscle ␣-actin antibody (MATERIALS AND METHODS). All of the cells were freshly dissociated ASMCs, as indicated by their elongated shapes and their cross-reactivity with this antibody (Fig. 1E). When SR Ca2⫹ stores were first unloaded with CPA in Ca2⫹-free medium, subsequent application of Caf (in the presence of CPA) was still able to induce a Ca2⫹ response, although its amplitude was smaller than that under control conditions without CPA pretreatment (Fig. 1, C and D). Furthermore, depletion of the IP3/CPA-sensitive store in Ca2⫹free medium abolished the response to ATP (Fig. 1F) and phenylephrine (not shown), which trigger IP3 synthesis, but not to Caf (Fig. 1F). This suggests that at least some RyRs are located on stores that have few, if any, IP3Rs not only in cultured ASMCs (25) but also in freshly dissociated myocytes (Fig. 1). Independent regulation of SOCE activated by the depletion of IP3/CPA- and Caf/Ry-sensitive stores. Depletion of IP3sensitive stores is the major trigger for activation of SOCE in nonmuscle cells. Thus the presence of SOCE in smooth muscle cells was previously considered to be associated with their partial reliance on IP3-sensitive Ca2⫹ stores. We addressed two questions. Does depletion of Caf/Ry-sensitive SR store also activate SOCE in ASMCs? If so, is the Caf/Ry-activated SOCE altered during cell phenotype modulation? Depletion of CAF/RY-sensitive Ca2⫹ store in either freshly dissociated (Fig. 2A) or cultured (Fig. 2B) ASMCs activates SOCE. The resting [Ca2⫹]cyt level in freshly dissociated cells was significantly lower than in primary cultured ASMCs (Fig.

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Fig. 1. Two types of functionally distinct sarcoplasmic reticulum (SR) Ca2⫹ stores in freshly dissociated arterial myocytes. A: effect of ryanodine (Ry) on caffeine (Caf)- and cyclopiazonic acid (CPA)-induced cytosolic Ca2⫹ concentration ([Ca2⫹]cyt) transients in arterial smooth muscle cells (ASMCs). Representative record showing the time course of spatially averaged changes in [Ca2⫹]cyt in the cell indicated by the circle in the fura-2 image in Ea (bar, 25 ␮m). B: summarized data showing the CPA-induced transient Ca2⫹ release under control conditions without Caf/Ry pretreatment [(⫺) Caf/Ry] and after depletion of the Caf/Ry-sensitive stores by repeated application of Caf (10 mM) in the presence of 1 ␮M Ry [(⫹) Caf/Ry] (n ⫽ 83). Values are means ⫾ SE. C and F: representative records showing the effect of CPA on Cafinduced [Ca2⫹]cyt transient in the presence of external Ca2⫹ (C) and on ATP- and Caf-induced [Ca2⫹]cyt transients in Ca2⫹-free medium (F). Comparable results were obtained in 32 (C) and 29 other cells (F) dissociated from mesenteric arteries of 8 rats. Caf (10 mM), Ry (1 ␮M), CPA (10 ␮M), ATP (5 ␮M), and Ca2⫹-free media were applied during the times indicated by the horizontal bars. D: summarized data showing the Caf-induced Ca2⫹ response in the absence [(⫺) CPA] and presence of 10 ␮M CPA [(⫹) CPA] (n ⫽ 32). Values are means ⫾ SE. *P ⬍ 0.05 vs. the amplitude of Caf-induced Ca2⫹ response under control conditions without CPA pretreatment. A pseudocolor Ca2⫹ image (Eb) shows resting [Ca2⫹]cyt in freshly dissociated ASMCs. At the end of the Ca2⫹ experiments, the same cells were labeled for ␣-actin (Ec).

2, A–C). The application of 10 mM Caf in Ca2⫹-free medium induced a rapid, large initial Ca2⫹ transient increase in freshly dissociated ASMCs but only a small, slow [Ca2⫹]cyt rise without an initial Ca2⫹ transient in cultured ASMCs (Fig. 2, A and B). The Ca2⫹ response in Ca2⫹-free medium is a manifestation of the unloading of the Caf-sensitive Ca2⫹ store. Subsequent restoration of external Ca2⫹ in the presence of Caf evoked a secondary rise in [Ca2⫹]cyt associated with SOCE (Fig. 2, A and B). To eliminate the contribution of voltagegated Ca2⫹ channels to Caf-induced Ca2⫹ entry, all solutions in these experiments contained 10 ␮M nifedipine, which at this concentration blocks not only L-type but also T-type Ca2⫹ channels (1). AJP-Cell Physiol • VOL

SR Ca2⫹ stores communicate with the plasma membrane to modulate Ca2⫹ entry as a function of [Ca2⫹]SR (33). Although the Caf-evoked Ca2⫹ release in cultured myocytes was approximately seven times smaller than in freshly dissociated ASMCs, the amplitude of subsequent SOCE was not significantly changed (Fig. 2, A–C). This indicates that the relationship between SR Ca2⫹ stores and plasma membrane SOCs might be more complex during ASMC phenotype modulation. We have to take into account not only the Ca2⫹ level in the SR but also changes in expression of SR receptors and transporters as well as changes in SOC subunit composition and their differential expression during ASMC phenotype modulation. Figure 2D, for example, shows that the Caf/Ry-sensitive store

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Fig. 2. Comparison of resting [Ca2⫹]cyt, Caf-induced Ca2⫹ release, Caf-induced store-operated Ca2⫹ entry (SOCE), and expression of Ry receptors (RyRs) in freshly dissociated and primary cultured ASMCs. A and B: records showing the time courses of [Ca2⫹]cyt changes in freshly dissociated myocytes and ASMCs cultured for 7 days. Caf (10 mM) was applied in Ca2⫹-free and Ca2⫹ containing solutions. To eliminate any contribution made by Ca2⫹ influx through voltagegated Ca2⫹ channels, 10 ␮M nifedipine was added 10 min before the recordings shown and was maintained throughout the experiment. C: summarized data showing resting [Ca2⫹]cyt (open bars), the Caf-induced transient Ca2⫹ release in the absence of extracellular Ca2⫹ (shaded bars), and the secondary rise in [Ca2⫹]cyt when Ca2⫹ was added back (solid bars) in freshly dissociated and primary cultured ASMCs. Resting [Ca2⫹]cyt data are from 333 cells. Peak Caf-induced Ca2⫹ release transients and SOCE were studied in 74 freshly dissociated and 44 cultured cells; each bar corresponds to data from a total of 16 rats. Values are means ⫾ SE. *P ⬍ 0.05 vs. resting [Ca2⫹]cyt in freshly dissociated cells. **P ⬍ 0.001 vs. the amplitude of Caf-induced Ca2⫹ release in freshly dissociated myocytes. D: time course of changes in ASMC sensitivity to Caf (10 mM) during cell culture. The fraction of Caf-responsive cells on days 1, 2, 3, 4, and 7 is expressed as a percentage of the total number of cells in each group (66 – 88 cells/ group). All ASMCs were tested according the protocol shown in A and B. Values are means ⫾ SE. *P ⬍ 0.001 vs. freshly dissociated ASMCs. E–G: Western blot analysis of RyR-I (E), RyR-II (F), and RyR-III (G) protein expression in freshly dissociated and primary cultured ASMCs (20 ␮g/lane). Membrane proteins from skeletal muscle (SkM; 0.25 ␮g/ lane), heart (0.25 ␮g/lane), and brain (20 ␮g/lane) were used as controls. RyR proteins (⬃500 kDa) are indicated at left. Representative Western blots are shown; comparable results were obtained in 5 (E), 6 (F), and 6 (G) immunoblots. Dissoc, freshly dissociated ASMCs; Cult, primary cultured ASMCs.

gradually lost its sensitivity to Caf activation during cell culture. The percentage of ASMCs that responded to Caf was determined on days 1, 2, 3, 4, and 7 of culture. All freshly dissociated myocytes and ASMCs cultured for 24 h responded to Caf. On day 3, only 52% of cells were sensitive to Caf, and on day 7, only ⬃4% of cells had a response to Caf (Fig. 2D). Increasing the Caf concentration from 10 to 20 mM did not increase the number of responsive ASMCs (not shown). To determine whether the decline in Caf sensitivity during ASMC phenotype modulation is associated with changes in expression of RyRs, we compared expression of all three isoforms of RyRs in freshly dissociated and primary cultured ASMCs (Fig. 2, E–G). Freshly dissociated myocytes expressed only RyR type II, whereas after 7–10 days in culture, the myocytes expressed only RyR type III. In contrast to Caf-induced SOCE, which was not altered during phenotype modulation (Fig. 2C), CPA-activated SOCE was greatly increased in proliferating ASMCs in culture (Fig. 3). The peak amplitude of the CPA-induced Ca2⫹ transients in Ca2⫹-free medium, reflecting leakage of Ca2⫹ from the SR, was also significantly larger in cultured ASMCs than in freshly dissociated myocytes. The maximal responses to CPA in Ca2⫹-free solution were developed by days 4 and 5 in culture. Note that AJP-Cell Physiol • VOL

CPA-activated SOCE in freshly dissociated myocytes (Fig. 3C) was smaller than CAF-evoked SOCE (Fig. 2C). Under physiological conditions, Ca2⫹ mobilization from the IP3-sensitive store is mediated by a variety of vasoactive agonists, which are known to trigger IP3 synthesis (10). To activate the phosphoinositide/Ca2⫹ signaling cascade, ASMCs were stimulated with the purinergic receptor agonist ATP. Figure 4 shows that ATP (10 ␮M) induced transient, much larger elevations of [Ca2⫹]cyt in Ca2⫹-free medium than did CPA (Fig. 3) in both freshly dissociated and cultured ASMCs. Subsequent restoration of external Ca2⫹ evoked a secondary rise in [Ca2⫹]cyt, apparently associated with both SOCE and ROCE (14). This rise in [Ca2⫹]cyt was significantly larger in cultured than in freshly dissociated ASMCs (Fig. 4C). Similar results were obtained for phenylephrine (not shown). Augmentation of ATP- or phenylephrine-mediated Ca2⫹ release in cultured myocytes correlated with increased expression of all three isoforms of IP3R (Fig. 4, D, F, and G). Freshly dissociated myocytes expressed IP3R-I at a low level (Fig. 4, D and E), and expression of IP3R-II and IP3R-III was negligible (Fig. 4, F and G). The results indicate that expression of IP3R and RyR isoforms is regulated differently. After primary culture of differ-

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Fig. 3. Augmented CPA-induced Ca2⫹ release and SOCE in primary cultured ASMCs. A and B: representative records showing the time course of changes in [Ca2⫹]cyt in freshly dissociated and cultured myocytes. CPA (10 ␮M) was applied in Ca2⫹-free and Ca2⫹-containing solutions. Nifedipine (10 ␮M) was added 10 min before the records shown and was maintained throughout the experiment. C: summarized data showing the CPA-induced transient Ca2⫹ peak in the absence of extracellular Ca2⫹ (shaded bars) and the secondary rise in [Ca2⫹]cyt when Ca2⫹ was added back (solid bars) in freshly dissociated and cultured ASMCs. Peak CPA-induced Ca2⫹ release transients and SOCE were studied in 42 freshly dissociated and 96 cultured cells. Values are means ⫾ SE of 12 experiments (1 rat/experiment). *P ⬍ 0.001 vs. the amplitudes of CPA-induced Ca2⫹ release and SOCE in freshly dissociated myocytes.

entiated ASMCs, expression of all three types of IP3R is increased and expression is switched from RyR-II to RyR-III. This may help to explain differences in ATP- and Caf-induced Ca2⫹ release in freshly dissociated and cultured ASMCs. Enhanced SOCE and upregulated expression of TRPC1/4/5, Orai1/2/3, and STIM1 proteins in cultured proliferating ASMCs. The preceding results show that modulation in phenotype alters expression of RyRs and IP3Rs and their respective sensitivities to Caf and IP3. To activate SOCE independently of receptor activation, we used the Ca2⫹ ionophore ionomycin (12, 37) to completely discharge SR Ca2⫹ stores (46). Freshly dissociated and cultured ASMCs were first treated with ionomycin (0.5 ␮M) in Ca2⫹-free medium to release stored Ca2⫹. The peak amplitude of this ionomycinevoked Ca2⫹ transient was greatly increased in cultured proliferating myocytes (Fig. 5, A and B). Once the [Ca2⫹]cyt had returned to baseline, external Ca2⫹ was restored. This Ca2⫹evoked Ca2⫹ transient (SOCE), too, was greatly augmented during proliferation (Fig. 5, A and B). Augmentation of SR Ca2⫹ stores in cultured proliferating ASMCs correlated with a significant increase of SERCA2b protein expression (Fig. 5, C and D), whereas expression of SERCA2a was not changed (Fig. 5, E and F). Like SOCE, TRPC1 and TRPC5, which are believed to form subunits of SOCs, were upregulated in cultured ASMCs (Fig. 6, A, B, D, and E). TRPC4, also a component of SOCs, was not detected in freshly dissociated cells but was readily detected in proliferating myocytes (Fig. 6C). TRPC2 and TRPC7 were not detected in freshly dissociated and cultured mesenteric and cerebral artery myocytes (6, 32) and, therefore, were not studied. STIM1, a key regulator of SOC, CRAC, and TRPC channels (36, 60, 75), was barely detected in freshly dissociated myocytes but was abundantly expressed in cultured cells (Fig. 6F). Specific antibodies recognizing rat Orai proteins are currently not available. Therefore, we used human reactive antibodies to examine expression of Orai proteins in human aortic homogenate and in human primary cultured proliferating aortic cells. The latter have large CPA-induced SOCE (1,162 ⫾ 72 nM; n ⫽ 40; not shown). Expression of each of the three members of the Orai family in human aortic homogenate was negligible (Fig. 7, A–C). Orai1 was detectable in cultured aortic cells, AJP-Cell Physiol • VOL

although at a level that was ⬃150 times lower than in Jurkat T cells, used as a positive control (Fig. 7A). In contrast to Orai1, Orai2 and Orai3 were abundantly expressed in cultured aortic cells (Fig. 7, B and C). Increased ROCE and augmented expression of TRPC3 and TRPC6 in cultured proliferating ASMCs. Augmented ATPinduced Ca2⫹ influx in proliferating ASMCs (Fig. 4, A–C) could be attributable to both SOCE and ROCE. To determine whether ROCE is indeed increased during proliferation, freshly dissociated and cultured myocytes were stimulated with the cell-permeable diacylglycerol analog OAG. OAG opens TRPC3 and TRPC6 channels in a protein kinase C-independent manner (34). SOCs have high Ca2⫹ selectivity and, unlike ROCs, are virtually impermeable to other alkaline-earth cations, such as Ba2⫹ (72). Therefore, to distinguish ROCs from SOCs, we measured Ba2⫹ entry. Ba2⫹ is not transported by SERCA or plasma membrane Ca2⫹ pumps (43). In the presence of extracellular Ba2⫹, OAG (80 ␮M)-induced elevations of cytosolic Ba2⫹ (fura-2 340/380 ratio) were significantly larger in proliferating ASMCs than in freshly dissociated ASMCs (Fig. 8, A and B). These changes in proliferating myocytes correlated with augmented expression of TRPC3 and TRPC6 proteins (Fig. 8, C–F), which are obligatory components of endogenous ROCs in a variety of cell types (34, 38). DISCUSSION

Modulation of vascular myocytes from a contractile to a proliferative state is important for vascular development and adaptation, but it also contributes to vascular diseases such as hypertension, atherosclerosis, and restenosis following angioplasty (31, 61). A characteristic feature of vascular smooth muscle adaptation and proliferation is alteration in intracellular Ca2⫹ handling. This may precede altered contractile properties and can potentially play a role in the modulation process itself (6). Despite extensive research on vascular smooth muscle phenotype modulation (31, 54), little is known about intracellular Ca2⫹ signaling during modulation (42, 74). The present report describes a study of mechanisms involved in the functional changes of SR Ca2⫹ stores, SOCE, and ROCE associ-

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Fig. 4. Comparison of ATP-induced Ca2⫹ release, SOCE and receptor-operated Ca2⫹ entry (ROCE), and expression of inositol 1,4,5-trisphosphate receptors (IP3Rs) in freshly dissociated and primary cultured ASMCs. A and B: representative records showing the time courses of [Ca2⫹]cyt changes in freshly dissociated and cultured ASMCs. ATP (10 ␮M) was applied in Ca2⫹-free and Ca2⫹-containing solutions. Nifedipine (10 ␮M) was added 10 min before the records shown and was maintained throughout the experiment. C: summarized data showing ATP-induced Ca2⫹ release (shaded bars) and Ca2⫹ influx (solid bars) in freshly dissociated and cultured ASMCs. Peak amplitudes of the ATP-induced Ca2⫹ responses in the absence and presence of external Ca2⫹ were studied in 22 freshly dissociated and 25 cultured cells. Values are means ⫾ SE of 8 experiments (1 rat/experiment). *P ⬍ 0.001 vs. the amplitudes of ATP-induced Ca2⫹ responses in freshly dissociated myocytes in the presence and absence of external Ca2⫹. D, F, and G: Western blot analysis of IP3R-I (D), IP3R-II (F), and IP3R-III (G) protein expression in freshly dissociated and primary cultured ASMCs. Membrane proteins from freshly dissociated myocytes (10 ␮g/lane in D; 40 ␮g/lane in F and G) and primary cultured ASMCs (10 ␮g/lane in D; 20 ␮g/lane in F and G) were loaded and probed with specific anti-IP3Rs antibodies. Representative Western blots are shown. Membrane proteins from brain (2 ␮g/lane in D and 20 ␮g/lane in F and G) were used as controls. E: data normalized to the amount of GAPDH and expressed as means ⫾ SE from 8 Western blots (16 rats). *P ⬍ 0.001 vs. IP3R-I protein expression in freshly dissociated myocytes. Comparable results were obtained in 7 (F) and 8 (G) immunoblots.

ated with ASMC phenotypic modulation from a contractile to a proliferative state in culture. Differential modulation of Ry/Caf- and IP3/CPA-sensitive stores in cultured proliferating ASMCs. In this study, we demonstrated that freshly dissociated myocytes from rat mesenteric artery, like primary cultured ASMCs (25), have two functionally distinct SR Ca2⫹ stores (Fig. 1). This view is supported by the following observations: depletion of the Ry/Caf-sensitive store by repeated application of Caf in the presence of Ry did not affect the response to CPA (Fig. 1, A and B). Conversely, depletion of IP3/CPA-sensitive store abolished the response to ATP but did not eliminate the response to Caf, although its amplitude was reduced (Fig. 1, C, D, and F). Functionally distinct IP3- and CAF-sensitive stores also have been described in freshly dissociated canine pulmonary ASMCs, whereas in canine renal ASMCs, IP3Rs and RyRs release Ca2⫹ from overlapping stores (39). The arrangements of the IP3Rs and RyRs on either a common SR store or two separate stores might reflect the complexity of the underlying vascular smooth muscle function and can vary from tissue to tissue (39, 51). AJP-Cell Physiol • VOL

Modulation of ASMCs from a contractile to a proliferative state in culture was associated with an increase in basal [Ca2⫹]cyt (Fig. 2C) and stored [Ca2⫹] (Figs. 3 and 5, A and B). Maintenance of sufficient [Ca2⫹]SR is required for ASMC growth, and depletion of SR Ca2⫹ stores with thapsigargin inhibits ASMC proliferation (26, 63, 74). We attribute the augmentation of SR Ca2⫹ storage capacity in proliferating ASMCs to the increased SERCA2b protein expression (Fig. 5, C and D), a widely expressed housekeeping isoform (71). Expression of SERCA2a, a muscle-specific isoform, however, was not changed. This is in apparent contrast to some findings in aortic myocytes. For instance, downregulation of SERCA2a has been described in proliferating rat vascular smooth muscle cells (49, 71), whereas increased expression of SERCA2a protein has been observed in porcine aortic myocytes after 16 –34 h in culture in the presence of platelet-derived growth factor (50). Differences in vascular smooth muscle preparations and experimental conditions are likely to account for these discrepancies. The increase in Ca2⫹ release in proliferating ASMCs was associated primarily with IP3/CPA-sensitive store. CPAevoked Ca2⫹ transients in Ca2⫹-free medium (Fig. 3) and ATP-

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Fig. 5. Elevated ionomycin-induced Ca2⫹ release, SOCE, and augmented expression of sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA) 2b in cultured ASMCs. A: representative records showing the time course of [Ca2⫹]cyt changes induced by 0.5 ␮M ionomycin in the absence and presence of extracellular Ca2⫹ in freshly dissociated and cultured ASMCs. B: summarized data showing the ionomycin-induced transient Ca2⫹ peak in the absence of extracellular Ca2⫹ (shaded bars) and SOCE (solid bars) in freshly dissociated and cultured myocytes. Data are means ⫾ SE of 78 freshly dissociated and 74 cultured cells from a total of 6 rats. **P ⬍ 0.001 vs. freshly dissociated ASMCs. C and E: Western blot analysis of SERCA2b and SERCA2a expression in freshly dissociated and primary cultured ASMCs. Membrane proteins (5 ␮g/ lane) were loaded and probed with specific antibodies. Representative Western blots are shown. D and F: data normalized to the amount of GAPDH and expressed as means ⫾ SE from 5 (D) and 4 (F) immunoblots (12 rats). *P ⬍ 0.001 vs. freshly dissociated cells.

induced Ca2⫹ release (Fig. 4, A–C) were significantly larger in proliferating ASMCs. Conversely, Caf-induced Ca2⫹ release was much smaller in cultured proliferating myocytes (Fig. 2, A–C). The roles of IP3Rs and RyRs in regulating vascular smooth muscle phenotype are unclear (71, 73, 74). If IP3Rs and RyRs are implicated in regulating ASMC phenotypic modulation, proliferating vascular myocytes should exhibit changes in IP3R and RyR isoform expression. Indeed, we observed dramatic augmentation of expression of all three IP3R isoforms in cultured proliferating ASMCs (Fig. 4, D–G). This promotes depletion of augmented IP3/CPA-sensitive Ca2⫹ stores (Fig. 4,

A–C), leading to increased activation of SOCE, crucial for cell proliferation (22). Complete knockdown of IP3R-I (dominant isoform with higher affinity for IP3) but not IP3R-III expression abolished IP3-evoked Ca2⫹ release, SOCE, and arrested proliferation of A7r5 vascular smooth muscle cells (73). Increased expression of IP3R-II and IP3R-III was also observed in cultured rat aortic myocytes compared with aortic homogenate (69). Moreover, neonatal rat blood vessels, in which vascular myocytes are predominantly in a proliferating state, expressed more IP3R-III than fully differentiated blood vessels from adult rats (68).

Fig. 6. Augmented expression of transient receptor potential C (TRPC1, TRPC4, and TRPC5) and stromal interacting molecule 1 (STIM1) proteins in proliferating ASMCs. A, C, D, and F: Western blot analysis of TRPC1 (A), TRPC4 (C), TRPC5 (D), and STIM1 (F) expression in freshly dissociated and primary cultured ASMCs. Membrane proteins (10 ␮g/lane) were loaded and probed with specific antibodies. Representative Western blots are shown. B, E, and G: data normalized to the amount of GAPDH and expressed as means ⫾ SE from 9 (B), 7 (E), and 5 (G) immunoblots (18 rats). *P ⬍ 0.001 vs. freshly dissociated cells. Comparable results were obtained in 8 (C) immunoblots.

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Fig. 7. Augmented expression of Orai proteins in proliferating ASMCs. Western blot analysis of Orai1 (A), Orai2 (B), and Orai3 (C) expression in human aortic homogenate (Homog; 20 ␮g/lane) and human cultured aortic cells (Cult; 20 ␮g/lane). Jurkat whole cell lysate (20 ␮g/lane) was used as a positive control. Representative Western blots are shown. Comparable results were obtained in 4 (A), 3 (B) and 3 (C) immunoblots.

In contrast to augmented expression of IP3Rs in proliferating ASMCs in culture, induction of proliferation with serum or platelet-derived growth factor reduces expression of RyRs, leading to loss of the Caf/Ry-sensitive Ca2⫹ pool (71). Vascular smooth muscle cells express mainly RYR-II and RYR-III (62). We observed a switch from the RYR-II to RYR-III expression on conversion to the proliferating phenotype (Fig. 2, F and G). Smooth and cardiac muscles share the expression of the ␣1C L-type Ca2⫹ channel isoform and RyR-II. The latter is required for Ca2⫹ release induced by Ca2⫹ entering through voltage-gated channels. Since the voltage-gated Ca2⫹ channels

Fig. 8. Enhancement of ROCE correlates with augmented expression of TRPC3 and TRPC6 proteins in proliferating ASMCs. A: representative records showing the time course of the ratio (F340/F380) signal induced by 80 ␮M 1-oleoyl-2-acetyl-sn-glycerol (OAG) in freshly dissociated and cultured ASMCs. Extracellular Ca2⫹ was replaced by 1 mM Ba2⫹ at the time indicated by the horizontal bar. Nifedipine (10 ␮M) was applied 10 min before the record shown and was maintained throughout the experiment. B: summarized data showing OAG-induced ROCE in freshly dissociated (n ⫽ 46) and cultured ASMCs (n ⫽ 38). Values are means ⫾ SE. **P ⬍ 0.001 vs. freshly dissociated ASMCs. Each bar corresponds to data from a total of 8 rats. C and E: Western blot analysis of TRPC3 and TRPC6 expression in freshly dissociated and cultured myocytes (10 ␮g/lane). D and F: data normalized to the amount of GAPDH and expressed as means ⫾ SE from 6 (D) and 8 (F) immunoblots (18 rats). *P ⬍ 0.001 vs. freshly dissociated cells. AJP-Cell Physiol • VOL

lose their function in proliferating vascular myocytes (59), the requirement for RYR-II in cultured cells should be diminished, as we observed. In contrast to RyR-II, RyR-III is not involved in Ca2⫹-induced Ca2⫹ release and is less sensitive to activation by Caf (10 mM) when extracellular solution contains 1.7 mM Ca2⫹ (15). This may explain why proliferating ASMCs, which express mainly RyR-III (Fig. 2G), are less responsive to Caf activation (Fig. 2, B and D). SOCE and ROCE are augmented in proliferating ASMCs. Modulation of ASMCs from a contractile to a proliferative phenotype in culture was marked by greatly augmented Ca2⫹ entry through SOCs. SOCE was activated by depletion of either Ry/Caf- or IP3/CPA-sensitive store in both freshly dissociated and primary cultured rat ASMCs (Figs. 2, A and B, 3, and 4, A and B). IP3Rs and RyRs are both implicated in triggering SOCE in porcine airway myocytes as well (3). Kiselyov et al. (40) demonstrated that RyRs, like IP3Rs, can interact with and gate store-operated human TRP3 (hTRP3) and CRAC current (ICRAC) channels. Augmentation of SOCE in proliferating ASMCs was mostly associated with depletion of IP3/CPA-sensitive store, which was also significantly augmented (Figs. 3 and 4, A and B). Caf-induced SOCE during phenotype modulation was, however, unchanged, although the peak amplitude of the Caf-induced Ca2⫹ release was much smaller (Fig. 2, A–C). Furthermore, Caf-activated SOCE in freshly dissociated myocytes was larger than CPA-induced SOCE. The results provide evidence for differential regulation of SOCE activated by the depletion of the IP3/CPA- and Ry/Caf-sensitive stores in freshly dissociated and cultured ASMCs. Different amplitudes of SOCE activated by depletion of IP3/CPA- and Ry/Caf-sensitive stores may also indicate that they are functionally independent. Moreover, we demonstrated for the first time that the transition from a contractile to a proliferative state in culture greatly increased ROCE, which was measured as extracellular Ba2⫹ influx activated by the cell-permeable diacylglycerol analog OAG (Fig. 7, A and B). In proliferating ASMCs, which have downregulated L-type voltage-gated Ca2⫹ channels, SOCE and ROCE may serve as compensatory Ca2⫹ entry pathways to provide the intracellular Ca2⫹ required for cell proliferation. Plasticity of TRPC, Orai, and STIM1 protein expression during phenotypic modulation of ASMCs in culture. The majority of functional TRPC channels in vivo are heterotetrameric complexes of different TRPC subunits (2, 5). TRPC1 associates with TRPC4 and TRPC5, whereas TRPC6 can associate with TRPC3 and TRPC7 (21, 35). When one of the TRPC subunits is suppressed, compensatory upregulation of other subunits can be observed (17). We found that all three potential partners in SOCs (TRPC1, TRPC4, and TRPC5) are upregulated in cultured proliferating myocytes (Fig. 6). In contrast to

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TRPC1 and TRPC5, expression of TRPC4 is negligible in freshly dissociated ASMCs. This indicates that the heterotetrameric TRPC channel structure might be different in freshly dissociated and cultured myocytes. Upregulated expression of TRPC1/4/5 proteins in proliferating ASMCs is consistent with the observed increase in SOCE. Which specific TRPC channels are involved in Ca2⫹ entry following the unloading of IP3/ CPA- or Caf/Ry-sensitive store is, however, unknown and requires further study. Molecular candidates for ROCs, TRPC3 and TRPC6 are also upregulated in cultured proliferating ASMCs. Notably, this is the first study to directly correlate increased TRPC3/TRPC6 protein expression with the augmented OAG-induced ROCE during modulation of arterial myocytes from a contractile to a proliferative phenotype in culture. Augmentation of TRPC6 protein expression is not observed in serum-free organ culture of intact vessels, which preserves contractility, although TRPC6 mRNA is increased (6). Implication of Orai proteins in SOCE in vascular smooth muscle is unknown. SOCs in vascular smooth muscle cells and ICRAC in nonexcitable cells have striking differences in biophysical properties, permeability to Ca2⫹, and activation mechanisms (2, 26, 47). This indicates that molecular composition of SOCs and ICRAC may be different. We demonstrated that native arteries readily express TRPC1 and TRPC5 proteins (Fig. 6) but do not express Orai proteins, which, on the contrary, are abundantly expressed in Jurkat T cells (Fig. 7). SOCE in native arterial myocytes might rather be attributable to the activity of TRPC channels. In cultured proliferating aortic cells, which abundantly express Orai2, Orai3, and much less Orai1, the role of these proteins in SOCE may be more significant. Augmented expression of Orai proteins in proliferating aortic myocytes might also implicate them in phenotype modulation. The essential role of STIM1, the Ca2⫹ sensor in the SR/ER, for SOCE was recently identified in a variety of cell types, including cultured vascular smooth muscle cells (56, 67). After store depletion, STIM1 relocalizes and accumulates within “junctional” ER structures located 10 –25 nm from the plasma membrane (76). Notably, TRPC channels are also confined to the plasma membrane microdomains adjacent to the underlying “junctional” ER (23). The molecular interactions involved in activation of ICRAC and SOCs by STIM1 remain, however, unknown. Suppression of STIM1 with small interfering RNA greatly reduces thapsigargin- or CPA-activated SOCE (56, 67). Our study revealed that STIM1 protein expression is markedly upregulated in cultured proliferating ASMCs (Fig. 6F). These data are in agreement with a previous study demonstrating that downregulation of STIM1 suppresses phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth in culture (67). In this report we identified mechanisms involved in the functional changes of IP3/CPA- and Caf/Ry-sensitive Ca2⫹ stores and of SOCE and ROCE during the arterial myocyte switch from a contractile to a proliferative phenotype in culture. The phenotype-dependent alterations in SR Ca2⫹ release and augmented Ca2⫹ entry through SOCs and ROCs are consistent with the observed changes in expression of RyRs and upregulation of IP3Rs, SERCA2b, STIM1, and various TRPC proteins. Similar alterations in Ca2⫹ signaling associated with arterial myocyte proliferation may apparently occur AJP-Cell Physiol • VOL

under some pathological conditions, including vascular injury, atherosclerosis, and hypertension. ACKNOWLEDGMENTS We thank Dr. M. P. Blaustein for insightful discussion. Present address of R. Berra-Romani: School of Medicine, Beneme`rita Universidad Auto`noma de Puebla, 72000 Puebla, Mexico. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grant PO1-HL-078870 Project 2 (to V. A. Golovina) and by funds from the University of Maryland School of Medicine. REFERENCES 1. Akaike N, Kostyuk PG, Osipchuk YV. Dihydropyridine-sensitive lowthreshold calcium channels in isolated rat hypothalamic neurones. J Physiol 412: 181–195, 1989. 2. Albert AP, Saleh SN, Peppiatt-Wildman CM, Large WA. Multiple activation mechanisms of store-operated TRPC channels in smooth muscle cells. J Physiol 583: 25–36, 2007. 3. Ay B, Prakash YS, Pabelick CM, Sieck GC. Store-operated Ca2⫹ entry in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 286: L909 –L917, 2004. 4. Beech DJ. Emerging functions of 10 types of TRP cationic channel in vascular smooth muscle. Clin Exp Pharmacol Physiol 32: 597– 603, 2005. 5. Beech DJ, Muraki K, Flemming R. Non-selective cationic channels of smooth muscle and the mammalian homologues of Drosophila TRP. J Physiol 559: 685–706, 2004. 6. Bergdahl A, Gomez MF, Wihlborg AK, Erlinge D, Eyjolfson A, Xu SZ, Beech DJ, Dreja K, Hellstrand P. Plasticity of TRPC expression in arterial smooth muscle: correlation with store-operated Ca2⫹ entry. Am J Physiol Cell Physiol 288: C872–C880, 2005. 7. Berra-Romani R, Blaustein MP, Matteson DR. TTX-sensitive voltagegated Na⫹ channels are expressed in mesenteric artery smooth muscle cells. Am J Physiol Heart Circ Physiol 289: H137–H145, 2005. 8. Berridge MJ. Calcium signalling and cell proliferation. Bioessays 17: 491–500, 1995. 9. Berridge MJ. Capacitative calcium entry. Biochem J 312: 1–11, 1995. 10. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 361: 315–325, 1993. 11. Beskina O, Miller A, Mazzocco-Spezzia A, Pulina MV, Golovina VA. Mechanisms of interleukin-1␤-induced Ca2⫹ signals in mouse cortical astrocytes: roles of store- and receptor-operated Ca2⫹ entry. Am J Physiol Cell Physiol 293: C1103–C1111, 2007. 12. Bishara NB, Murphy TV, Hill MA. Capacitative Ca2⫹ entry in vascular endothelial cells is mediated via pathways sensitive to 2-aminoethoxydiphenyl borate and xestospongin C. Br J Pharmacol 135: 119 –128, 2002. 13. Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 59: 1– 61, 1979. 14. Chin TY, Chueh SH. Distinct Ca2⫹ signalling mechanisms induced by ATP and sphingosylphosphorylcholine in porcine aortic smooth muscle cells. Br J Pharmacol 129: 1365–1374, 2000. 15. Coussin F, Macrez N, Morel JL, Mironneau J. Requirement of ryanodine receptor subtypes 1 and 2 for Ca2⫹-induced Ca2⫹ release in vascular myocytes. J Biol Chem 275: 9596 –9603, 2000. 16. Dietrich A, Chubanov V, Kalwa H, Rost BR, Gudermann T. Cation channels of the transient receptor potential superfamily: their role in physiological and pathophysiological processes of smooth muscle cells. Pharmacol Ther 112: 744 –760, 2006. 17. Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, Obst M, Yildirim E, Salanova B, Kalwa H, Essin K, Pinkenburg O, Luft FC, Gudermann T, Birnbaumer L. Increased vascular smooth muscle contractility in TRPC6⫺/⫺ mice. Mol Cell Biol 25: 6980 – 6989, 2005. 18. Dreja K, Bergdahl A, Hellstrand P. Increased store-operated Ca2⫹ entry into contractile vascular smooth muscle following organ culture. J Vasc Res 38: 324 –331, 2001. 19. Dreja K, Hellstrand P. Differential modulation of caffeine- and IP3induced calcium release in cultured arterial tissue. Am J Physiol Cell Physiol 276: C1115–C1120, 1999. 20. Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. A mutation in Orai1 causes

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