Orai1 and Ca²⁺-independent phospholipase A₂ are required for store-operated I_cat-SOC current, Ca²⁺ entry, and proliferation of primary vascular smooth muscle cells

Abstract

Store-operated Ca²⁺ entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca²⁺-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca²⁺-independent phospholipase A₂ (iPLA₂β, or PLA2G6) in activation of cat-SOC current (I_cat-SOC), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA₂β leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA₂β are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.

ca²⁺ influx is essential for a variety of vascular smooth muscle cell (SMC) functions and is known to be mediated by multiple mechanisms. Because of their phenotypic plasticity (46, 47), SMC are able to physiologically switch from contractile (excitable) to synthetic (nonexcitable) phenotype, which may share some important characteristics of excitable and nonexcitable cells. Store-operated Ca²⁺ entry (SOCE) mechanism was found to be present in both contractile and synthetic SMC (for reviews see Refs. 3, 9, 13, 26, 31, 33, 63). SOCE plays an important role in agonist-induced SMC constriction (1, 18, 28, 50, 55, 57) and nitric oxide-induced relaxation (18), which are the hallmarks of the contractile state of primary SMC. At the same time, SOCE was found to be upregulated in synthetic SMC (10, 15, 27) and was shown to play an important role in their proliferation (10, 27) and migration (52).

One of the most important recent discoveries was identification of Orai1 as a pore-forming subunit of the Ca²⁺-selective store-operated channel (CRAC) (24, 34, 35, 72). However, while there is an agreement on the ability of Orai1 to encode a Ca²⁺-selective SOC (CRAC), the role of Orai1 in primary vascular SMC remains an open question.

Because of the phenotypic plasticity and the ability of vascular SMC to share the features of excitable and nonexcitable cells (46, 47), the molecular nature of store-operated channels and determinants of SOCE in primary SMC remains obscure. On one hand, Orai1 was found to mediate SOCE in synthetic (proliferating) arterial myocytes (7) and in the rat aortic A7r5 cell line (52) and was shown to play an important role in neointima formation (76). Orai1-mediated I_CRAC current was recently reported in cultured SMC of high passages, or cell lines that represent SMC of synthetic (nonexcitable) phenotype (52). However, I_CRAC had not been found in primary differentiated SMC; in contrast to synthetic SMC lines, SOCE in primary SMC was found to be mediated by poorly cation-selective store-operated channels (cat-SOC) (2–4, 38, 43, 64, 65) that are thought to be advantageous for excitable cells. Indeed, cat-SOC channels in primary SMC poorly discriminate Ca²⁺ and Na⁺ ions (65), and under physiological conditions they not only provide Ca²⁺ to refill the stores, but also create a nonselective cation current (I_cat-SOC) that is capable of membrane depolarization, that in turn can activate voltage-gated L-type Ca²⁺ channels (51), and trigger agonist-induced SMC constriction (50). Because of a profoundly different Ca²⁺ selectivity of CRAC and cat-SOC channels, the role of Orai1 in I_cat-SOC in primary SMC cells remains obscure and requires further assessment.

It is now well established that STIM1 [a Ca²⁺ sensor protein in endoplasmic reticulum (ER)] triggers activation of Orai1-encoded channels (for most recent reviews, see Refs. 17, 22, 23, 25, 30, 48). In heterologous systems, overexpressed STIM1 and Orai1 were shown to colocalize in ER-plasma membrane junctions, where they can interact and form a molecular complex that allows transmission of the signal from STIM1 in depleted stores, to Orai1 in plasma membrane, which leads to activation of Orai1-encoded channels (for review, see Refs. 17, 22, 23, 25, 30, 48). Endogenous Orai1 is essential for Ca²⁺-selective store-operated (CRAC) current (I_CRAC) in nonexcitable cells, and its molecular knockdown was shown to inhibit I_CRAC current, SOCE, and cell proliferation.

Studies in primary SMC demonstrated that activation of endogenous SOCE requires not only STIM1, but also the presence and functional activity of Ca²⁺-independent phospholipase A₂ (iPLA₂β, or PLA2G6) (57–60). This requirement is not limited to SMC, as iPLA₂β was also found to be essential for SOCE in a wide variety of excitable and nonexcitable cells, including Jurkat T lymphocytes (61), rat basophilic leukemia (RBL) cells (19, 75), platelets (61), astrocytes (56), keratinocytes (54), skeletal muscle cells (12), fibroblasts (37), prostate cancer cells (66), endothelial cells (11), a neuroblastoma/glioma cell line (20), and others. Importantly, along with Orai1 and STIM1, iPLA₂β was identified as a crucial component of SOCE in a comprehensive screen of Drosophila melanogaster genes (68). Careful dissection of the signaling events leading to activation of endogenous store-operated channels in native cells (19, 29, 59) led us to a proposal that iPLA₂β can be an important component of signal transduction between endogenous ER-resident STIM1 and endogenous Orai1 in plasma membrane (14). Identification of the relative importance of endogenous Orai1 and iPLA₂β for activation of I_cat-SOC current and SOCE in primary vascular SMC is a focus of the present study, which will also address their mutual roles in SMC proliferation. So far, the role of iPLA₂β in proliferation of vascular SMC had not been established, and the importance of iPLA₂β for cell cycle and proliferation in other cell types (6, 36, 62) was attributed to regulation of membrane phospholipids that are required for cell growth/proliferation, rather than Ca²⁺ signaling.

Thus, specific goals of this study were to find out whether Orai1 can be responsible for I_cat-SOC current in primary SMC, to determine the relative roles of Orai1 and iPLA₂β in I_cat-SOC and SOCE, and to test whether molecular knockdown of iPLA₂β could mimic the inhibitory effects of knockdown of Orai1 on proliferation of primary SMC. The results of these studies were published as an abstract (71).

MATERIALS AND METHODS

Primary aortic SMC.

Primary SMC were isolated from either mouse (mSMC) or rabbit (rbSMC) aorta, as described in our earlier study (65). To briefly summarize, mSMC were freshly isolated from aortas of two mice using collagenase (type 2, 2 mg/ml, Worthington) and elastase (0.5 mg/ml, Roche). Dispersed mSMC were placed on glass coverslips in a 35-mm petri dish and kept in Dulbecco's modified Eagle's medium (DMEM, GIBCO) with 1% heat-inactivated FBS. Under low-serum concentrations, freshly isolated mSMC adhere to the surface (have spindle-like shape), do not proliferate, retain their contractile phenotype, and provide a model for electrophysiological and Ca²⁺ imaging studies of differentiated (contractile) SMC. Limitations of this model (in comparison with proliferating cells) include low cell counts, and lower efficiency of cell transfection that limits the ability to fully downregulate the proteins. For studies of primary synthetic SMC, we used a model of primary rbSMC described before (65). This model provided high numbers of cells, and high transfection rates, and allowed the studies of SMC proliferation. Briefly, thoracic aorta was removed from male New Zealand White rabbits (2–2.5 kg) and cleaned of adherent fat, connective tissue, and endothelium. The strips of rbSMC were removed from the medial layer, dispersed with collagenase (4 mg/ml, type 2, Worthington) and elastase (1 mg/ml, Roche), and placed on coverslips in 35-mm petri dishes. rbSMC were grown in M199 medium with 20% heat-inactivated FBS at 37°C in 5% CO₂ for up to 7 days. Only primary (nonpassaged) mSMC and rbSMC were used in all of the studies. All animal experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and were approved by the Institutional Animal Care and Use Committee of Boston University.

SMC transfection and molecular studies.

Lipofectamine Transfection Reagent (Invitrogen) was used for transfection of primary mSMC and rbSMC. Cells were transfected with 1) small interfering RNA (siRNA) against Orai1 (5′-GUCCACAACCUCAACUCCTT-3′), or scrambled RNA (Ambion); and 2) antisense (5′-fluorescein-CTCCTTCACCCGGAATGGGT-3′) or sense (5′-fluorescein-ACCCATTCCGGGTGAAGGAG-3′) specific to iPLA₂β. Orai1^GFP was used as described elsewhere (29).

For electrophysiological and imaging studies of mSMC, the cells were kept in 1% FBS and transfected 3 days after isolation from the vessel. Experiments were done 36–48 h after transfection. Construct encoding green fluorescent protein (GFP; 0.5 μg/ml, Lonza) was added for visual detection of transfected cells for patch-clamp and Ca²⁺ imaging experiments. Transfection rate was ∼50%, as determined by GFP expression in control experiments.

For proliferation studies, rbSMC were transfected on day 3 at ∼25% confluence. Transfection rate was 80–90% as determined by GFP expression in control experiments.

For assessment of endogenous Orai1 expression, total RNA was extracted from SMC freshly isolated from mouse, rabbit, and rat using High Pure RNA Isolation Kit (Roche). cDNA templates were created with a High Capacity RNA-to-cDNA kit (Applied Biosystems). Orai1 cDNA was amplified with PCR and analyzed in 2% agarose gel. Expected length of Orai1 products calculated on the basis of nucleotide sequence is 740 bp for mSMC and 785 bp for rSMC. Since the sequence of rbOrai1 has not been reported to date, the cDNA fragment corresponding to rbOrai1 was amplified using primers designed for its homologs, and was sequenced to confirm its identity. Quantitative PCR (qRT-PCR) was done using the rat TaqMan assay (Applied Biosystems).

Electrophysiology.

Whole cell currents were recorded in primary mSMC using standard whole cell (dialysis) patch-clamp technique as previously described (19, 65). An Axopatch 200B amplifier was used, and data were filtered at 1 kHz and digitized at 5 kHz. Pipettes were used with tip resistance of 2–4 MΩ. After breaking into the cell, the holding potential was 0 mV, and ramp depolarization (from −100 to +100 mV, 150 ms) was applied every 3 s. The amplitude of the current was expressed in pA/pF. The time course of current development was analyzed at −80 and +80 mV for each individual cell. Average current-voltage relationships (from 5–10 cells as specified on the figures) are shown during ramp depolarization after the current reached its maximum. Passive leakage current with zero reversal potential [at the moment of breaking into the cell, or after cat-SOC current inhibition with 10 μM diethylstilbestrol (74)] was subtracted. Standard intracellular (pipette) solution contained (in mM) 100 CsAspartate, 40 CsCl, 4.5 NaCl, 10 BAPTA, and 10 HEPES (pH 7.2). Standard extracellular solution contained (in mM) 130 NaCl, 2 CaCl₂, 5 HEPES, 3 CsCl, 1 MgCl₂, and 10 TEA (pH 7.4). Experiments were performed at 20–22°C.

Ca²⁺ influx studies.

mSMC and rbSMC were loaded with fura-2AM (Molecular Probes), and changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored as previously described (19, 59). A dual-excitation fluorescence imaging system (Intracellular Imaging) was used for studies of individual cells. The changes in intracellular Ca²⁺ were expressed as ΔRatio, which was calculated as the difference between the peak F₃₄₀/F₃₈₀ ratio after extracellular Ca²⁺ was added, and its level right before Ca²⁺ addition. Data were summarized from independent experiments done in at least three different cell preparations for each experimental condition.

Proliferation studies.

Proliferation of primary rbSMC was assessed using two methods: cell counting and 5-bromo-2-deoxyuridine (BrdU) incorporation analysis (see immunostaining methods below). Cells were freshly isolated from rabbit aorta and cultured in 20% FBS for up to 7 days. For cell counting, the number of cells in three different 0.25-mm² areas in two wells (total of 6 areas) was counted each day. The time course of cell proliferation is shown as cell number (×10³) per cm².

Imaging.

A Nikon TE2000 wide field system with a ×60 oil immersion objective (1.4 numerical aperture) was used for imaging of live and fixed primary rbSMC as previously described for human embryonic kidney 293 (HEK293) cells (29). Fluorescence was monitored using the following filter sets (Chroma): 1) for GFP: ET470/40 (excitation), ET525/50 (emission), and T495LP (dichroic), and 2) for Texas Red: ET560/40 (excitation), ET630/75 (emission), and T585LP (dichroic). Stack of images were taken at Z intervals of 0.3 μm and then deconvolved using the AutoQuant Module in the NIS-elements software (Nikon).

Immunostaining.

Primary rbSMC transfected with human (h)Orai1^GFP were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). Afterwards, cells were washed three times for 5 min using PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and rinsed in PBS for 5 min. PBS was subsequently removed and cells were treated with blocking solution (10% normal goat serum in PBS-0.1% Triton X-100) for 60 min. Samples were incubated with the primary antibody against αOrai1 (Sigma, O8264) diluted 1:100 in 1% bovine serum albumin (BSA) in PBS-0.1% Triton X-100 for 2 h at RT followed by an additional incubation of 5 min at 37°C. After being washed two times with PBS-0.1% Triton X-100 and once with PBS for 5 min, cells were incubated with the secondary antibody (Alexa594 goat anti rabbit, Invitrogen) diluted 1:1,000 in 1% BSA dissolved in PBS-0.1% Triton X-100 for 60 min at RT. Finally, cells were washed once with PBS for 5 min and stored in the dark at 4°C until imaging.

BrdU incorporation analysis was done using a standard immunostaining. Briefly, rbSMC were cultured in Lab-Tek Chamber glass-bottomed slides (Nalge Nunc). At 48 h after transfection, cells were treated with BrdU (20 μM, Sigma) for 24 h. Then, they were fixed 10 min in methanol at −20°C and treated with the primary monoclonal anti-BrdU antibody (1:100 dilution; Sigma) and secondary FITC-conjugated goat anti-mouse antibody (1:100 dilution; Jackson Laboratories, West Grove, PA). The slides were also stained with Vectashield Mounting Medium (Vector Laboratory), with propidium iodide (excitation at 535 nm and emission at 615 nm when bound to DNA), producing a red fluorescence signal that was used to locate nuclei of the cells.

Statistical analysis.

Summary data are presented as means ± SE. Student's t-test was used to determine the statistical significance of all of the data. Data were considered significant at P < 0.01.

RESULTS

Orai1 expression in primary aortic SMC.

To demonstrate that endogenous Orai1 is expressed in primary SMC, total RNA was purified from smooth muscle cells freshly isolated from aortas of rabbit (rbSMC), mouse (mSMC), and rat (rSMC), and RT-PCR was performed. Figure 1A shows that endogenous Orai1 cDNA is present and can be detected in primary freshly isolated SMC from all three species. However, in spite of Orai1 presence, we found it hard to visualize endogenous Orai1 protein in primary aortic SMC using polyclonal antibody for Orai1 (αOrai1, Cayman). In contrast, when hOrai1^GFP was overexpressed in these cells, it was easily recognized by αOrai1, and the specificity of overexpressed protein staining was confirmed by its overlapping with GFP fluorescence signal (Fig. 1B).

Fig. 1.

Expression of endogenous Orai1 and localization of overexpressed Orai1^GFP in primary aortic smooth muscle cells (SMC). A: agarose electrophoresis of Orai1 RT-PCR products from SMC. Total RNA was purified from primary SMC freshly isolated from aortas of rabbit (rbSMC), mouse (mSMC), and rat (rSMC). B: representative images of the bottom plane of the primary rbSMC overexpressing Orai^GFP showing 1) (red) immunostaining against Orai1 (αOrai1), 2) (green) green fluorescent protein (GFP) fluorescent signal from Orai1^GFP, 3) merged image of 1 and 2. C: representative image of live rbSMC (deconvolved middle plane of rbSMC, 5 μm from the cell bottom): zoomed image on the right corresponds to the area specified on the left image. D: representative image of bottom (adherent) plane of the live rbSMC overexpressing Orai1^GFP before and after treatment with thapsigargin (TG, 5 μM). Arrows point to Orai1 accumulation.

Figure 1C demonstrates plasma membrane localization of Orai1^GFP in live rbSMC. Furthermore, Fig. 1D shows that even when expressed alone (without additional expression of STIM1), Orai1^GFP can accumulate and form puncta upon thapsigargin (TG, 5 μM)-induced Ca²⁺ store depletion in primary SMC. Thus, correct targeting of Orai1 to plasma membrane in primary SMC and its ability to accumulate in puncta upon Ca²⁺ store depletion could be demonstrated in live cells overexpressing Orai1^GFP.

Orai1 and iPLA₂β are essential for cat-SOC and SOCE in primary SMC.

To determine the role of Orai1 in endogenous cat-SOC channel function, whole cell patch-clamp recordings were done in primary mSMC, which is a well-defined model for electrophysiological studies of nonproliferating primary vascular SMC (65). Dialysis of mSMC with 10 mM BAPTA (widely used for depletion of Ca²⁺ stores) resulted in the development of a nonselective cation current, which was identical to the current described as I_cat-SOC (65). Fig. 2, A and B, shows a representative time course of development of I_cat-SOC and its average current-voltage relationship in control mSMC transfected with scrambled RNA. In contrast to I_CRAC, the current in primary mSMC could be recorded at both negative and positive membrane potentials (Fig. 2A) and had reversal potential around 0 mV (Fig. 2B). Figure 2A also shows that it can be fully inhibited by diethylstilbestrol (10 μM), a known inhibitor of CRAC and cat-SOC channels and SOCE in excitable and nonexcitable cells (29, 74, 75).

Fig. 2.

Molecular downregulation of Orai1 or Ca²⁺-independent phospholipase A₂β (iPLA₂β) equally inhibits store-operated cation-selective (cat-SOC) current (I_cat-SOC) and store-operated Ca²⁺ entry (SOCE) in primary mSMC. A: time course of whole cell currents (pA/pF) developing at −80 mV (inward current) and +80 mV (outward current) during primary mSMC dialysis with 10 mM BAPTA. Representative traces from cells transfected with either scrambled RNA (control, open symbols) or small interfering RNA (siRNA) to Orai1 (−Orai1, closed symbols) are shown. Extracellular diethylstilbestrol (DES, 10 μM) was applied at the end of the experiment, as shown. B: summary current-voltage (I–V) relationships (means ± SE, n = 8 and n = 10, respectively) of the maximum cat-SOC currents in control and −Orai1 cells. C: summary I–V relationships (means ± SE, n = 4 and n = 5, respectively) of the maximum cat-SOC currents in control and −iPLA₂β cells. D: representative traces show intracellular Ca²⁺ (measured as F₃₄₀/F₃₈₀) recorded simultaneously in a number of individual nonproliferating mSMC transfected with either scrambled RNA (control) or siRNA against Orai1 (−Orai1). The cells were pretreated with TG (5 μM) for 3 min in Ca²⁺-free solution before 2 mM Ca²⁺ was added at the time indicated. E and F: bar graphs summarize the data from experiments similar to those shown in D and show maximum Ca²⁺ influx (means ± SE) after TG treatment in control and −Orai1 (E) or −iPLA₂β (F) cells. Basal Ca²⁺ influx was measured in cells treated with DMSO (0.5%, 3 min) instead of TG. The number of cells tested for each condition is shown in parentheses above the bars. Data were obtained from 3 independent sets of experiments. **P < 0.01.

Transfection of primary mSMC with siRNA specific to Orai1 resulted in significant 70% attenuation of I_cat-SOC. (Fig. 2, A and B): at −80 mV the maximum current density diminished from −2.4 ± 0.3 pA/pF (n = 8) in cells transfected with scrambled RNA to −0.7 ± 0.1 pA/pF (n = 7) in cells transfected with siRNA (P < 0.01).

Importantly, we found that Orai1-dependent I_cat-SOC in primary mSMC also required iPLA₂β, which we have previously shown to be involved in the activation of endogenous cat-SOC channels in vascular SMC (59). Figure 2C compares current-voltage relationships of I_cat-SOC in cells transfected with sense (control) and antisense to iPLA₂β (−iPLA₂β) and shows ∼75% attenuation: at −80 mV the maximum current densities were reduced from −2.8 ± 0.2 pA/pF (n = 5) to −0.8 ± 0.1 pA/pF (n = 5) in mSMC transfected with antisense and sense, respectively (P < 0.01).

To verify the relative roles of Orai1 and iPLA₂β in endogenous SOCE, we tested the effects of transfection of mSMC cells with siRNA to Orai1, or antisense to iPLA₂β on TG-induced Ca²⁺ influx. Figure 2, E and F, demonstrates that Ca²⁺ entry triggered by TG (5 μM for 5 min) in mSMC was indeed inhibited by ∼70% in cells transfected with siRNA to Orai1, and by ∼80% in cells transfected with antisense to iPLA₂β. These results are totally in line with the results obtained for I_cat-SOC inhibition in mSMC under the same transfection conditions.

Thus, endogenous cat-SOC current and SOCE in primary vascular SMC equally depend on both, Orai1 and iPLA₂β.

Orai1 and iPLA₂β are equally important for proliferation of primary SMC.

To test whether and to what extent Orai1 and iPLA₂β-dependent SOCE may be involved in proliferation of primary aortic SMC, rbSMC in primary culture were used. Primary rbSMC were earlier shown to have the same cat-SOC channels as mSMC (65) and have proved to be a better model for molecular and functional studies, as they yield significantly larger numbers of cells and have higher transfection rates than mSMC. Figure 3 shows that TG-induced SOCE in rbSMC is similar to that in mSMC (Fig. 2). qRT-PCR confirmed that Orai1 is expressed in primary rbSMC in culture, and transfection with siRNA effectively reduces Orai1 expression by 94 ± 1% in 48 h. Figure 3A demonstrates that molecular knockdown of Orai1 resulted in ∼90% inhibition of TG-induced SOCE in rbSMC, consistent with Orai1 being essential for SOCE in proliferating primary rbSMC. Importantly, Fig. 3B shows that molecular downregulation of iPLA₂β produced similar SOCE inhibition. Thus, Orai1 and iPLA₂β appear to be equally important for SOCE in nonproliferating and proliferating primary SMC.

Fig. 3.

Knockdown of either Orai1 or iPLA₂β equally impairs SOCE in primary rbSMC. A and B: representative traces comparing intracellular Ca²⁺ (measured as F₃₄₀/F₃₈₀) recorded simultaneously in 5–10 individual proliferating rbSMC 48 h after transfection with scrambled RNA (control) or siRNA against Orai1 (−Orai1) (A) and sense (control) or antisense to iPLA₂β (−iPLA₂β) (B). The cells were pretreated with TG (5 μM) for 3 min in Ca²⁺-free solution before 2 mM Ca²⁺ was added at the time indicated. C and D: summary data from 3–4 independent experiments described in A and B, respectively. Maximum Ca²⁺ influx (means ± SE) is shown after TG treatment in control cells and cells in which either Orai1 (C) or iPLA₂β (D) was knocked down. Basal Ca²⁺ influx was tested by application of 0.5% DMSO instead of TG. The number of cells for each condition is shown in parentheses above the bars. Summary of 3–4 independent experiments for each condition is shown. **P < 0.01.

To compare the roles of Orai1 and iPLA₂β in proliferation of primary SMC, we followed the rate of rbSMC proliferation in 20% FBS starting 1 day after their isolation from rabbit aorta (Fig. 4). On day 3 after isolation the cells were transfected with either siRNA to Orai1 or antisense to iPLA₂β with control cells transfected with either scrambled RNA, or sense oligonucleotides, respectively, and cell numbers were monitored over up to 7 days in primary culture. We found that molecular knockdown of either Orai1 (Fig. 4A) or iPLA₂β (Fig. 4B) results in a significant impairment of proliferation of rbSMC. In 48 h after transfection, the increase in the number of cells transfected with Orai1 siRNA (−Orai1) was nearly 70% less than in matching control cells transfected with scrambled RNA. Similar inhibition of rbSMC proliferation was evident also in iPLA₂β-deficient cells, in which the increase in cell number during the same 48 h period was reduced by >80%. To confirm these results, DNA synthesis in proliferating rbSMC was assessed by BrdU incorporation. Figure 5 shows that molecular knockdown of either Orai1 or iPLA₂β resulted in 69% and 65% reduction in the number of BrdU-positive cells, respectively. Thus, Orai1 and iPLA₂β appeared to be equally important for division and proliferation of primary vascular SMC.

Fig. 4.

Knockdown of either Orai1 or iPLA₂β inhibits proliferation of primary rbSMC. A and B: time course of proliferation of rbSMC. The numbers of cells (means ± SE) in primary culture were measured from day 1 to day 7 after their isolation from aorta. On day 3, the cells were transfected with scrambled RNA (control, open symbols) or siRNA to Orai1 (−Orai1, closed symbols) (A) and sense (control, open symbols) or antisense to iPLA₂β (−iPLA₂β, closed symbols) (B). Results are representative of three separate experiments. **P < 0.01.

Fig. 5.

The effects of molecular downregulation of Orai1 (A and B) or iPLA₂β (C and D) on 5-bromo-2-deoxyuridine (BrdU) incorporation into the nuclei in primary rbSMC. A: representative images showing immunostaining with anti-BrdU antibody (to detect BrdU incorporation, green) and propidium iodine (to detect nuclei, red) of rbSMC transfected with either scrambled RNA or siRNA against Orai1. Yellow nuclei in merged images show dividing cells that stain with both markers. B: summary data showing the percentage of BrdU-positive cells in control and −Orai1 rbSMC. Results are representative of three independent experiments. **P < 0.01. C and D: similar to A and B, but comparing control and −iPLA₂β rbSMC transfected with sense and antisense specific to iPLA₂β, respectively.

DISCUSSION

To summarize our findings, we confirmed the presence of Orai1 in primary SMC and demonstrated its essential role for I_cat-SOC current and SOCE. We found that Orai1 and iPLA₂β are equally important for I_cat-SOC and SOCE in primary SMC and are both essential for their proliferation.

Thus, Orai1 appears to be essential not only for CRAC channels (I_CRAC) in nonexcitable cells (including synthetic SMC), but also for cat-SOC channels (I_cat-SOC) that are typical for primary vascular SMC. This duality may be physiologically justified. Indeed, differentiated vascular SMC have many features of excitable cells, including voltage-gated Ca²⁺ channels that can be activated by depolarization created by cat-SOC channels and SOCE mechanism (51). When they are exposed to growth hormones in vivo (or serum in cell culture conditions), differentiated SMC are known to quickly switch to a synthetic phenotype that resembles nonexcitable cells (16, 46, 47). This switch may be associated with increase in Ca²⁺ selectivity of SOC channels, and appearance of Orai1-mediated CRAC-like current that was found only in synthetic SMC.

Our finding that Orai1 is an essential component of cat-SOC further supports the idea that there may be a phenotype-dependent switch in Orai1 channel selectivity that will make it possible for Orai1 to encode SOC channels that display different selectivity, and yet mediate the same type of Ca²⁺ entry. The molecular nature of such a switch is yet to be determined, but several options may be considered.

First, mutations in the selectivity filter of Orai1 were shown to reduce its high Ca²⁺ selectivity (24, 67, 70). Expression of such mutants in heterologous systems was shown to create cat-SOC-like channels, but such mutations have not been reported in native Orai1. Another possibility is a posttranslational modification of Orai1 that may change its selectivity in response to a phenotypic switch, which may be the easiest and fastest way to change selectivity of Orai1-encoded channels in vascular SMC without changing the mechanism of its activation/regulation. Further studies are needed to test these and potentially other possibilities.

Another way to change the channel selectivity is to change the composition and nature of its subunits. For example, while Orai1 homo-tetramer forms Ca²⁺-selective CRAC channel (39), heteromeric channel encoded by Orai1 and Orai3 subunits overexpressed in HEK293 model cells was shown to form a much less Ca²⁺-selective channel, and was proposed to provide a concept for plasticity of Ca²⁺ selectivity of cat-SOC channels. However, clear demonstration of formation of native Orai1/Orai3 channels with low Ca²⁺ selectivity is still missing. Instead, Orai1 and Orai3 were found to be essential components of endogenous arachidonate-regulated Ca²⁺-selective (ARC) channel (40–42), which is not store-operated and does not require iPLA₂β (41). Another cat-SOC candidate is TRPC1 channel (for review, see Refs. 44, 49, 53). While some studies suggested cat-SOC in SMC to be encoded by TRPC1 (5, 8, 32), or a complex of Orai1 and TRPC1 subunits (5, 45, 69, 73), there is a significant line of evidence that contradicts these ideas (for review, see Refs. 13, 14). Side-by-side comparison of endogenous Orai1 and TRPC1-encoded channels (75) showed a totally different mechanism of their activation: in contrast to Orai1, which requires store depletion and depends on iPLA₂β, TRPC1 channel depends on the inositol 1,4,5-trisphosphate receptor and totally lacks iPLA₂β dependence. The unlikely role of TRPC1 in cat-SOC in primary SMC was further demonstrated by the finding of a totally normal SOCE and normal contractile function of vascular SMC in TRPC1 knockout mice (21).

It is important to emphasize the significance of our present findings that showed dependence of I_cat-SOC and SOCE on both Orai1 and iPLA₂β in primary SMC. Indeed, despite striking differences in Ca²⁺ selectivity of CRAC and cat-SOC channels in SMC, the mechanism of their iPLA₂β-dependent activation seems to be identical (14). Our new finding that proliferation of primary SMC equally depends on Orai1 and iPLA₂β further extends the physiological importance of iPLA₂β-dependent activation of Orai1-encoded channels, and demonstrates that this mechanism is essential for both contractile and synthetic activity of vascular SMC.

GRANTS

This work was supported by National Institutes of Health (NIH) Grants R01HL071793 and R01HL054150. B. Yang was supported by NIH training grant T32 HL007224.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: V.M.B. conception and design of the research; B.Y., T.G., and J.D.-G. performed the experiments; B.Y., T.G., and J.D.-G. analyzed the data; T.G., J.D.-G., and V.M.B. interpreted the results of the experiments; B.Y., T.G., J.D.-G., and V.M.B. prepared the figures; V.M.B. drafted the manuscript; B.Y. and V.M.B. edited and revised the manuscript; B.Y., T.G., and V.M.B. approved the final version of the manuscript.