Nonsteroidal Anti-inflammatory Drugs Inhibit Vascular Smooth Muscle Cell Proliferation by Enabling the Ca²⁺-dependent Inactivation of Calcium Release-activated Calcium/Orai Channels Normally Prevented by Mitochondria

Abstract

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca²⁺ homeostasis and Ca²⁺-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca²⁺ entry (SOCE) and voltage-operated Ca²⁺ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca²⁺ released-activated Ca²⁺ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca²⁺-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca²⁺ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca²⁺ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca²⁺ uptake, suggesting that they favor the Ca²⁺-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca²⁺ buffering conditions but not in strong Ca²⁺ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca²⁺-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca²⁺.

Introduction

Increased vascular smooth muscle cell (VSMC)³ proliferation, a process controlled by Ca²⁺ channel switching, is a key event in the development of atherosclerosis, restenosis, and other occlusive and proliferative disorders of the vasculature (1, 2). Salicylate, the major aspirin metabolite, and other nonsteroidal anti-inflammatory drugs (NSAIDs) may induce direct, platelet-independent effects on the vascular wall (3–5). For example, salicylate effectively inhibits VSMC proliferation and DNA synthesis in vivo and in vitro without inducing cellular toxicity or apoptosis (4).

A series of NSAIDs, including aspirin, ibuprofen, indomethacin, and sulindac, induce a dose-dependent inhibition of proliferation in A10 cells (6, 7), a VSMC cell line derived from embryonic rat aorta. The effects of NSAIDs occur in the absence of cytotoxicity and are independent of cyclooxygenase (7). Aspirin treatment also inhibits neointimal proliferation in dogs fed a cholesterol-enriched diet (8) and prevents the development of atherosclerosis in rabbits (9). Therefore, NSAIDs inhibit VSMC proliferation and show salutary effects in the treatment of vascular proliferative disorders by a yet unknown mechanism of action unrelated to anti-inflammatory activity.

Intracellular Ca²⁺ is a major trigger for vasoconstriction and a stimulus for VSMC proliferation (1, 2, 10). Several Ca²⁺ channels participate in regulating intracellular Ca²⁺, including voltage-operated and store-operated Ca²⁺ channels (10, 11). SOCE is activated after the emptying of intracellular Ca²⁺ stores by physiological stimuli and is involved in cell proliferation in several cell types, including T cells (12, 13). In these cells, SOCE requires not only the activating signal from the empty store but also the close proximity of functional mitochondria acting as Ca²⁺ sinks to prevent the strong Ca²⁺-dependent inactivation of SOC channels (14–18). It is unknown whether mitochondria control SOCE in VSMCs or not. Recently, two important proteins involved in SOCE have been discovered: Stim1, a sensor of the Ca²⁺ content of the store (19), and Orai1, a plasma membrane store-operated Ca²⁺ channel (20). Both proteins have been recently involved in SOCE in VSMCs (21–25) although other proteins, including members of the TRPC family of cation channels, might be involved in SOCE as well (26–28). SOCE and the novel proteins Stim1 and Orai1 may be involved in VSMC proliferation in vitro and in vivo. Knockdown of Stim1 decreases SOCE, inhibits cAMP response element-binding protein transcription factor activation, and prevents human coronary artery VSMC proliferation (25, 29).

Ca²⁺ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype. In the proliferative phenotype, the cells show increased SOCE, and Stim and Orai proteins are up-regulated (30). Furthermore, proliferating arterial myocytes have up-regulated Stim1, and its knockdown prevents nuclear factor of activated T cell-dependent transcription activity and growth factor-induced proliferation (31). Stim1 knockdown also prevents neointima formation and restenosis in animal models of injured carotid artery (31, 32). Rat aortic VSMCs display a SOC quite similar to the classic Ca²⁺ release-activated Ca²⁺ current (Icrac; 33), and the knockdown of either Stim1 or Orai1 (but not Orai2, Orai3, TRPC1, 4, or 6) inhibits SOCE, Icrac, and VSMC proliferation and migration (29). Thus, Orai1- and Stim1-dependent SOCE may play an important role in VSMC proliferation. It is not surprising, therefore, that these proteins have been proposed as candidate targets for proliferative disorders of the vascular wall (31, 32).

We have investigated whether salicylate and other NSAIDs might prevent VSMC proliferation acting on signals controlling SOCE activity. First, we have characterized SOCE and its contribution to cell proliferation in A10 rat aortic cells, a model of neointimal VSMCs (34). Second, we have asked whether NSAIDs prevent A10 cell proliferation acting on SOCE. Finally, the possible mechanism of SOCE inhibition was investigated.

EXPERIMENTAL PROCEDURES

Materials

Fura-2/AM, tetramethyl rhodamine methyl ester (TMRM), and coelenterazines are from Invitrogen. LaCl₃, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), and 2-aminoethoxydiphenyl borate (2-APB) are from Calbiochem. Thapsigargin and nifedipine are from Alomone Labs (Jerusalem, Israel). Media and sera are from Lonza (Basel, Switzerland). Other chemicals are from Sigma-Aldrich or Merck. Mitochondria-targeted GFP-aequorin and A10 cells were kindly donated by Profs. Philippe Brulet (CNRS, Paris, France) and Santiago Lamas (Consejo Superior de Investigaciones Científicas, Madrid, Spain), respectively.

Cell Culture

A10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with glutamine and antibiotics. RBL-2H3 cells were cultured in MEM-α (Invitrogen, 22561-021) supplemented with 15% fetal bovine serum (FBS; Invitrogen, 10270-106), 1% GlutaMAX (Invitrogen, 35050-038) and 1% PenStrep (Invitrogen, 15140-122), which contains 10,000 units/ml penicillin and 10,000 μg/ml streptomycin. Cells were maintained continuously in log-phase growth at 37 °C with 5% CO₂. Cells were prepared at a concentration of 3000 cells/well 24–48 h before patch clamp and imaging experiments.

Store-operated and Voltage-operated Ca²⁺ Entry

SOCE was monitored as reported earlier (35) by imaging the rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) that follows Ca²⁺ addition to cells with depleted Ca²⁺ stores. VOCE was measured by imaging the rise in [Ca²⁺]_cyt induced by depolarization with high K⁺ (75 mm) instead of Na⁺. A10 cells were plated at about 17 × 10⁴ cells/ml (10 × 10³ cells/60 μl) on 12-mm glass coverslips coated with 0.01 mg/ml poly-l-lysine. After 24 h, cells were loaded with 4 μm Fura-2/AM for 1 h at room temperature, incubated with thapsigargin (1 μm) for 10 min in Ca²⁺-free medium containing 145 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 0.5 mm EGTA, 10 mm glucose, 10 mm Hepes/NaOH, pH 7.42, and placed on the stage of an inverted microscope (Zeiss Axiovert S100 TV). Subsequently, cells were perfused with prewarmed (37 °C) Ca²⁺-free medium and illuminated alternately at 340 and 380 nm before test solutions. Light emitted at wavelengths longer than 520 nm was recorded with a Hamamatsu OrcaER digital camera through a 40 × oil lens (NA 1.3). Pixel-by-pixel ratios of consecutive frames were captured and analyzed using the Aquacosmos® software.

Electrophysiology

Patch clamp experiments were performed in the tight seal whole cell configuration at 21–25 °C. Membrane currents were acquired with an EPC-9 patch clamp amplifier (HEKA). Voltage ramps of 200-ms duration spanning a range of −150 to +100 mV were delivered from a holding potential of 0 mV at a rate of 0.5 Hz over a period of 400 s. All voltages were corrected for a liquid junction potential of −12 mV between internal and bath solutions. Currents were filtered at 2.9 kHz and digitalized at a sampling rate of 10 kHz. To display the current recordings, currents were digitally filtered offline at 1 kHz. Pipette and cell capacitance were electronically canceled before each voltage ramp. Current amplitudes at −130 mV from individual voltage ramp current were used to depict the temporal development of currents and analyze current inactivation. Statistical errors of averaged data are given as means ± S.E. analyzing n cells. Standard external solution was as follows: 120 mm NaCl, 2 mm MgCl₂, 10 mm CaCl₂, 10 mm Tetraethylammonium-Cl, 10 mm Hepes, 10 mm glucose, pH 7.2 with NaOH, 300 mosmol liter⁻¹. Ibuprofen, indomethacin, and salicylate were added to the external solution at a final concentration of 10 and 100 μm. Cells were preincubated with each compound for 5 min before patching them. The standard pipette solution for whole cell patch clamp recordings contained 0.05 mm inositol trisphosphate, 5 × 10⁻⁸ mm thapsigargin, 120 mm Cs-glutamate, 8 mm NaCl, 10 mm Cs-BAPTA, 3 mm MgCl₂, 4 mm CaCl₂, 10 mm Hepes, pH 7.2 with CsOH, 300 mm mosmol liter⁻¹ (resulting in 150 nm free Ca²⁺ as calculated with WebMaxC). To weakly buffer Ca²⁺ in the pipette, the following solution was used: 1.2 mm EGTA, 0.05 mm inositol trisphosphate, 5 × 10⁻⁸ mm thapsigargin, 145 mm Cs-aspartate, 3 mm MgCl₂, 8 mm NaCl, 10 mm Cs-Hepes, pH 7.2 with CsOH, 280 mosmol liter⁻¹. The mitochondrial mixture to preserve mitochondrial respiration contained 5 mm Mg-ATP, 0.5 mm Tris-GTP, 2.5 mm malic acid, 2.5 mm Na⁺-pyruvate, 1 mm NaH₂PO₄. The mitochondrial mixture was added to the weak Ca²⁺ buffer solution just before starting the experiment. Data were analyzed using Igor Pro (Wavemetrics), Pulse (HEKA), FitMaster (HEKA), and Excel (Microsoft). All values are given as mean ± S.E. (number of cells). Three or more independent experiments were performed for each experimental condition. In case, data points were normally distributed, an unpaired two-sided Student's t test was used. If normal distribution could not be confirmed, a nonparameterized test (Mann-Whitney) was carried out. p values are stated in the figure legends.

RT-PCR

Total RNA was extracted from A10 cells using TRIzol reagent (Invitrogen). cDNA was made from 2 μg of RNA by a high capacity cDNA Reverse Transcription kit (Applied Biosystems). The sense and antisense primers targeting Stim1 and Orai1 were designed using PRIMER 3 software. PCR was performed by using the following primers: rat Stim1 sense, 5′-TAA CTG GAC CGT GGA TGA GG-3′ and antisense, 3′-GTC CAC TAA CAC CGC TCA G-5′; rat Orai1 sense, 5′-TGG TAG CGA TGG TGG AAG TC-3′ and antisense 3′-TGC CTC AAC TCC AAC ACC TG-5′. Primers were synthesized by VWR International Eurolab (Barcelona, Spain). Amplification was started with initial denaturation at 94 °C for 3 min, then 25–30 cycles with denaturation at 94 °C for 60 s, annealing at 60 °C for 1 min, and extension at 72 °C for 30 s and was followed by a final extension at 72 °C for 10 min. Gel electrophoresis was used to identify the PCR products in a 1% agarose gel using ethidium bromide staining.

Cell Proliferation

Cells were cultured in DMEM containing 10% FBS and antibiotics. Cells were plated in wells at about 10 × 10³ in 3 ml and incubated with test solutions for 15 days. Cell number was determined at day 1 and at day 15 using a hemocytometer. Cell death was estimated using trypan blue staining.

Mitochondrial Potential (Δψ)

A10 cells were loaded with the Δψ probe TMRM (100 nm) for 30 min at room temperature, placed on the perfusion chamber of a Zeiss Axiovert S100 TV inverted microscope and superfused continuously with prewarmed (37 °C) standard medium. Fluorescence images were taken at 10-s intervals with a Hamamatsu OrcaER camera. At the end of the recording, the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 10 μm) was perfused for 5 min to collapse Δψ. The fluorescence image of TMRM after collapse of Δψ was used as background fluorescence in conditions of total uncoupling. Fluorescence recordings from individual cells were expressed as the percent value of the value just before treatment and averaged as reported previously (36).

Bioluminescence Imaging of Mitochondrial Ca²⁺

A10 cells were nucleofected (Amaxa) with a plasmid containing mitochondria-targeted, GFP-aequorin. 24 h later, cells were incubated in standard medium (see above) containing 4 μm coelenterazine h or n for 2 h at room temperature. Then, the coverslips were placed in the stage of an inverted microscope (Zeiss Axiovert S100 TV) equipped with a bottom-port attached, Hamamatsu VIM photon counting camera handled with an Argus 20 image and the Aquacosmos® software. Cells were perfused continuously with warm (37 °C) standard medium and subjected to photon counting imaging at 10-s intervals. For experiments in intact cells, cells were perfused with standard medium. The effects of SOCE on [Ca²⁺]_mit were imaged after presentation of 1 mm extracellular Ca²⁺ to cells previously treated with thapsigargin in Ca²⁺-free medium. The effects of VOCE on [Ca²⁺]_mit were imaged after depolarization with high K⁺ medium. For experiments in permeated cells, A10 cells were permeated with 20 μm digitonin in “intracellular” medium (130 mm KCl, 10 mm NaCl, 1 mm MgCl₂, 1 mm K₃PO₄, 0.2 mm EGTA, 1 mm ATP, 20 μm ADP, 2 mm succinate, 20 mm Hepes/KOH, pH 6.8). Then, the cells were incubated with the same medium containing 200 nm Ca²⁺ (buffered with EGTA) with or without NSAIDs for 5 min. Finally, perfusion was switched to “intracellular” medium containing 10 μm Ca²⁺ (with or without NSAIDs) for 1 min. Photonic emissions were converted to mitochondrial Ca²⁺ concentration ([Ca²⁺]_mit) values as detailed elsewhere (37, 38).

Cell ATP Levels

Cells were seeded in 6-well plates and cultured in medium containing either vehicle or different NSAIDs. After 3 days, cells were washed twice with phosphate-buffered saline (PBS) at 37 °C, and 1 ml of boiling 20 mm Tris, pH 7.75, 4 mm EDTA solution was added. After 2 min, samples were centrifuged for 5 min at 10,000 × g. ATP was measured later from the supernatant by the luciferin-luciferase assay using a Cairn luminometer (Cairn Research, Kent, UK).

Statistics

When only two means were compared, Student's t test was used. For >2 groups, statistical significance of the data was assessed by ANOVA and compared using Bonferroni's multiple comparison tests. Differences were considered significant at p < 0.05.

RESULTS

We have characterized SOCE in A10 rat aorta VSMCs. Fluorescence imaging experiments show that cells with intact Ca²⁺ stores (Fig. 1A) undergo no change in [Ca²⁺]_cyt when extracellular Ca²⁺ is added. However, cells with depleted stores displayed large increases in [Ca²⁺]_cyt after the addition of extracellular Ca²⁺ revealing SOCE. A second pulse induced a similar rise in [Ca²⁺]_cyt (Fig. 1B). Plasma membrane depolarization with medium containing a high K⁺ concentration also increases [Ca²⁺]_cyt revealing VOCE (Fig. 1C). Interestingly, the relative abundance of cells showing Ca²⁺ entry and the size of the rise in [Ca²⁺]_cyt was larger for SOCE than for VOCE (Fig. 1D).

FIGURE 1.

A10 cells show SOCE and VOCE. A10 cells were loaded with Fura-2/AM and subjected to fluorescence imaging of cytosolic Ca²⁺. A, in intact cells the addition of extracellular Ca²⁺ does not change the ratio of fluorescences excited at 340 and 380 nm (Ratio 340/380) reflecting [Ca²⁺]_cyt. B, in thapsigargin-treated cells, readdition of Ca²⁺ increased this ratio in all cells (n = 5 experiments) reflecting SOCE. A further Ca²⁺ pulse evoked the same response. Pictures on top show representative ratio images (from 0 to 1) coded in pseudocolor. C, depolarization with medium containing a high concentration (75 mm) of K⁺ (in exchange for Na⁺) induced a lower increase in the ratio in a fraction of cells revealing VOCE (n = 3 experiments). D, top bars show the size of rise in ratio induced by SOCE and VOCE in responsive cells. Bottom bars show percent of cells showing SOCE and VOCE, respectively (mean ± S.E. (error bars), *, p < 0.05).

To characterize SOCE further in A10 cells and its role in cell proliferation, we investigated expression of Orai1 and Stim1, recently involved in SOCE in other cell types including VSMC (22–25). Supplemental Fig. 1 shows that A10 cells expressed both Stim1 and Orai1 mRNAs as determined by RT-PCR. Second, the effects of a series of antagonists on SOCE and VOCE were tested in A10 cells. Fig. 2A shows that classic SOCE antagonists including LaCl₃ (La³⁺) and 2-APB inhibited SOCE. BTP2, a novel Icrac antagonist (39), also inhibited SOCE but at a much lower concentration (Fig. 2A). SOCE antagonists did not inhibit VOCE in A10 cells (Fig. 2B), and dihydropyridines that block VOCE did not inhibit SOCE (supplemental Fig. 2). Third, we studied the effects of SOCE antagonists on A10 cell proliferation. La³⁺, 2-APB, and BTP2 inhibited A10 cell proliferation at the same concentrations that prevent SOCE (Fig. 2C), consistent with a role for SOCE in A10 cell proliferation.

FIGURE 2.

SOCE antagonists inhibit A10 cell proliferation. A, SOCE measurements were carried out as in Fig. 1. Panels show representative experiments for each antagonist tested. Average (mean ± S.E. (error bars)) recordings of all cells in the same fields (n = 7–9 cells) are shown (n = 3). B, effects of antagonists on VOCE were investigated as in Fig. 1. Antagonists were added at the indicated concentrations after depolarization (n = 3). C, A10 cells were cultured for 15 days in vitro, and the effects of antagonists on cell proliferation were tested. La³⁺ and 2-APB were used at 10 μm and BTP2 at 10 nm (*, p < 0.05 versus control, n = 3). Antagonists had no effect on cell viability (data not shown).

As reported above, salicylate and other NSAIDs show salutary effects in the vascular wall and prevent VSMC proliferation by a yet unknown mechanism unrelated to anti-inflammatory activity (3–9). Fig. 3 shows that salicylate inhibits A10 cell proliferation in a dose-dependent manner. Other NSAIDs, including ibuprofen, sulindac, and indomethacin, also inhibited A10 cell proliferation at therapeutic concentrations. (R)-flurbiprofen, an optic enantiomer lacking anti-inflammatory activity, inhibits A10 cell proliferation in a dose-dependent manner. In search for the anti-proliferative mechanism, we have investigated the effects of NSAIDs on SOCE in A10 cells. Salicylate, ibuprofen, indomethacin, and sulindac inhibited SOCE significantly in A10 cells (Fig. 4). Similar results were obtained with (R)-flurbiprofen (supplemental Fig. 3). Inhibition of SOCE by salicylate is also observed even when salicylate is added after the rise in [Ca²⁺]_cyt induced by Ca²⁺ pulse in store-depleted cells (Fig. 5A). NSAIDs could inhibit A10 cell proliferation acting also on VOCE. However, none of the NSAIDs tested inhibited VOCE in A10 cells (Fig. 5, B–F). Interestingly, the addition of NSAIDs during the depolarizing pulse promoted rather a small rise in [Ca²⁺]_cyt. Thus, NSAIDs inhibit Ca²⁺-dependent cell proliferation in A10 cells acting on SOCE but not on VOCE. Consistent with the role of Ca²⁺ entry in proliferation, chelating extracellular Ca²⁺ also largely inhibited A10 cell proliferation whereas the equimolar addition of excess Ca²⁺ did not inhibit proliferation, indicating that EGTA had no toxic effects (supplemental Fig. 4). In addition, an NSAID (indomethacin) inhibited Ca²⁺-dependent proliferation but had no further effect in the absence of extracellular Ca²⁺ (supplemental Fig. 4).

FIGURE 3.

NSAIDs inhibit A10 cell proliferation. A, dose-dependent effects of salicylate (100–2000 μm) on A10 cell proliferation. B, effects of different NSAIDs including ibuprofen, sulindac, and indomethacin, all tested at 100 μm, on A10 cell proliferation. C, dose-dependent effects of (R)-flurbiprofen (1–100 μm) on A10 cell proliferation. *, p < 0.05 versus control (n = 3). Results are mean ± S.E. (error bars).

FIGURE 4.

NSAIDs inhibit SOCE in A10 cells. SOCE was measured in A10 cells as in Fig. 1. A, Ca²⁺ recordings are mean ± S.E. (error bars; n = 9,9,18 cells, respectively) and representative of n = 3. B, bars show the dose-dependent effects of salicylate on SOCE (mean ± S.E. values, n = 3). C, bars show the effects of ibuprofen, indomethacin, and sulindac, all tested at 100 μm, on SOCE. *, p < 0.05.

FIGURE 5.

NSAIDs do not inhibit VOCE in A10 cells. SOCE was measured in A10 cells as in Fig. 1. Panels show Ca²⁺ recordings of individual cells in experiments representative of at least three similar ones. A, salicylate (500 μm) decreases [Ca²⁺]_cyt after the Ca²⁺ readdition to thapsigargin-treated cells. B–F, effects of NSAIDs on VOCE were tested by recording the effect of NSAIDs perfused after depolarization with high K⁺ medium. Neither 500 μm salicylate (B), 100 μm sulindac (C), 100 μm indomethacin (D), nor 100 μm ibuprofen decreased [Ca²⁺]_cyt when perfused during depolarization. F shows mean ± S.E. (error bars) values of the levels of [Ca²⁺]_cyt before (control) and after NSAID treatment (p > 0.05, n = 3).

NSAIDs may inhibit SOCE acting directly on CRAC channels or modulating a SOCE regulatory mechanism. To address this issue we have investigated the effects of NSAIDs on Icrac in RBL cells. We used RBL cells for two reasons. First, Icrac is large and well characterized in these cells (15). Second, a strong mechanism of regulation of Icrac and SOCE by mitochondria in RBL cells is also well characterized (15, 16). Consistently, we found that RBL cells displayed a robust SOCE that was nearly abolished by mitochondrial uncoupling (supplemental Fig. 5). We tested the effects of salicylate, ibuprofen, indomethacin, and sulindac on SOCE in RBL cells. We found that all tested NSAIDs inhibited SOCE in RBL cells just as they did in A10 cells (supplemental Fig. 5). Next, we tested the effects of NSAIDs on CRAC/Orai channel activity in RBL cells directly using the whole cell patch clamp configuration. Fig. 6 shows the typical inward rectifying I-V relationship for Icrac at 2, 100, and 400 s after starting recording in control cells. Neither salicylate, ibuprofen, nor indomethacin inhibited Icrac in RBL cells studied under conditions of strong Ca²⁺ buffering (10 mm BAPTA). Under this condition, the inflowing Ca²⁺ is rapidly buffered by the Ca²⁺ chelator and thereby reducing the Ca²⁺-dependent channel inactivation efficiently (Fig. 6, B and C). The statistical analysis shows that in these conditions NSAIDs have no direct effects on CRAC/Orai channel activity (Fig. 6D). However, under weak Ca²⁺ buffering condition (1.2 mm EGTA), mitochondrial Ca²⁺ uptake has been reported to be essential for reducing the accumulation of incoming Ca²⁺ close to sites that govern Ca²⁺-dependent CRAC channel inactivation (14–16). Indeed, mitochondrial depolarization induces significantly inhibition of Icrac (13–16). To keep the capability of mitochondria to take up Ca²⁺ during Icrac recording, a mixture of several compounds was supplied to the pipette solution (see “Experimental Procedures”). This mitochondrial mixture help to maintain the Δψ and the subsequent long lasting activity of CRAC/Orai channel activity. Fig. 6E shows the time course of Icrac in weak Ca²⁺ buffer in the presence of mitochondrial mixture. However, in presence of protonophore CCCP or specific mitochondrial uniporter blockers ruthenium red or ruthenium 360, the long lasting Icrac in cells dialyzed with mixture was significantly reduced (Fig. 6E). The same is true for indomethacin-, salicylate-, and ibuprofen-treated cells (Fig. 6F). Thus, only under weak Ca²⁺ buffering condition is CRAC significantly prevented by the uncoupler CCCP, ruthenium derivatives, and NSAIDs (Fig. 6G).

FIGURE 6.

NSAIDs accelerate slow inactivation of CRAC currents in RBL cells under weak Ca²⁺ buffering conditions. A–C, average current-voltage (I/V) relationship of CRAC currents from RBL-2H3 cells patched with the strong buffer solution (10 mm BAPTA) at 2 (black trace), 100 (red trace), and 400 (blue trace) s after establishing the whole cell configuration and current kinetics in control cells (B, n = 16) and in cells exposed (C) to ibuprofen (n = 7), salicylate (n = 11), indomethacin (n = 6), all tested at 10–100 μm is shown. D, average CRAC currents were obtained from cells as shown in B and C, respectively. Currents sizes were extracted at −130 (black trace) and +80 (red trace) mV, normalized to the cell size, averaged, and plotted versus time. Currents were leak-corrected by subtracting averages of the currents from first three voltage ramps before CRAC channel activation. E, CRAC currents were studied in control cells with the weakly buffering solution (1.2 mm EGTA) and the mitochondrial mixture (see “Experimental Procedures”) except that cells were preincubated with CCCP for 5 min to disrupt mitochondrial Ca²⁺ uptake. F, same conditions were used as in E except that cells were preincubated with indomethacin (red trace), salicylate (black trace), or ibuprofen (blue trace). G, statistical analysis of CRAC currents was performed 6 min after establishing the whole cell configuration as a fraction of the maximal current for the control conditions and for experiments in the presence of R360 + RR. Levels of significance are indicated (**, p < 0.01; ***, p < 0.001). Errors bars indicate S.E.

The above observations provide important clues regarding the mechanism of inhibition of SOCE by NSAIDs. They suggest that NSAIDs promote the Ca²⁺-dependent inactivation of CRAC channels by preventing the ability of mitochondria to take up Ca²⁺. To test this possibility we have investigated whether mitochondria controls SOCE in A10 cells just as they do in Jurkat T and RBL cells (13–16). In the first place we studied whether Ca²⁺ entry induced by SOCE induces mitochondrial Ca²⁺ uptake. For this end, A10 cells were transfected with mitochondria-targeted aequorin and subjected to bioluminescence imaging for monitoring of [Ca²⁺]_mit. Fig. 7A shows that Ca²⁺ pulses to cells with depleted stores induced a rise in [Ca²⁺]_mit, indicating that mitochondria are sensitive to rises in [Ca²⁺]_cyt induced by SOCE. However, depolarization with high K⁺ failed to increase [Ca²⁺]_mit in any of the A10 cells tested (Fig. 7B). These data suggest, but do not prove, that mitochondria are not sensitive to [Ca²⁺]_cyt rises induced by VOCE in A10 cells. This effect is probably due to the low rise in [Ca²⁺]_cyt induced by VOCE (Fig. 1). Next, we investigated the effects of mitochondrial depolarization on SOCE. Fig. 8A shows that Δψ collapse induced by FCCP (+oligomycin) inhibits SOCE in a reversible manner in A10 cells. Similar results were obtained with antimycin A (+oligomycin) to depolarize mitochondria (Fig. 8B). Salicylate did not increase the effect of FCCP on SOCE further (supplemental Fig. 6), indicating that both FCCP and NSAID act by the same mechanism. FCCP also decreased SOCE when presented after Ca²⁺ readdition (Fig. 8C). Interestingly, addition of FCCP after depolarization with high K⁺ induced a small rise in [Ca²⁺]_cyt, a behavior resembling the effects of NSAIDs (Fig. 5). Thus, mitochondrial uncoupling or depolarization inhibits SOCE but not VOCE in A10 cells.

FIGURE 7.

SOCE, but not VOCE, induces mitochondrial Ca²⁺ uptake in A10 cells. A10 cells were transfected with GFP-aequorin targeted to mitochondria, loaded with coelenterazine, and subjected to bioluminescence imaging of mitochondrial [Ca²⁺] in single cells. Left (SOCE), A10 cells were treated with 1 μm thapsigargin for 10 min in Ca²⁺-free medium to deplete intracellular Ca²⁺ stores and perfused with extracellular Ca²⁺ containing medium to induce SOCE, and the effects on photonic emissions reflecting mitochondrial Ca²⁺ uptake were imaged. Pictures on top show a typical fluorescence image of transfected cells (left) and the accumulated photonic emissions during SOCE. Top traces show calculated [Ca²⁺]_mit values in individual cells. Bottom traces reflect percent of remaining photonic emissions. Right (VOCE), cells were perfused with high K⁺-containing medium to induce VOCE, and the effects on photonic emissions reflecting mitochondrial Ca²⁺ uptake were imaged. Top pictures are representative fluorescence and bioluminescence images. Traces show [Ca²⁺]_mit recordings in individual cells and percent remaining counts. Data are representative of at least three independent experiments of each kind. Pseudocolor scale goes from 0 to 10 photons/pixel.

FIGURE 8.

Mitochondrial depolarization inhibits SOCE but not VOCE in A10 cells. SOCE was estimated in A10 cells as shown in Fig. 1. A, 10 μm FCCP (added in the presence of 0.12 μm oligomycin) inhibits SOCE in a reversible manner. B, mitochondrial depolarization with antimycin A (0.5 μg/ml) + oligomycin (0.12 μm) also inhibits SOCE in A10 cells. C, 10 μm FCCP added during SOCE decreases [Ca²⁺]_cyt. D, FCCP added after depolarization with high K⁺ did not decrease [Ca²⁺]_cyt but rather increased it. All data are single-cell recordings representative of 8–17 cells studied in at least three independent experiments for each panel.

To support further the view that NSAIDs inhibit SOCE acting on a mitochondria-dependent regulatory mechanism, we tested the effects of NSAIDs on Δψ. Fig. 9, A and B, shows that salicylate decreases Δψ in A10 cells in a dose-dependent manner. Similar results were obtained with ibuprofen, sulindac, and indomethacin at the same concentrations that inhibit SOCE and cell proliferation (Fig. 9C). To exclude any possible metabolic influence, we studied the effects of NSAIDs on cell ATP levels in A10 cells. Fig. 9D shows that treatment with NSAIDs did not affect the cell ATP levels significantly. Because Δψ is the driving force for mitochondrial Ca²⁺ uptake, we investigated next whether changes in Δψ induced by NSAIDs were sufficient to inhibit mitochondrial Ca²⁺ uptake. Toward this end, A10 cells were transfected with an aequorin plasmid fused to the GFP and targeted to mitochondria (37). After 24 h, cells were incubated with coelenterazine n and subjected to photon-counting imaging for monitoring [Ca²⁺]_mit in individual A10 VSMCs. Transfected cells were selected by their GFP fluorescence (Fig. 10) where increases in [Ca²⁺]_mit promote the release of photonic emissions. Transfected A10 cells were permeated with low concentrations of digitonin (34) and then stimulated with internal medium containing 10 μm Ca²⁺ to evoke a rise in [Ca²⁺]_mit (Fig. 10A), which was prevented by FCCP (Fig. 10B). NSAIDs, including salicylate at 500 μm (Fig. 10C) and 100 μm ibuprofen (Fig. 10D) and indomethacin (Fig. 10E), also inhibited the rise in [Ca²⁺]_mit in permeated A10 cells in a significant manner (Fig. 10F).

FIGURE 9.

NSAIDs depolarize mitochondria in A10 cells. The effects of NSAIDs on mitochondrial potential were tested by fluorescence microscopy of cells loaded with TMRM. A, effects of vehicle, salicylate (100–2000 μm), or FCCP (10 μm) on TMRM fluorescence were normalized to the value before addition of treatment and averaged (arrow). B, mean ± S.E. (error bars) values of three independent experiments are shown (*, p < 0.05). C, effects of vehicle (control), FCCP (10 μm), ibuprofen, indomethacin, and sulindac (all tested at 100 μm) on TMRM fluorescence were normalized to the value before addition of treatment (arrow). Results are representative of n = 3 experiments. D, effects of NSAIDs including salicylate, (R)-flurbiprofen, indomethacin, sulindac sulfide, and ibuprofen on ATP levels in A10 cell are shown. All NSAIDs were tested at 100 μm except salicylate which was tested at 500 μm. None of the treatments changed cell ATP levels in A10 cells (n = 3, p > 0.05).

FIGURE 10.

NSAIDs inhibit mitochondrial Ca²⁺ uptake in permeated A10 cells. A10 cells were transfected with GFP-aequorin targeted to mitochondria and subjected to bioluminescence counting imaging to estimate mitochondrial Ca²⁺ uptake in permeated, single cells. Pictures show a typical brightfield image, the GFP fluorescence image (GFP Fluor.), and photonic emissions released after a Ca²⁺ pulse (AEQ Biolum). Cells were permeated with low concentrations of digitonin in internal medium. A, perfusion with internal medium containing 10 μm Ca²⁺ induced a large rise in [Ca²⁺]_mit. B, 10 μm FCCP abolished the rise in [Ca²⁺]_mit induced by 10 μm Ca²⁺. C–E, salicylate (C, 500 μm), ibuprofen (D, 100 μm), and indomethacin (E, 100 μm) also inhibited [Ca²⁺]_mit rises induced by 10 μm Ca²⁺. Traces are recordings representative of 4–7 cells studied in at least three independent experiments. F, bars show mean ± S.E. (error bars) values of [Ca²⁺]_mit increases (n = 3; *, p < 0.05).

DISCUSSION

We show that NSAIDs inhibit proliferation of rat aortic A10 cells, a model of neointimal VSMC, and that this effect is mediated by inhibition of SOCE, an important Ca²⁺ entry pathway involved in cell proliferation. We also show that NSAIDs do not inhibit SOCE directly acting on CRAC channels but target an important regulatory mechanism of SOCE by mitochondria. The results confirm the importance of SOCE in VSMC proliferation and provide an action mechanism for the antiproliferative and salutary effects of NSAIDs in the vasculature. In support of this view, we demonstrated first that A10 cells display both SOCE and VOCE, the former being quantitatively larger than the latter, consistent with a proliferative VSMC phenotype (1, 2). Interestingly, SOCE activation in VSMC favors immediate-early gene expression and growth whereas VOCE promotes rather VSMC differentiation (40). SOCE in A10 cells resembles the one best characterized in T and RBL cells in the following aspects: (i) It is prevented by classic SOCE antagonists La³⁺ and 2-APB and the novel antagonist BTP2, (ii) A10 cells express Orai1 and Stim1 recently involved in CRAC in other cell types including VSMCs, (iii) SOCE is important in A10 cell proliferation just as in T cell clonal expansion (13), and (iv) SOCE is tightly modulated by mitochondria because inhibition of mitochondrial Ca²⁺ uptake prevents this pathway. Thus, SOCE in A10 VSMCs is regulated by mitochondria and involved in cell proliferation.

Our data agree with recent reports indicating that SOCE and novel proteins Stim1 and Orai1 are critical in VSMC proliferation. For example, proliferating VSMCs show increased SOCE and up-regulated Stim and Orai proteins (30), and their knockdown decreases SOCE and cell proliferation in coronary artery VSMCs (25) and in rat aortic VSMCs (29). Orai and Stim knockdown prevents restenosis in rat injured carotid artery (32). Increased SOCE is also involved in vascular pathology. For example, SOCE mediates pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension (42). Therefore, targeting SOCE may contribute to preventing VSMC proliferation in occlusive and proliferative disorders of the vessel wall.

As stated above, NSAIDs inhibit VSMC proliferation and DNA synthesis in vivo and in vitro without cellular toxicity and independently of cyclooxygenase (4, 6, 7). Consistently, our results show that NSAIDs diminish A10 cell proliferation at roughly the same concentrations that inhibit SOCE. The little disconnect between both parameters in some cases could be related to the facts that acute and chronic effects of NSAIDs could not be entirely similar and that a little component of A10 cell proliferation is independent of Ca²⁺ entry. The effects of NSAIDs cannot be explained by cell death, anti-inflammatory activity, or VOCE inhibition because cell viability was not decreased, structural analogs lacking anti-inflammatory activity ((R)-flurbiprofen) mimicked inhibition, and NSAIDs did not prevent VOCE. Therefore, SOCE inhibition underlies the antiproliferative effects of NSAIDs in VSMCs.

NSAIDs may inhibit SOCE acting directly on channels or, alternatively, by targeting a regulatory mechanism. Our results favor this latter option for several reasons. First, NSAIDs do not inhibit CRAC directly. This conclusion is based in our studies with RBL cells. NSAIDs inhibit SOCE in RBL cells but do not prevent Icrac in strong Ca²⁺ buffer. However, in weak Ca²⁺ buffer, where mitochondrial Ca²⁺ uptake is essential for sustaining CRAC/Orai channels, NSAIDs significantly reduced Icrac. Second, SOCE (but not VOCE) promotes mitochondrial Ca²⁺ uptake in A10 cells. Third, SOCE (but not VOCE) is largely inhibited when mitochondrial Ca²⁺ uptake is impaired by mitochondrial depolarization with protonophores. These compounds are not selective to mitochondria, and they may collapse the proton gradient in any acidic organelle. As some of these acidic organelles have been reported to act as agonist-sensitive Ca²⁺ stores, protonophore effects should be regarded with caution. Finally, NSAIDs depolarize mitochondria and impair mitochondrial Ca²⁺ uptake in A10 cells, an action consistent with previous reports on the effects of salicylate and NSAIDs as mitochondrial uncouplers in both isolated mitochondria and intact cells (35, 41). Interestingly, NSAIDs added after VOCE produce the same effects than FCCP. It has been reported that mitochondria may regulate CRAC channels by production of ATP which supposedly acts as a calcium buffer (43). However, the finding that ruthenium red blocks CRAC confirms that mitochondrial Ca²⁺ buffering is an important factor. Moreover, ATP levels do not change upon exposure to NSAIDs.

Taken together, our results suggest that NSAIDs prevent mitochondrial Ca²⁺ uptake, thus facilitating the Ca²⁺-dependent inactivation of SOC channels rather than inhibiting SOC channels directly. As SOCE is clearly involved in VSMC proliferation, this mechanism may underlie the antiproliferative and salutary effects of NSAIDS on proliferative disorders of the vascular wall.