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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Complex regulation of store-operated Ca²⁺ entry pathway by PKC- in vascular SMCs

¹Ion Channel and Calcium Signaling Unit, Boston University School of Medicine, Boston, Massachusetts; and ²Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen de Roció/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla/Laboratorio de Investigación Cardiovascular, Sevilla, Spain

Submitted 15 August 2007 ; accepted in final form 16 April 2008

	ABSTRACT

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The role of PKC in the regulation of store-operated Ca²⁺ entry (SOCE) is rather controversial. Here, we used Ca²⁺-imaging, biochemical, pharmacological, and molecular techniques to test if Ca²⁺-independent PLA₂β (iPLA₂β), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca²⁺ stores and triggered Ca²⁺ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA₂β and Ca²⁺ entry that were absent in SMCs in which iPLA₂β was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca²⁺ entry only in proliferating SMCs, which correlates with the expression of the specific PKC- isoform. Molecular downregulation of PKC- impaired TG- and OAG-induced Ca²⁺ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca²⁺ stores as well as direct PKC--dependent activation of iPLA₂β, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

protein kinase C-; Ca²⁺-independent phospholipase A₂; diacylglycerol; smooth muscle cells

Recently, we (15, 31, 32) found and others (16–19) confirmed that iPLA₂β is a major determinant for the activation of SOCE. We (9) demonstrated that upon depletion of intracellular Ca²⁺ stores, a diffusible Ca²⁺ influx factor (CIF) is produced that displaces inhibitory calmodulin (CaM) from iPLA₂β, leading to its activation and the production of lysophospholipids, which, in turn, activate plasma membrane Ca²⁺ influx channels and SOCE in SMCs. Upon refilling of the stores and the termination of CIF production, CaM binds back to iPLA₂β, inhibiting its activity and terminating SOCE. Thus, iPLA₂β emerged as a crucial transducer of the signal from depleted stores to plasma membrane channels and a potential target for the regulation and fine tuning of SOCE by other signaling cascades, including those that involve PKC. In previous studies, Albert and Large (3, 4) demonstrated 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate can cause the activation of Ca²⁺-conducting channels that can be also activated by norepinephrine in SMCs dispersed from the portal vein (3, 4). Also, Su and others (35) presented evidence that OAG can activate cation current in Jurkat cells that may be similar to that activated by store depletion. In addition, in multiple studies, DAG has been shown to activate some transient receptor potential C channels, which is thought to be a direct effect on the channel itself (for a review, see Ref. 18). Thus, DAG (and OAG) may have multiple effects, and the mechanism of DAG and PKC involvement in the activation of the specific SOCE pathway remains unclear.

This study was designed to test which steps in the SOCE pathway may be affected by DAG and whether iPLA₂β may be involved in the regulation of SOCE by PKC in SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca²⁺ stores as well as direct PKC--dependent activation of iPLA₂β, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

	MATERIALS AND METHODS

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Cells. Primary cultured SMCs from rabbit aortas were prepared following the same protocol as previously described (31, 32). All animal experiments were carried out in accordance with protocols and guidelines established by the United States National Institutes of Health.

Intracellular Ca²⁺ measurements. SMCs were loaded with fura-2 AM (2 µM fura-2 AM for 30 min at 37°C, followed by a 15-min wash), and changes in intracellular Ca²⁺ [fluorescence at 340/380 nm (F₃₄₀/F₃₈₀)] were monitored as previously described (15, 31–33). A dual-excitation fluorescence-imaging system (InCyt Im2, Intracellular Imaging, Cincinnati, OH) was used for experiments using individual cells. Experiments were done in 0 mM Ca²⁺ solution [containing (in mM) 120 NaCl, 4.7 KCl, 4 MgCl₂, 0.2 EGTA, and 10 HEPES], and the Ca²⁺ influx was determined from changes in fura-2 fluorescence after the readdition of Ca²⁺ (2 mM). Changes in intracellular Ca²⁺ were expressed as a change in ratio (Ratio), which was calculated as the difference between the peak F₃₄₀/F₃₈₀ ratio after extracellular Ca²⁺ was added and its level right before Ca²⁺ addition. The basal Ca²⁺ influx in the primary culture of SMCs was Ratio = 0.29 ± 0.10 (n = 130), which was subtracted from summary data shown by the bar graphs but not from original traces. Data were summarized from the large number of individual cells (20–40 cells in a single run, with 3–9 identical experiments done in at least 3 cell preparations).

SMC transfection. Transfection was done using Lipofectamine plus (Invitrogen) and following standard protocols. The oligonucleotides for antisense and scrambled PKC- were as specified in Ref. 36. Briefly, SMCs on days 6 and 7 (60–70% confluence) and 95% confluent SMCs were transfected with antisense and scrambled oligonucleotides [the same as successfully used in a previous study (36)] directed against PKC-: antisense oligonucleotide 5'-CATGAGGGCCGATGTGACCT-3' and scrambled oligonucleotide 5'-TACGCATAACGCGCTGGTGG-3'. Experiments were conducted 60–72 h after transfection. The oligonucleotides were labeled with fluorescein, and its presence in the cells was verified by imaging (excitation at 480 nm and emission at 515 nm) before the experiments.

Western blot analysis. Protein samples were incubated with Laemmli sample buffer at 95°C for 2 min, and 30 µg of total protein were loaded on a 7.5% SDS-polyacrylamide gel for electrophoresis. Proteins were transferred to nitrocellulose membranes in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) at 40 V overnight. The membrane was then blocked in PBS containing 0.05% Tween 20 (PBST) with 3% milk for 1 h and then incubated with primary (anti-PKC-) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. The primary antibody was removed, and blots were washed three times for 10 min with milk-PBST. Blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:2,000 in milk-PBST, washed three times in PBST, and treated with enhanced chemiluminescence reagents (Super ECL, Pierce) for 1 min. Blots were then exposed to photographic films, and the optical density was determined using Un-scan-it analysis software (Silk Scientific).

iPLA₂ assay. iPLA₂ Activity was performed using a modified commercial assay kit originally designed for the cytosolic PLA₂ (cPLA₂; Cayman Chemical). To detect the activity of iPLA₂ instead of cPLA₂, the assay buffers were modified to contain no Ca²⁺ (Ca²⁺ is needed for cPLA₂ but not for iPLA₂ activity). Phospholipase activity was assayed by incubating the samples with the substrate (arachidonoyl thio-PC) for 1 h at room temperature in a modified Ca²⁺-free assay buffer of the following composition: 300 mM NaCl, 10 mM HEPES, 8 mM Triton X-100, 4 mM EGTA, 60% glycerol, and 2 mg/ml BSA (pH 7.4). The generated free thiols were visualized by the addition of DTNB for 5 min, and the absorbance was determined at 405 nm using a standard microplate reader. The background iPLA₂-independent component of basal lipase activity was determined in control samples when all specific iPLA₂ activity had been inhibited with bromenol lactone (BEL; 10 µM for 5 min) and was subtracted from all the readings. The specific activity of iPLA₂ was expressed in absorbance units per milligram of protein.

Immunostaining. Cells grown in the Lab-Tek Chamber glass-bottomed slide system (Nalge Nunc) were fixed for 5 min in methanol at –20°C and air dried at room temperature. SMCs were then treated for 1 h with 1% heat-inactivated goat serum containing 0.1% Triton X-100 in PBS (for blocking) and incubated in a humidified chamber overnight a 4°C with primary antibody solution (rabbit anti-PKC-, Santa Cruz Biotechnology, 1:25 dilution). After washes with PBS, slides were incubated for 1 h in the dark with FITC-conjugated goat anti-rabbit antibody (1:200 dilution, Jackson Laboratories, West Grove, PA). Slides were mounted with Vectashield medium with Texas red (Vector Labs), and cell samples were analyzed using a high-resolution imaging system with the Openlab software package from Improvision (Coventry, UK). Raw images of SMCs (showing the localization of FITC conjugated with an anti-PKC- antibody within the whole cell) were visualized at 488-nm excitation and 510-nm emission. The nucleus (labeled by Texas red) was visualized at 544-nm excitation and 620-nm emission. As negative controls, cells were incubated with secondary antibodies only.

Drugs and treatments. Drugs were purchased from Sigma. Fura-2 and fura-2 AM were from Invitrogen. Chiral enantiomers of BEL (S-BEL and R-BEL) were separated by HPLC utilizing a Chirex column of 3,5-dinitrobenzoyl-R-phenylglycine attached to a silica matrix (Phenomenex) as previously described (21). It is important to notice that BEL and its enantiomers are suicidal substrates for iPLA₂; inhibition is irreversible, requires basal activity of this enzyme, and strongly depends on the temperature, duration of treatment, and concentration used (8, 15, 21, 31–33). In experiments with intact cells, the optimal conditions for BEL treatment (to ensure complete inhibition of iPLA₂) are as follows: intact cells need to be pretreated (in bath solution not containing BSA or serum) with 10–25 µM BEL for 30 min at 37°C, and BEL can then be washed away before the beginning of the experiments. In cases where iPLA₂ is already active, significantly shorter (1–5 min) treatments may fully inhibit the enzyme.

Statistical analysis. Group data are presented as means ± SE. A single or paired Student's t-test was used to determine the statistical significance of the obtained data. The significance between multiple groups was evaluated using ANOVA followed by Turkey's test. Data were considered significant at P < 0.05.

	RESULTS

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To test the potential effects of DAG and the role of PKC in SOCE, we used a variety of pharmacological and molecular tools and examined the effects of PKC activation and inhibition on Ca²⁺ influx in proliferating (or confluent) rabbit aortic SMCs. Ca²⁺ influx was triggered either by depletion of the stores with thapsigargin (TG) or via different shortcuts in the iPLA₂β and lysophospholipid-mediated SOCE pathway (32), which is shown in Fig. 1. By consecutive activation/inhibition of different components of this pathway (with the tools shown in Fig. 1), we started to look at which specific components and step(s) in the signal transduction pathway from the stores to ion channels may be regulated by PKC (and activated by OAG).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Scheme of the store-operated Ca2+ (SOC) entry (SOCE) pathway mechanism. The scheme summarizes the experimental procedures used to highlight the different facets and regulation of the SOCE pathway in smooth muscle cells (SMCs). 2-APB, 2-aminoethoxydiphenyl borate; DES, diethylstilbestrol; BEL, bromenol lactone; PM, plasma membrane; LPC, lysophosphatidylcholine; iPLA2, Ca2+-independent PLA2; CaM, calmodulin; CIF, Ca2+ influx factor; OAG, 1-oleoyl-2-acetyl-sn-glycerol; IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; TG, thapsigargin.

View larger version (12K): [in this window] [in a new window]   Fig. 2. PKC inhibition impairs TG- and OAG-induced Ca2+ influx. A, left: representative traces showing the changes in intracellular Ca2+ concentration [presented as the ratio of fluorescence at 340 to 380 nm (F340/F380)] in fura-2-loaded aortic SMCs. TG (2 µM) was applied in the absence of extracellular Ca2+. To test for Ca2+ influx, Ca2+ (2 mM) was added at the time shown (arrow). TG-induced responses are shown in a representative control cell and the cell to which PKC inhibitor [chelerythrine (Cheler); 10 µM] was applied 2 min before Ca2+, as shown (*). The bar graph on the right shows the average amplitude of TG-induced Ca2+ influx (ratios ± SE) from control and Cheler-treated SMCs and from SMCs where OAG was applied after the addition of extracellular Ca2+ to TG-treated SMCs (TG + OAG). Each bar summarizes the result from 70–105 cells (numbers shown above each bar). B: similar to A, but OAG (50 µM) was applied to SMCs instead of TG. Bar graphs represent means ± SE of Ca2+ influx from 83–93 cells.

View larger version (11K): [in this window] [in a new window]   Fig. 3. OAG and PMA-induced Ca2+ influx are prevented by SOC channel inhibitors. A, left: representative traces showing the changes in intracellular Ca2+ concentration in fura-2-loaded SMCs. OAG (50 µM) was applied in the absence of extracellular Ca2+, and 2 mM Ca2+ was then added as indicated (arrow). Traces are for SMCs treated with OAG (control) and when 2-APB (100 µM) or DES (10 µM) were added 2 min before Ca2+, as shown (*). Bar graph on the right shows the average amplitudes of OAG-induced Ca2+ influx (ratios ± SE) from the experiments as show on the left. Each bar summarizes the result from 32–121 cells (numbers shown above each bar). B, left: representative traces showing PMA-induced changes in intracellular Ca2+ concentration in fura-2-loaded SMCs. PMA (1 µM) was applied in the absence of extracellular Ca2+, and 2 mM Ca2+ was then added as shown (arrow). Traces are for SMCs treated with PMA (control) and when 2-APB (100 µM) was added 1 min before Ca2+, as shown (*). Bar graph on the right shows average amplitudes of PMA-induced Ca2+ influx (ratios ± SE) from control, 2-APB-treated, and Cheler (10 µM)-treated SMCs. n = 23–40 cells, as indicated above each bar.

View larger version (7K): [in this window] [in a new window]   Fig. 4. LPC evoked a Cheler-insensitive Ca2+ influx. Left: representative traces show intracellular Ca2+ changes in fura-2-loaded SMCs treated with LPC (300 nM). LPC was added in Ca2+-free solution for 4–5 min before Ca2+ (2 mM) administration. Traces are for cells exposed to LPC alone (–Cheler), cells treated for 3 min with 10 µM Cheler before Ca2+ addition (+Cheler), and cells treated with LPC and 2-APB (100 µM) added as shown (*). The bar graph on the right shows average amplitudes of LPC-induced Ca2+ influx (ratios ± SE) from control, 2-APB-treated, and Cheler (10 µM)-treated SMCs. n = 20–27 cells, as indicated above each bar.

View larger version (15K): [in this window] [in a new window]   Fig. 5. OAG-induced Ca2+ influx is inhibited by S-BEL but not by R-BEL. A, left: representative traces showing the changes in intracellular Ca2+ in SMCs induced by TG (2 µM) in control cells and in cells pretreated with BEL enantiomers. S-BEL was used to inhibit iPLA2β or R-BEL to block iPLA2 (25 µM for 30 min at 37°C). Extracellular Ca2+ (2 mM) was added at the time shown (arrow). The bar graph on the right shows the dose dependence of the effects of S-BEL and R-BEL on TG-induced Ca2+ influx in SMCs. The best fit was generated with a Hill equation with an IC50 for S-BEl of 3 µM. n = 60–140 cells. B, left: representative traces of OAG-induced Ca2+ influx in control cells and in SMCs pretreated with S-BEL to block iPLA2β (25 µM for 30 min at 37°C) or R-BEL to block iPLA2 (25 µM for 30 min at 37°C). The bar graph on the right summarizes data from 40–50 cells of the experiments on the left.

View larger version (11K): [in this window] [in a new window]   Fig. 6. OAG-induced a PKC-dependent activation of iPLA2 in intact and homogenized SMCs. A: summary data showing the activity of iPLA2 in samples from intact untreated SMCs (basal), from cells treated for 5 min with 5 µM TG, from SMCs treated for 3 min with 50 µM OAG, from cells pretreated for 5 min with 10 µM Cheler and then 50 µM OAG, and from SMCs pretreated with 25 µM BEL (30 min at 37°C) and then 50 µM OAG. Data are from 12–15 measurements from 3 different cultures. B: average activity of iPLA2 in samples from untreated SMCs that were first homogenized and then treated as in A. Data are from 9–12 measurements from 4 different cultures. *Significant increases in activity compared with basal levels (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 7. OAG induced a partial depletion of intracellular Ca2+ stores and Ca2+ influx that declined within 5–10 min. A, left: representative traces showing the changes in intracellular Ca2+ concentration (F340/F380) in control SMCs or those pretreated with 50 µM OAG in Ca2+-free conditions. Ionomycin (IM; 100 nM) was applied to release Ca2+ from the stores, and Ca2+ (2 mM) was then added at the time indicated (arrow) to test for Ca2+ influx. Summary data are shown on the right. The bar graph presents the average Ca2+ release and Ca2+ influx tested in the absence (n = 85 cells) and presence of OAG (n = 140 cells). B, left: overlay of representative traces showing Ca2+ responses in SMCs when OAG (50 µM) was applied in the absence of extracellular Ca2+ and Ca2+ (2 mM) was then added at different times after OAG application, as shown (* and arrows). Summary data on the right show the time course of Ca2+ influx induced by OAG. Each bar is an average from 42–80 cells.

Role of PKC- in the activation of SOCE in proliferating SMCs.

View larger version (13K): [in this window] [in a new window]   Fig. 8. OAG and TG induced Ca2+ influx in confluent SMCs. A, left: representative traces showing the changes in intracellular Ca2+ concentration in fura-2-loaded aortic SMCs. OAG (50 µM) was applied in the absence of extracellular Ca2+, and 2 mM Ca2+ was then added at the time shown (arrow). TG (2 µM) was added at the end of the experiments. The bar graph on the right shows average amplitudes (ratios ± SE) of TG-induced Ca2+ influx, OAG-treated SMCs, and from SMCs where TG was applied after the addition of extracellular Ca2+ in OAG-treated SMC, as shown on the left. Summary data are from 42–60 cells, as shown above each bar. B: bar graph showing the changes in intracellular Ca2+ concentration (ratios ± SE) in confluent SMCs. Bars are for SMCs treated with TG (2 µM) for 4–5 min in the absence of extracellular Ca2+, and Ca2+ (2 mM) was then added (control); for SMCs treated with TG when 2-APB (100 µM), Cheler (10 µM), calphostin C (1 µM), or KN-62 (10 µM) were added 2 min before Ca2+; and for cells pretreated with 25 µM of either R-BEL or S-BEL for 30 min. Numbers of cells are indicated above each bar. C: same as in B except that OAG was applied to induce Ca2+ influx. Numbers of cells are indicated above each bar.

View larger version (31K): [in this window] [in a new window]   Fig. 9. Distribution of PKC- in proliferating and confluent SMCs. A: immunostaining of proliferating (left) and confluent (right) SMC with PKC- antibody conjugated to FITC. B: Western blot (top) and densitometry analysis (bottom) showing PKC- expression in samples from proliferating and confluent SMCs. Average data were from 3 different Western blots.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Effect of SMC transfection with antisense nucleotides for PKC- on TG- and OAG-induced Ca2+ influx. A, left: representative traces showing the changes in intracellular Ca2+ concentration in proliferating SMCs (40–60%). Cells were treated with TG (2 µM for 5 min in the absence of extracellular Ca2+), and Ca2+ then was added at the time shown (arrow). Traces are for nontransfected SMCs (control) and SMCs transfected with PKC- sense (s) or antisense (a/s) oligonucleotides 60–72 h before the experiments. Insert, representative Western blot of PKC- expression from SMCs transfected with sense and antisense oligonucleotides. Summary data on the right show TG-induced Ca2+ influx from the experiments on the left. The average was from a total of 75–150 cells. B: same as in A except that OAG (50 µM) was applied to induce Ca2+ influx. The average was from a total of 50–60 cells. C: bar graphs showing summary data of TG-induced (left) and OAG-induced (right) Ca2+ influx in SMCs confluent at 95% under same conditions as in A and B. The average was from 108–135 cells for TG-treated SMCs and 30–35 cells for OAG-treated SMCs.

	DISCUSSION

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Several lines of evidence suggest that OAG may produce the depletion of Ca²⁺ stores in SMCs. Indeed, OAG application resulted in significant (but not complete) Ca²⁺ loss from stores, which may be due to the OAG-induced uncoupling of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) function that was demonstrated earlier by Cardoso and colleagues (12) in muscle cells and platelets. Transient inhibition of SERCA by OAG may easily explain the transient activation of Ca²⁺ entry that we found to peak within the first minute and then fade away, leaving 25% of the maximum Ca²⁺ entry after 5 min. The transient nature of the OAG-induced effect was similar to what we earlier observed in platelets upon their activation with thrombin, which may involve naturally produced DAG (37). Some of the controversies in earlier reports related to the ability of DAG (or OAG) to activate SOCE may be explained by the time dependence of its effect on store depletion. One will expect that the time window when OAG effects could be observed and the level of the residual SOCE may significantly vary, being longer lasting and more pronounced in some cell types, whereas being hardly detectible in other cell types and experimental conditions. Importantly, OAG-induced activation of single SOC channels (3) could be a result of OAG-induced store depletion (in the case of cell-attached membrane patches in intact SMCs) or direct PKC-dependent activation of iPLA₂β when SOC channels were studied in outside-out membrane patches in which iPLA₂β (and PKC) may remain present and fully functional (32).

The fact that OAG was able to activate SOCE in both proliferating and confluent SMCs, which had different levels of expression of PKC- and different sensitivities of SOCE to general PKC inhibitors, strongly suggests that OAG may mimic the effects of TG and activate SOCE via depletion of stores, which does not seem to involve PKC. However, the important role of PKC- in both TG- and OAG-induced SOCE and their equal sensitivity to PKC inhibitors in proliferating SMCs strongly suggest that PKC- can regulate some other step(s) in the SOCE pathway. We found that lysophospholipid-induced SOCE activation (a part of the pathway downstream of iPLA₂) is not sensitive to PKC inhibition, whereas a PKC inhibitor can significantly impair iPLA₂ activation in cell homogenates. This suggests that iPLA₂β itself can be a target for PKC regulation in proliferating SMCs. In vitro activation of iPLA₂β by PKC-dependent phosphorylation was reported earlier in the membrane fraction of ventricular myocytes and macrophage-like P388D₁ cells (1, 34). Such PKC-induced activation of iPLA₂β may either originate a "shortcut" signal in the SOCE pathway or may amplify the signal originating in depleted stores, in both cases resulting in SOCE activation. Our new finding that PKC plays a role only in proliferating but not confluent SMC suggests that the contribution of the PKC-dependent mechanism may vary in different cell types (or even in the same cells, but in different stages of their growth and proliferation). It also suggests that the major effect of OAG on SOCE may be via direct PKC-independent inhibition of SERCA function, as suggested by Cardoso et al. (12).

Thus, the role of DAG (OAG) and PKC in SOCE regulation may be a rather complex and flexible phenomenon that may physiologically adjust to the needs and expression patterns of different types of cells and different levels of their proliferation and differentiation. We do not believe that PKC is crucial for SOCE, because chelerythrine, which is known to block the majority of PKC isoforms, did not have any effect in confluent SMCs, where SOCE works just fine with or without it. The fact that chelerythrine blocks significant (but noticeably not all) SOCE in proliferating cells makes us believe that PKC can be an important modulator of SOCE, and our study provides evidence that iPLA₂β can be a target for such PKC-dependent (and OAG/DAG-dependent) modulation. When one thinks about PKC and Ca²⁺ regulation, many additional processes within the cell should be taken into consideration. As one of many examples, SERCA inhibitors (which are widely used as a specific method to trigger the activation of SOCE) may produce a significant Ca²⁺ rise in the cytosol (due to passive Ca²⁺ release), which, in the absence of efficient intracellular Ca²⁺ buffering, may lead to the Ca²⁺-induced activation of PLC (20). This will result in the production of DAG, which can trigger the activation and reported translocation of PKC to the plasma membrane (23, 24, 34, 39), where it can modulate iPLA₂β activity and SOCE. This nonspecific pathway can cross talk with the SOCE pathway, and its ability to modulate SOCE will greatly depend on the ability of the cell to effectively buffer the passive Ca²⁺ leak from the stores (which will translate into whether PLC can get activated or not and if DAG will be produced) and the availability of DAG-dependent PKC that can translocate to the plasma membrane and regulate iPLA₂β activity. All these can vary in different cell types as well as in the same cells at different stages of their proliferation and may be one of the reasons why PKC can play an important role in proliferating but not confluent SMCs. Further studies are needed to assess the potential ability of some other protein kinases to modulate SOCE through iPLA₂β and to discover the whole net of complex interactions between different signaling cascades that may be involved in the regulation of the SOCE pathway.

	GRANTS

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This work was supported by American Heart Association Grant 04252860; Fondos de Investigación Sanitaria del Gobierno de España Grant PI050396 (to T. Smani), and National Heart, Lung, and Blood Institute Grants HL-54150 and HL-71793 (to V. M. Bolotina). T. Smani is an investigator of the "Ramon y Cajal" program supported by the Spanish Ministry of Education.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Smani, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen de Roció, Quirofano Experimental, Laboratorio de Investigación Cardiovascular, Avenida Manuel Siurot s/n, Sevilla 41013, Spain (e-mail: tasmani{at}us.es)

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.

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