Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

Kristen M. Park, Mario Trucillo, Nicolas Serban, Richard A. Cohen, Victoria M. Bolotina


Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (CaL2+) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a CaL2+ inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and CaL2+ channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2β totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by CaL2+ channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of CaL2+ channels and lead to Ca2+ entry and vasoconstriction.

  • store-operated calcium entry
  • smooth muscle cells
  • constriction

in cerebral arteries and other small-diameter vessels, voltage-gated L-type Ca2+ (CaL2+) channels are known to be the primary channels responsible for agonist-induced constriction, in as much as their inhibition was shown to fully relax most microvessels (19, 20). On the other hand, depletion of intracellular Ca2+ stores by different agonists [or by inhibitors of sarco(endo)plasmic reticulum Ca2+-ATPase] activates store-operated (SOC) channels and store-operated Ca2+ entry (SOCE) in isolated smooth muscle cells (SMC) (for review see Refs. 1, 3, 26). In conduit vessels, nonselective cation SOC (rather than CaL2+ channels) play a major role in agonist-induced constriction and nitric oxide-induced relaxation (8, 27), but the role of SOC in constriction of microvessels, especially in the cerebral circulation, remains unclear. In SMC of cerebral arterioles, activation of SOCE increases Ca2+ but fails to generate constriction (11, 12), suggesting that SOC may be mediating Ca2+ influx into restricted space (or cellular compartments), which may be spatially separated from the contractile apparatus. In interlobular arteries, SOC are present but do not significantly contribute to Ca2+ entry after agonist stimulation (10). In numerous other types of SMC, Ca2+ store depletion was reported to increase Ca2+ and cause constriction, although it was studied only using poorly selective SOC inhibitors (for review see Refs. 3 and 14). Thus the following questions remain. 1) By what mechanism might SOC be involved in the physiological regulation of agonist-induced constriction in microvessels? 2) How might their role be related to activation of CaL2+ channels. Because of the nonselective cation permeability of SOC in SMC, SOC activation should cause a significant membrane depolarization, which may result in cross talk between SOC and CaL2+ channels (7); this theory is supported by studies in isolated SMC from guinea pig gallbladder (18) and in the A7r5 cell line (21), but little is known about their possible cross talk in intact vessels.

Recently, the Ca2+-independent phospholipase A2 (iPLA2) β (group VI) (2, 30) was found to be a crucial determinant of SOCE activation in isolated aortic SMC, but the role of iPLA2β in intact cerebral and other microvessels is unknown. Functional inhibition of iPLA2 with its suicidal substrate bromoenol lactone (BEL) or specific antisense oligonucleotides against iPLA2β has been shown to produce identical impairment of SOCE in isolated SMC (24, 25), as well as astrocytes (23), keratinocytes (22), skeletal muscle (4), fibroblasts (16), prostate cancer (28), and other cell types (24, 25). The crucial role of iPLA2β in SOCE activation was recently confirmed in a screen of Drosophila melanogaster genes (29), where molecular knockdown of the CG6718 gene (a Drosophila homolog of human iPLA2β) significantly affected SOCE. In isolated vascular SMC, we discovered that single-cation SOC and SOCE can be activated by lysophospholipids, which are produced by the plasma membrane-bound iPLA2β (24). Activation of iPLA2β can be physiologically achieved by Ca2+ influx factor, which is produced in endo(sarco)plasmic reticulum on depletion of Ca2+ stores (5) and is capable of displacing inhibitory calmodulin from its binding site on iPLA2β. Thus agonist-induced depletion of the stores can activate SOC and SOCE in SMC via an iPLA2β-dependent pathway (24).

Discovery of iPLA2β as a crucial component of the SOCE mechanism in SMC and development of a chiral-specific functional inhibitor of iPLA2β (9, 15) have opened new avenues for studying the role of the SOCE mechanism in vascular constriction. In the present study, we have examined the roles of SOC and CaL2+ channels in microvessels and tested the hypothesis that agonist-induced iPLA2-dependent activation of SOC may be required for secondary activation of CaL2+ and Ca2+ entry, which trigger contractile responses in cerebral, mesenteric, and carotid arteries. Using simultaneous recording of Ca2+ in SMC and vessel diameter, we demonstrate that phenylephrine (PE)-induced constriction fully depends on the functional activity of iPLA2β and SOC, as well as CaL2+ channels. These new findings establish the crucial role of iPLA2β-dependent SOC in vascular responses to agonist stimulation and provide a new important mechanism of regulation of tone in different microvascular beds.


Drugs and materials.

Unless otherwise indicated, all drugs were purchased from Sigma. BEL was purchased from Biomol; S- and R-BEL from Cayman Chemicals; and fura 2-AM from Invitrogen. Physiological salt solution (PSS) consisting of (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11 glucose was bubbled with 95% O2-5% CO2 to ensure pH 7.4.

Isolation of mouse cerebral and mesenteric and rabbit carotid arteries.

Posterior cerebral arteries (PCA) and mesenteric arteries were obtained from male mice (C57BL/6, 8–10 wk old). First-order PCA and second-order mesenteric arteries (∼120 μm diameter) were dissected free from the mesenteric arterial bed or brain and connective tissue in ice-cold PSS. Dissected arterial segments (200 μm long) were cannulated in a chamber connected to a pressure myograph (Living Systems Instrumentation, Burlington, VT) and immediately used for experiments.

Carotid arteries were obtained from male New Zealand rabbits (3.1 ± 0.4 kg body wt), cleaned of all connective tissue in cold PSS, and mounted onto a myograph (see below).

All research was performed in accordance with the American Physiological Society “Guiding Principles in the Care and Use of Animals” and with Institutional Animal Care and Use Committee approval.

Simultaneous recording of Ca2+ and vessel diameter.

Mircovessels were maintained at 37°C throughout the experiment by superfusion with prewarmed, bubbled PSS at a rate of 5 ml/min. Before the experiments, vessels were equilibrated for 20 min at 10 mmHg, then pressure was raised to 60 mmHg; intravascular pressure was controlled by a pressure servo and peristaltic pump (Living Systems Instrumentation). The chamber with the microvessel was mounted on an Olympus microscope equipped with the IonOptix data acquisition system (IonOptix, Milton, MA), which allowed simultaneous measurement of 1) intraluminal diameter using the Softedge Acquisition Subsystem and 2) intracellular Ca2+ concentration ([Ca2+]i) using a ratiometric photomultiplier system. Fura 2-AM loading of SMC was accomplished by incubation of the mounted arteries in external PSS containing 5 μM fura 2-AM for 50 min at room temperature in the dark. The fura 2-AM ratio (ratio of fura 2 fluorescence at 340-nm excitation to that at 380-nm excitation, with emission at 510 nm) was recorded at a sampling rate of 5 Hz. Arterial diameter and fluorescence ratio were simultaneously recorded and plotted over time.

Isometric tension measurements.

Isometric tension was measured as previously described (6, 8). Briefly, 5-mm-long rings of rabbit carotid artery segments were cleaned of connective tissue and mounted on metal stirrups in organ chambers, and isometric tension was recorded. Rings were maintained at 37°C in bubbled (95% O2-5% CO2) PSS. Rings were equilibrated and stretched to an optimal tension of 7 g. Pharmacological agents were added to the bath as indicated.

Statistical analysis.

Summary data are presented as means ± SE. Student's t-test was used to determine the statistical significance of the data. P < 0.05 was considered significant.


To investigate the role of SOC in contractile responses of microvessels, we first tested and compared the effects of 2-aminoethyl diphenylborinate (2-APB, a widely used SOC inhibitor) and nimodipine (Nim, an inhibitor of CaL2+ channels) on responses to PE or high K+. [Ca2+]i in SMC was measured simultaneously with intraluminal vessel diameter in PCA (Fig. 1). The high-K+-induced Ca2+ rise and vessel constriction were inhibited by 5 μM Nim but were not affected by 75 μM 2-APB [which is known to fully inhibit SOC and SOCE in SMC (12)], consistent with the involvement of only CaL2+ channels in depolarization-induced [Ca2+]i rise and vessel constriction (Figs. 1 and 2). On the other hand, PE (10 μM)-induced [Ca2+]i rise and vessel constriction were inhibited by Nim and 2-APB (Figs. 1 and 2), suggesting the involvement of SOC and CaL2+ channels in agonist-induced responses.

Fig. 1.

Simultaneous Ca2+ rise in smooth muscle cells (SMC) and constriction caused by phenylephrine (PE) and high K+ in intact cerebral artery. Representative traces show changes in intracellular Ca2+ [as ratio of fura 2-AM fluorescence at 340 nm to 380 nm (top trace)] and intraluminal diameter of intact pressurized vessel (bottom trace) during bath application of 10 μM PE, 75 μM 2-aminoethyl diphenylborinate (2-APB), high (60 mM) K+, and 5 μM nimodipine (Nim). Vessel was washed for 3 min between PE and K+ applications.

Fig. 2.

Effects of 2-APB and Nim on PE- and high-K+-induced simultaneous Ca2+ rise and constriction in intact cerebral arteries. A: changes in intracellular Ca2+ (top trace) and intraluminal diameter (bottom trace) during application of 10 μM PE, 5 μM Nim, 60 mM K+, and 75 μM 2-APB. B: changes in Ca2+ (top) and simultaneous changes in intraluminal vessel diameter (bottom). Values are means ± SE from 8 experiments. *P < 0.05 vs. control.

To provide new mechanistic insights into the role of SOC activation and the iPLA2-mediated SOCE pathway in microvessel constriction, the effect of functional inhibition of iPLA2 on PE- and K+-induced Ca2+ influx and constriction of cerebral, mesenteric, and carotid arteries was examined. Simultaneous changes in [Ca2+]i and vessel diameter in response to consecutive application of K+ (60 mM) and PE (10 μM) to a cerebral artery are shown in Fig. 3. After the responses were tested under control conditions, the vessels were treated for 30 min with 25 μM BEL, which was previously shown to irreversibly impair iPLA2 function in SMC (25). After the vessels were treated with BEL and washed for 10 min, K+ and PE were reapplied (Fig. 3), and the vessels retained a normal response to high K+, but not PE. As shown in Fig. 4A, inhibition of iPLA2 did not affect Ca2+ influx and vessel constriction induced by high K+ (and mediated by CaL2+ channels) but dramatically impaired agonist-induced responses in cerebral arteries. Similar results in mouse mesenteric arteries (Fig. 4B) and rabbit carotid artery (Fig. 5) extended our findings to these additional microvascular beds as well as different types of animals. Figure 5 also demonstrates that inhibition of iPLA2 not only could prevent agonist-induced constriction but also could relax the preconstricted artery.

Fig. 3.

Effect of irreversible Ca2+-independent phospholipase A2 (iPLA2) inhibition by bromoenol lactone (BEL) on K+- and PE-induced responses in intact cerebral artery. Results from a representative experiment show effects of application of high (60 mM) K+ and 10 μM PE on Ca2+ (top trace) and vessel diameter (bottom trace) before and after 30 min of treatment with 25 μM BEL. Addition of PE after BEL treatment dramatically reduced intracellular Ca2+ rise and completely abolished constriction; K+ did not affect Ca2+ or vessel diameter. Traces represent results from 4 experiments.

Fig. 4.

Inhibition of iPLA2 by BEL results in inhibition of Ca2+ influx and vessel constriction on stimulation by agonist, but not high K+, in cerebral (A) and mesenteric (B) arteries. Summary data are from experiments similar to those described in Fig. 3. Values are means ± SE for 6 (A) and 8 (B) experiments. *P < 0.05 vs. control.

Fig. 5.

Inhibition of iPLA2 by BEL results in vessel relaxation and irreversible inhibition of agonist-induced, but not depolarization-induced, constriction in carotid artery SMC. Carotid artery ring was constricted with 0.1 μM PE, and 25 μM BEL was added in the continuous presence of PE. After complete vessel relaxation, PE and BEL were washed out for 20 min, and vessel was treated with high (30 mM) K+ and then with PE. Values are means ± SE from 4 experiments. *P < 0.05 vs. control.

To confirm that iPLA2β, and not iPLA2γ, is required for agonist-induced Ca2+ entry and constriction in microvessels, we compared the effects of S-BEL and R-BEL [chiral enantiomers of this suicidal substrate that can discriminate these iPLA2 isoforms (9, 15)] on PE-induced responses in cerebral arteries. Although vessels pretreated with R-BEL (which is specific to iPLA2γ) responded normally to PE, S-BEL (which is specific to iPLA2β) totally impaired PE-induced Ca2+ entry and constriction in the same vessels (Fig. 6).

Fig. 6.

Inhibition of iPLA2β by S-BEL results in inhibition of agonist-induced Ca2+ influx and vessel constriction, whereas inhibition of iPLA2γ by R-BEL has no effect on agonist responses. Representative traces and summary data show PE (10 μM)-induced Ca2+ (top trace) and diameter (bottom trace) responses of cerebral vessels after treatment with R-BEL (25 μM for 25 min) and then again after treatment with S-BEL (25 μM for 25 min).


Our data in intact cerebral, mesenteric, and carotid arteries demonstrate for the first time that iPLA2-dependent activation of SOC is essential for agonist-induced Ca2+ entry and constriction in a wide variety of arteries, suggesting that this is a widespread phenomenon in microvessels.

To account for our finding of the equally important role of iPLA2β, as well as SOC and CaL2+ channels, we propose a model for agonist-induced responses in microvessels (Fig. 7). On the basis of our earlier findings in isolated SMC (8, 24), we propose that agonist stimulation causes depletion of Ca2+ stores and activation of iPLA2, which produces lysophospholipids that activate SOC. Nonselective cation SOC (27) can produce significant membrane depolarization and secondary activation of CaL2+ channels, which cause Ca2+ entry and, in turn, lead to vessel constriction. In this way, CaL2+ channels and SOC may be equally important for agonist-induced Ca2+ entry in SMC and constriction, as we found in microvessels. In contrast to smaller arteries, in the aorta we found that CaL2+ channels are not essential for agonist-induced Ca2+ influx and constriction (inhibition of CaL2+ has little to no effect on aortic tone) (8, 27). The difference may be explained by an ability of Ca2+ entering through SOC to directly cause contraction in aortic SMC and/or SOC-induced depolarization being insufficient for secondary activation of CaL2+ channels in SMC of these vessels. There is little doubt about the role of CaL2+ channels in responses of resistance vessel to agonists, in as much as selective inhibition of CaL2+ channels in all vessels tested in the present study impaired Ca2+ entry and constriction. This result was consistent with CaL2+ channels, not SOC, mediating Ca2+ entry, which triggers vessel constriction. However, SOC still plays a crucial role in agonist-induced responses, if not as a major Ca2+ entry path (as in aortic SMC), then as a depolarizing trigger for secondary activation of CaL2+ channels. Interestingly, Figs. 3 and 4 show that PE-induced constriction may be fully abolished, even though some residual Ca2+ rise in SMC could be detected after BEL treatment. Stronger inhibition of contractile responses may reflect the involvement of iPLA2 in agonist-induced Ca2+ sensitization of contractile proteins (13), which may amplify the effect of the reduction on agonist-induced Ca2+ influx.

Fig. 7.

A model for agonist-induced Ca2+ entry and constriction of conduit and microvessels. Agonist stimulation triggers inositol trisphosphate (IP3) production and depletion of Ca2+ stores, leading to production of Ca2+ influx factor (CIF). CIF displaces inhibitory calmodulin (CaM) from iPLA2, causing its activation and production of lysophospholipids (LysoPL) and, in turn, activates SOC. Nonselective cation SOC in SMC allow Ca2+ and Na+ entry. In conduit vessels, SOC-mediated Ca2+ entry may cause vessel constriction; in microvessels, this pool of Ca2+ may be spatially separated from the contractile apparatus. Instead, in microvessels, SOC-induced depolarization may trigger activation of L-type Ca2+ (CaL2+) channels, which allow Ca2+ entry and, in turn, lead to vessel constriction. Thus SOC may play an important, but slightly different, role in constriction of different vessels: in conduit vessels, SOC allow Ca2+ influx, which is sufficient for constriction; in resistance vessels, SOC serve as a trigger for secondary activation of CaL2+ channels, which mediate Ca2+ influx and, in turn, cause vessel constriction. IP3R, IP3 receptor; PLC, phospholipase C; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase. R, G:G-coupled receptor.

The use of functional inhibition of iPLA2β in our study (in addition to a widely used SOC inhibitor) introduces a new effective way to selectively impair the SOCE pathway in microvascular SMC and provides a more reliable method for assessing the role of SOC in physiology and pathology of microcirculation. Indeed, direct SOC inhibitors (e.g., 2-APB) may not exclude the possibility of involvement of additional non-SOC, in as much as 2-APB is known to inhibit agonist-induced Ca2+ release (17), as well as other non-SOC, which could also participate in agonist-induced responses. Recently, we demonstrated (31) that a TRPC1-encoded inositol trisphosphate (IP3) receptor-operated channel (which can be clearly distinguished from SOC) may be activated by agonist-induced IP3 rise and, potentially, may be involved in SMC and vessel responses to agonists. Importantly, because 2-APB is able to block both channels (SOC and IP3 receptor-operated channels), it is impossible to distinguish their role in agonist-induced responses solely on the basis of 2-APB-induced inhibition. In our present study, in addition to 2-APB, we used BEL-induced inhibition of iPLA2 as a selective way to impair SOC activation, in as much as only SOC require its functional activity (4, 9, 16, 2225, 28). BEL (and its chiral enantiomers) provides new specific tools for assessing the role of iPLA2β and SOC activation in intact vessels, in which molecular knockdown of proteins poses significant difficulties.

In summary, we have demonstrated that iPLA2-dependent activation of SOC, as well as CaL2+ channels, is crucial for agonist-induced Ca2+ entry and constriction in conduit and microvessels (Fig. 7).


This work was supported by National Heart, Lung, and Blood Institute Grants HL-054150 and HL-071793. K. M. Park was supported by National Heart, Lung, and Blood Institute Fellowship HL-007969.


We thank Dr. Gokina for help in establishing the method for simultaneous measurement of Ca2+ and vessel diameter.

Present address of N. Serban: Department of Physiology, University of Medicine and Pharmacy, Universitatii 16, Iasi 700115, Romania.


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