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

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     

Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C

The mechanism of receptor-induced activation of the ubiquitously expressed family of mammalian canonical transient receptor potential (TRPC) channels has been the focus of intense study. Primarily responding to phospholipase C (PLC)-coupled receptors, the channels are reported to receive modulatory input from diacylglycerol, endoplasmic reticulum inositol 1,4,5-trisphosphate receptors and Ca2+ stores. Analysis of TRPC5 channels transfected within DT40 B cells and deletion mutants thereof revealed efficient activation in response to PLC-beta or PLC-gamma activation, which was independent of inositol 1,4,5-trisphoshate receptors or the content of stores. In both HEK293 cells and DT40 cells, TRPC5 and TRPC3 channel responses to PLC activation were highly analogous, but only TRPC3 and not TRPC5 channels responded to the addition of the permeant diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG application or elevated endogenous DAG, resulting from either DAG lipase or DAG kinase inhibition, completely prevented TRPC5 or TRPC4 activation. This inhibitory action of DAG on TRPC5 and TRPC4 channels was clearly mediated by protein kinase C (PKC), in distinction to the stimulatory action of DAG on TRPC3, which is established to be PKC-independent. PKC activation totally blocked TRPC3 channel activation in response to OAG, and the activation was restored by PKC-blockade. PKC inhibition resulted in decreased TRPC3 channel deactivation. Store-operated Ca2+ entry in response to PLC-coupled receptor activation was substantially reduced by OAG or DAG-lipase inhibition in a PKC-dependent manner. However, store-operated Ca2+ entry in response to the pump blocker, thapsigargin, was unaffected by PKC. The results reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation, reflecting a likely universal feedback control on TRPCs, and that DAG-mediated PKC-independent activation of TRPC channels is highly subtype-specific. The profound yet distinct control by PKC and DAG of the activation of TRPC channel subtypes is likely the basis of a spectrum of regulatory phenotypes of expressed TRPC channels.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.     

Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

The ER Ca2+ sensor STIM1 and the Ca2+ channel Orai1 are key players in store-operated Ca2+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca2+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca2+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca2+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca2+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca2+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca2+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca2+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.     

Bromoenol lactone, an inhibitor of Group V1A calcium-independent phospholipase A2 inhibits antigen-stimulated mast cell exocytosis without blocking Ca2+ influx

Calcium-independent phospholipase A2 (iPLA2beta) has recently been suggested to regulate Ca2+ entry by activating store-operated Ca2+ channels. These studies have been conducted in mast cells using thapsigargin to deplete intracellular stores. In RBL 2H3 and bone marrow-derived mast cells (BMMCs), Ca2+ entry is critical for exocytosis and therefore we have examined whether the proposed mechanism would be relevant when a physiological stimulus is applied to these cells. Using an iPLA2beta antibody, we demonstrate that the 84kDa iPLA2beta is expressed in these mast cells. As bromoenol lactone (BEL) is a suicide-based irreversible inhibitor of iPLA2beta it was used to probe this potential mechanism. We observe inhibition of exocytosis stimulated either with antigen or with thapsigargin. However, BEL also inhibits exocytosis when stimulated using a Ca2+ ionophore A23187, which passively transports Ca2+ down a concentration gradient and also in permeabilised mast cells where Ca2+ entry is no longer relevant. Moreover, BEL has only a minor effect on antigen- or thapsigargin-stimulated Ca2+ signalling, both the release from internal stores and sustained elevation due to Ca2+ influx. These results cast doubt on the proposed mechanism involving iPLA2beta required for Ca2+ entry. Although inhibition of exocytosis by BEL could imply a requirement for iPLA2beta activation for exocytosis, an alternative explanation is that BEL inactivates other target proteins required for exocytosis.     

Capacitative cation entry in human myometrial cells and augmentation by hTrpC3 overexpression

Transient receptor potential (Trp) channels have been implicated in mediating store- and receptor-activated Ca2+ influx. Different properties of this influx in various cell types may stem from the assembly of these Trp proteins into homo- or heterotetramers or association with other regulatory proteins. We examined the properties of endogenous capacitative Ca2+ entry in PHM1 immortalized human myometrial cells that express endogenous hTrpCs 1, 3, 4, 6, and 7 mRNA and in primary human myocytes. In PHM1 cells, activation of the oxytocin receptor or depletion of intracellular Ca2+ stores with the endoplasmic reticulum calcium pump-inhibitor thapsigargin induced capacitative Ca2+ entry, which was inhibited both by SKF 96365 and gadolinium (Gd3+). Whereas unstimulated cells did not exhibit Sr2+ entry, oxytocin and thapsigargin enhanced Sr2+ entry that was also inhibited by SKF 96365 and Gd3+. In contrast, Ba2+, a poor substrate for Ca2+ pumps, accumulated in these cells in the absence of the capacitative entry stimulus and also after oxytocin and thapsigargin treatment. Both types of entry were markedly decreased by SKF 96365 and Gd3+. The membrane-permeant derivative of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), elicited oscillatory increases in PHM1 intracellular Ca2+ that were dependent on extracellular Ca2+. These properties were also observed in primary human myocytes. Overexpression of hTrpC3 in PHM1 cells enhanced thapsigargin-, oxytocin-, and OAG-induced Ca2+ entry. These data are consistent with the expression of endogenous hTrpC activity in myometrium. Capacitative Ca2+ entry can potentially contribute to Ca2+ dynamics controlling uterine smooth muscle contractile activity.     

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.     

Activation mechanism for CRAC current and store-operated Ca2+ entry: calcium influx factor and Ca2+-independent phospholipase A2beta-mediated pathway

Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2beta and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2beta when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2beta prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2beta, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2beta activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2beta, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry.     

Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells overexpressing the calcium sensing receptor

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Go6983, or Go6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms

In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.     

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

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+ (Ca2+L) 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 Ca2+L) inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca2+L channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2beta totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by Ca2+L 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 Ca2+L channels and lead to Ca2+ entry and vasoconstriction.     

Role of protein kinase C in chick embryo skeletal myoblast fusion

The involvement of Ca2+ and PGE1 in myoblast fusion has been well documented. Extracellular Ca2+ is essential for myoblast adhesion, alignment, and fusion. There is an obligatory increase in Ca2+ influx immediately preceding fusion and the Ca2+ ionophore A23187 promotes precocious fusion. PGE1 receptors appear just prior to fusion, and an antagonist of PGE1 binding to cell surface receptors blocks fusion when added prior to Ca2+ influx. Finally, exogenous PGE1 induces precocious fusion. The present study was an initial test of the hypothesis that membrane protein phosphorylation by protein kinase C (PKC) links PGE1 receptor occupancy and the increase in Ca2+ influx. Our conclusion that PKC is an essential component in the regulation of myoblast fusion is based in part on the following evidence: (1) an activator of PKC, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), at low concentration and for a brief exposure period, induces precocious fusion and stimulates Ca2+ influx; (2) 4 alpha-phorbol-12,13-didecanoate, an inactive analog of TPA, has no discernible effect on fusion or Ca2+ influx; (3) 1-oleoyl-2-acetyl glycerol, an analog of endogenous diacylglycerol (DAG) which activates PKC, promotes precocious fusion, as does the DAG kinase inhibitor R59022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl]ethyl]-7- methyl-5H-thiazole-[3,2 alpha]-pyrimidin-5-one) which raises the level of endogenous DAG by inhibiting its catabolism; (4) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a highly potent PKC inhibitor, reversibly blocks myogenesis at a point between alignment and fusion; and (5) H-7 also blocks the normal increase in Ca2+ influx preceding fusion.     

CIF and other mysteries of the store-operated Ca2+-entry pathway

The molecular mechanism of the store-operated Ca2+-entry (SOCE) pathway remains one of the most intriguing and long lasting mysteries of Ca2+ signaling. The elusive calcium influx factor (CIF) that is produced upon depletion of Ca2+ stores has attracted growing attention, triggered by new discoveries that filled the gap in the chain of reactions leading to activation of store-operated channels and Ca2+ entry. Ca2+-independent phospholipase A2 emerged as a target of CIF, and a major determinant of the SOCE mechanism. Here, we present our viewpoint on CIF and conformational-coupling models of SOCE from a historical perspective, trying to resolve some of the problem areas, and summarizing our present knowledge on how depletion of intracellular Ca2+ stores signals to plasma membrane channels to open and provide Ca2+ influx that is required for many important physiological functions.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Smooth muscle cell arachidonic acid release, migration, and proliferation are markedly attenuated in mice null for calcium-independent phospholipase A2beta

Pharmacologic evidence suggests that the lipid products generated by one or more calcium-independent phospholipases A(2) (iPLA(2)s) participate in the regulation of vascular tone through smooth muscle cell (SMC) Ca(2+) signaling and the release of arachidonic acid. However, the recent identification of new members of the iPLA(2) family, each inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, has rendered definitive identification of the specific enzyme(s) mediating these processes difficult. Accordingly, we used iPLA(2)beta(-/-) mice to demonstrate that iPLA(2)beta is responsible for the majority of thapsigargin and ionophore (A23187)-induced arachidonic acid release from SMCs. Both thapsigargin and A23187 stimulated robust [(3)H]arachidonate (AA) release from wild-type aortic SMCs that was dramatically attenuated in iPLA(2)beta(-/-) mice (>80% reduction at 5 min; p < 0.01). Moreover, iPLA(2)beta(-/-) mice displayed defects in SMC Ca(2+) homeostasis and decreased SMC migration and proliferation in a model of vascular injury. Ca(2+)-store depletion resulted in the rapid entry of external Ca(2+) into wild-type aortic SMCs that was significantly slower in iPLA(2)beta-null cells (p < 0.01). Furthermore, SMCs from iPLA(2)beta-null mesenteric arterial explants demonstrated decreased proliferation and migration. The defects in migration and proliferation in iPLA(2)beta-null SMCs were restored by 2 mum AA. Remarkably, the cyclooxygenase-2-specific inhibitor, NS-398, prevented AA-induced rescue of SMC migration and proliferation in iPLA(2)beta(-/-) mice. Moreover, PGE(2) alone rescued proliferation and migration in iPLA(2)beta(-/-) mice. We conclude that iPLA(2)beta is an important mediator of AA release and prostaglandin E(2) production in SMCs, modulating vascular tone, cellular signaling, proliferation, and migration.     

Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization

In this study, we report the molecular cloning of cDNAs encoding three distinct isoforms of rat (r) TRP6 Ca(2+) channels. The longest isoform, rTRP6A, contains 930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. Transient transfection of COS cells with expression vectors encoding rTRP6A or rTRP6B increased Ca(2+) influx and gave rise to a novel Ba(2+) influx after activation of M(5) muscarinic acetylcholine receptors. By contrast, passive depletion of intracellular Ca(2+) stores with thapsigargin did not induce Ba(2+) influx in cells expressing rTRP6 isoforms. Ba(2+) influx was also stimulated in rTRP6A-expressing cells after exposure to the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B-expressing cells failed to show OAG-induced Ba(2+) influx. Expression of a rTRP6 N-terminal fragment of rTRP6B or rTRP6A antisense RNA blocked M(5) muscarinic acetylcholine receptor-dependent Ba(2+) influx in COS cells that were transfected with rTRP6 cDNAs. Together these results suggest that rTRP6 participates in the formation of Ca(2+) channels that are regulated by a G-protein-coupled receptor, but not by intracellular Ca(2+) stores. In contrast to the results we obtained with rTRP6A and rTRP6B, cells expressing rTRP6C showed no increased Ca(2+) or Ba(2+) influxes after stimulation with carbachol and also did not show OAG-induced Ba(2+) influx. Glycosylation analysis indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but that rTRP6C is mostly not glycosylated. Together these results suggest that the N terminus (3-56 amino acids) is crucial for the activation of rTRP6A by diacylglycerol and that the 735-802 amino acid segment located just downstream from the 6th transmembrane segment may be required for processing of the rTRP6 protein.     

Contribution of store-operated Ca2+ entry to pHo-dependent changes in vascular tone of porcine coronary smooth muscle

Vascular smooth muscle contracts on increases of extracellular pH (pH(o)) and relaxes on pH(o) decreases possibly resulting from changes in transsarcolemmal Ca(2+) influx. Therefore, we studied store-operated Ca(2+) entry (SOCE; i.e. capacitative Ca(2+) entry (CCE)) during acidification (pH(o)=6.5) and alkalinization (pH(o)=8.0) in isolated porcine coronary smooth muscle cells (SMCs) by monitoring cytoplasmic Ca(2+) ([Ca(2+)](i)) and divalent cation entry (Mn(2+) quench) with fura-2/AM-fluorometry. Additionally, we evaluated the contribution of SOCE to pH(o)-dependent changes in isometric tension of porcine coronary smooth muscle strips. SOCE elicited in SMCs by the SERCA inhibitor BHQ was strongly modulated by pH(o) showing a decrease upon acidification and vice versa an increase upon alkalinization. BHQ-mediated tension of smooth muscle strips also revealed strong pH(o) dependence. In contrast, L-VOC-dependent tension ([K(+)](o)=20 and 40 mmol l(-1)) was remarkably less affected by pH(o) changes. Moreover, refilling of depleted Ca(2+) stores after repeated M(3)-cholinergic receptor stimulation could be almost completely inhibited by SKF 96365 and was markedly reduced by acidification and considerably enhanced by alkalinization pointing to a major role of SOCE in refilling. We conclude that vascular tone particularly responds to alterations in pH(o) whenever SOCE substantially contributes to the amount of activator Ca(2+) for contraction.