The inhibition of receptor-mediated and voltage-dependent calcium entry by the antiproliferative L-651,582.

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1,2,3-triazole-4- carboxamide, an antiproliferative and antiparasitic agent previously shown to affect 45Ca2+ uptake into mammalian cells, inhibits both receptor-mediated and voltage-dependent calcium entry in well characterized in vitro systems. Indo 1 fluorescence measurements of cytosolic calcium levels indicate that the drug has no effect on the initial transient release of internal stores of calcium stimulated by fMet-Leu-Phe in rat polymorphonuclear leukocytes. It does decrease the levels maintained subsequently, however, indicating blockage of calcium influx through receptor-operated channels. L-651,582 also blocks the stimulation of leukotriene B4 (LTB4) production by fMet-Leu-Phe with an IC50 = 0.5 micrograms/ml equal to that for calcium entry inhibition. The LTB4 inhibition is likely due to calcium entry inhibition since L-651,582 does not inhibit calmodulin or enzymes producing arachidonate metabolites. L-651,582 also inhibits potassium-stimulated 45Ca2+ influx into GH3 cells with an IC50 of 0.5 microgram/ml, indicating a block of voltage-gated L-type calcium channels. Patch voltage clamp measurements of current through L- and T-type calcium in guinea pig atrial cells also indicate that L-651,582 is a calcium antagonist. Block of L-type calcium channels is voltage-dependent, and the apparent dissociation constant for the high affinity state is 0.2 micrograms/ml. The IC50 for block of T-type calcium channels is 1.4 micrograms/ml. The inhibition of cellular proliferation and the production of arachidonate metabolites by L-651,582 may be the result of the nearly equipotent block of receptor-operated and voltage-gated calcium 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.     

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Epidermal growth factor-activated calcium and potassium channels.

The earliest responses to activation of the epidermal growth factor (EGF) receptor include a transient increase in calcium influx and a transient membrane hyperpolarization. The underlying mechanisms are, however, not well understood as yet. In the present study, we have applied patch clamp recording in the cell-attached and the outside-out mode, and fluorimetric cytosolic Ca2+ determinations, to identify the nature of the ion channels involved, to characterize their properties at the level of single channels, and to unravel their mechanism of activation. We provide evidence that activation of the EGF receptor results initially in the activation of voltage-independent Ca2+ channels that can be defined as direct receptor-operated channels. This in turn causes the activation of Ca(2+)-dependent K+ channels, which results in a (delayed) membrane hyperpolarization and then leads to the activation of a second class of Ca2+ channels that are sensitive to hyperpolarization. An autocatalytic generation of further hyperpolarization and Ca2+ influx is the predicted outcome of this ionic cascade. Based on the observed inhibitory effects of protein kinase C activation on the activity of Ca(2+)-dependent K+ channels, we propose that protein kinase C is involved in the negative regulation of this cascade, which explains the transient nature of these responses.     

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.     

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.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx.

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45- HPB-ALL T cells were transfected with cDNA encoding the CD45RA+B+C- isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45- cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub-clones. In CD45- cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45-transfected cells. Co-aggregation of CD4- or CD8-p56lck kinase with the TCR in CD45- cells restored TCR-induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor-mediated calcium signals were largely due (60-90%) to Ca2+ influx, and only a minor component (10-40%) was caused by Ca2+ release from intracellular stores. Maximal CD3-mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non-detectable. These results indicate that CD45-regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4- or CD8-p56lck can only restore signal transduction coupling in CD45- cells when brought into close association with the TCR.     

Phorbol esters modulate thrombin-operated calcium mobilisation and dense granule release in human platelets

Human alpha-thrombin-induced elevation of cytosolic free calcium ([Ca2+]i) and dense granule release was examined in platelets preincubated with either activators or an inhibitor of protein kinase C. 12-O-Tetradecanoylphorbol 13-acetate (TPA) or two 12-deoxy analogues of TPA, when added alone to platelets, did not elevate [Ca2+]i, as monitored by quin2 fluorescence, though small amounts of dense granule release were detected. Preincubation of the platelets with either TPA or 12-deoxyphorbol 13-phenylacetate, but not the parent, 4-beta-phorbol, produced a dose-dependent inhibition of the elevation of [Ca2+]i and 5-hydroxytryptamine release induced by human alpha-thrombin. Furthermore, this phorbol ester-mediated inhibition of human alpha-thrombin-induced activation could be prevented by H7 (1-[5-isoquinolinesulphonyl]-2-methylpiperazine), the recently described inhibitor of protein kinase C. These results suggest a role for protein kinase C as a modulator of receptor-operated calcium fluxes in human platelets.     

Phorbol ester blocks the coupling of GTP-binding protein with receptor-controlled calcium channels

Using the intracellular Ca2+-specific indicator, Quin 2, it was demonstrated that an addition to platelet suspensions of the GTP-binding protein activator, sodium fluoride, stimulates the Ca2+ and Ba2+ influx from the incubation medium into the cytoplasm via receptor-operated Ca2+ channels (Ca-ROC). The fluoride-induced Ca2+ influx is blocked by the protein kinase C activator, phorbol myristate acetate as well as by the platelet adenylate cyclase activator, prostaglandin E1. A two-dimensional electrophoretic analysis of platelet phosphoproteins revealed that the phorbol ester enhances the phosphorylation of proteins with molecular masses of about 20 and 40 kDa. The experimental results suggest that the participation of the GTP-binding protein in the receptor coupling to Ca-ROC. The mechanism of the blocking effect of phorbol esters and prostaglandin E1 on Ca-ROC consists in an impaired coupling of these channels to the GTP-binding protein that activates them.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.     

Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.     

The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine

Recombinant monocyte-chemotactic and activating factor (rMCAF; alternative acronyms MCP-1, TDCF, human JE) induced migration of human monocytes across polycarbonate or nitrocellulose filters. Maximal induction of migration was observed at a concentration of 10 ng/ml (10(-9) M). Checkerboard analysis revealed that rMCAF elicited true gradient-dependent chemotactic migration, although a gradient independent chemokinetic effect was observed at low concentrations (1-5 ng/ml). rMCAF caused a rapid (less than 5 s) and transient (approximately 1.5 min) increase of free cytosolic Ca2+ ions, as assessed by the fura-2 probe. No Ca2+ increase was detected in neutrophils or lymphocytes stimulated by rMCAF. Studies conducted in the absence of extracellular Ca2+ or in the presence of Ni2+ (an inhibitor of Ca2+ influx) suggested that the increase of intracellular Ca2+ induced by rMCAF is dependent on the influx of extracellular Ca2+ through plasma membrane channels. Bordetella pertussis toxin inhibited the intracellular Ca2+ elevation and chemotaxis caused by rMCAF. The possible involvement of Ca(2+)-dependent protein kinases in rMCAF signaling pathway(s) was explored using inhibitors. Inhibitors of GMP-dependent kinase and myosin L chain kinase had no effect on rMCAF-induced monocyte migration. In contrast, protein kinase C/cAMP-dependent kinase inhibitors (such as, C-I, H-7, HA-1004, KT5720, and Staurosporine) markedly decreased rMCAF induced chemotaxis suggesting the involvement of a serine/threonine protein kinase, possibly protein kinase C, in rMCAF signaling pathway.     

Effects of the antithrombitic agent PCA 4230 on agonist-induced Ca2+ entry and Ca2+ release in human platelets.

We have studied the effects of the antithrombitic agent PCA 4230 on the entry of Mn2+, used here as a Ca2+ surrogate for Ca2+ channels, and on the release of Ca2+ from the intracellular stores in stimulated human platelets loaded with fura-2. PCA 4230 prevented receptor-operated calcium entry activated by thrombin, ADP and collagen with no modification of the Ca2+ release from the intracellular stores. PCA 4230 also inhibited cytochrome P-450-mediated O-dealkylase activity with the same concentration-dependence as the thrombin-induced Mn2+ entry. These results suggest that the inhibitory effects of PCA 4230 on Ca2+ influx may be due to its interaction with cytochrome P-450, which has been proposed recently to be involved in the activation of receptor-operated Ca2+ channels. In addition, PCA 4230 inhibited both PAF-induced Ca2+ entry and Ca2+ release, behaving as a PAF-antagonist. All these effects contribute to explain the antithrombitic action of PCA 4230.     

Enzymological analysis of mutant protein kinase Cgamma causing spinocerebellar ataxia type 14 and dysfunction in Ca2+ homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cgamma (PKCgamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCgamma kinase activity. To determine the effect of these mutations on PKCgamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCgammas. We found that wild-type PKCgamma, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCgamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCgamma. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCgammas was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

The role of endogenously formed diacylglycerol in the propagation and termination of platelet activation. A biochemical and functional analysis using the novel diacylglycerol kinase inhibitor, R 59 949

The putative roles for the second messenger, diacylglycerol, were investigated in intact platelets using a novel diacylglycerol kinase inhibitor, R 59 949, or (3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2,3- dihydro-2-thioxo-4(1H)-quinazolinone). The compound inhibited the diacylglycerol kinase in a concentration-dependent manner (10(-8) to 10(-5) M) in isolated platelet membranes and in intact platelets. When platelets were stimulated with vasopressin in the presence of the compound, protein kinase C activity was markedly increased; the formation of inositol phosphates, the increase in intracellular Ca2+ and shape-change reaction were antagonized while the vasopressin-induced polyphosphoinositide synthesis was amplified, and this in a distinct inositolphospholipid pool. In the presence of R 59 949, vasopressin- as well as collagen-induced release reaction and aggregation was strongly increased, independently of the formation of arachidonate metabolites. It is concluded that diacylglycerol formed after receptor activation, likely by activating the protein kinase C, plays an important role in the propagation of platelet functional responses in casu aggregation and secretion and controls the termination of the primary receptor coupled responses.     

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.     

ATP promotes NCX-reversal in aortic smooth muscle cells by DAG-activated Na+ entry

Reversal of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) has been shown to mediate Ca(2+) influx in response to activation of G-protein linked receptors. Functional coupling of reverse-mode NCX with canonical transient receptor potential channels (TRPC), specifically TRPC6, has recently been demonstrated by our laboratory to mediate Ca(2+) influx in rat aortic smooth muscle cells (RASMCs) following ATP stimulation. In this communication, we provide further detail of this functional coupling by indirectly measuring NCX reversal. We found that NCX reversal, induced by the removal of extracellular Na(+), was increased following stimulation with ATP and the diacylglycerol analog 1-Oleoyl-2-acetyl-sn-glycerol. This increased NCX reversal was attenuated by SKF-96365, an inhibitor of non-selective cation channels, and by activation of protein kinase C with phorbol ester 12-tetradecanoylphorbol-13 acetate. These data are consistent with the known properties of TRPC6 and further support that functional coupling of TRPC6 and NCX occurs via a receptor-operated, rather than store-operated, cascade in RASMCs.     

Receptor-Operated Calcium Influx Mediated by Protein Tyrosine Kinase Pathways

Abstract

Calcium influx from the extracellular space elicited by activation of heterotrimeric G protein-coupled and heptahelical receptors plays a critical role in transmembrane signal transduction in a wide variety of cell systems. In nonexcitable cells, the precise voltage-independent mechanism by which calcium enters the cell remains unknown. Multiple mechanisms appear to be operating in different cell types (1–3): 1. G protein-operated calcium influx, 2. second messenger-operated calcium influx, 3. capacitative calcium influx, and 4. phosphorylation of calcium channels. Receptor-operated calcium channels have a fundamental role in stimulus-secretion coupling in many different cells, but these channels remain to be purified and cloned. This review proposes that receptor-operated calcium influx is mediated by protein tyrosine kinase pathways. The function of protein tyrosine kinase pathways and their interactions with other receptor-operated calcium influx mechanisms are described.