Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Oxytocin stimulates prostaglandin F2alpha secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C

The mechanism for oxytocin's (OT) stimulation of PGF2alpha secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca2+ and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca2+ by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF2alpha release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF2alpha secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF2alpha secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. G?6976, G?6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF2alpha release. These results are consistent with the hypothesis that OT mobilizes Ca2+ to activate a Ca2+-dependent PKC pathway to promote PGF2alpha secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.     

B cell activation. VII. Independent and synergistic effects of mobilized calcium and diacylglycerol on membrane potential and I-A expression

Although cross-linking of murine B cell membrane Ig (mIg) has been shown to induce a rapid increase in intracellular free calcium [Ca++)i), both the source and the function of the Ca++ in lymphocyte activation is unclear. Toward elucidation of its function, we investigated the relationship between the initial (Ca++)i response and other cell physiologic changes that occur early after mIg cross-linking, apparently as a linear cascade, leading to increased membrane I-A expression. Results suggest that the (Ca++)i response results from polyphosphoinositol hydrolysis induced by mIg cross-linking. The (Ca++)i response cannot be induced by activation of protein kinase C (PKC) with phorbol diesters (e.g., PMA) or synthetic diacylglycerol (DAG), suggesting that this response precedes the PKC activation. However, inhibition of phosphatidylinositol turnover by exposure of cells to dbcAMP during anti-Ig stimulation significantly inhibits the (Ca++)i response, suggesting that phosphatidylinositol turnover may be causally related to Ca++ mobilization. The ability of exogenous phospholipase C to induce the (Ca++)i response also supports this conclusion. Of the products of mono- and poly-phosphatidylinositol hydrolysis, the inositol phosphates (InsP, InsP2, InsP3) are implicated as promoters of Ca++ mobilization, because exogenous synthetic diacylglycerol is without effect on (Ca++)i. In light of recent evidence obtained with other systems, we suggest that InsP3 is responsible for mIg cross-linking-induced Ca++ mobilization from intracellular stores in B lymphocytes. Both depolarization and increased I-A expression are induced by increasing (Ca++)i with the Ca++ ionophores A23187 and ionomycin. These events can also be induced by the activation of PKC with high doses of PMA. When suboptimal doses of both A23187 and PMA are present, these reagents synergize in the induction of depolarization. This suggests that one role for the initial rise in (Ca++)i is to act with the DAG liberated from PtdIns turnover, possibly by enhancing translocation of cytosolic PKC to the plasma membrane, and thereby promote changes in ion transport that are apparent as a decrease in the membrane potential.     

Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore

In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2-)release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated "protein kinase C." PMA enhanced O2- release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2- release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2-.     

Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.     

The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-1 09203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.     

Inhibitory and stimulatory effects of phorbol ester on vasopressin-induced cellular responses in cultured rat aortic smooth muscle cells

In rat aortic smooth muscle cells, vasopressin (AVP) induces prostacyclin (PGI2) production, probably as the consequence of phospholipase C activation. Our study analyzes the effects of phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation on AVP-induced inositol 1,4,5-trisphosphate formation, cytosolic free Ca2+ concentration [( Ca2+]c), and PGI2 production. PMA rapidly decreased PKC activity in the cytosol of smooth muscle cells, while increasing it transiently in the membranes with a maximum around 20 min. Prior exposure of the cells to PMA resulted in a transient inhibition of both AVP-induced inositol 1,4,5-trisphosphate formation and [Ca2+]c rise. This was inversely correlated with membraneous PKC activity and partially reversed by the PKC inhibitor staurosporine. In contrast, pretreating the cells with PMA markedly potentiated A23187 or AVP-induced PGI2 production. Under those conditions, AVP-induced PGI2 production did not correlate either with PMA-induced membranous PKC activity or with AVP-induced PLC activation. However, this potentiating effect of PMA was reversed by staurosporine and was not mimicked by the 4 alpha-phorbol, an inactive analogue of PMA. Thus, the possibility is raised that, while inhibiting AVP-induced PLC activation, PMA-induced PKC activation increases the Ca2+ sensitivity of the cellular signaling system leading to PGI2 production.     

Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells.

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca2+ release from internal stores and a subsequent sustained Ca2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca2+ inflow as inferred with Mn2+ quench method was blocked by Ni2+ and a receptor-operated Ca2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKC beta, whereas longer treatment was needed for down-regulation of PKC alpha. Taken together, it was suggested that the BK-induced initial Ca2+ peak and the sustained Ca2+ inflow through the activation of a receptor-operated Ca2+ channel, are differentially regulated by PKC isozymes alpha and beta, respectively, in osteoblast-like MC3T3-E1 cells.     

Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.     

钙离子和冈田酸对亚胺环己酮诱导小鼠卵子孤雌激活的影响

哺乳动物卵母细胞在排卵后停滞在第二次减数分裂中期,受精和多种物理或是化学刺激可以克服这一阻滞使卵母细胞活化。蛋白合成抑制剂亚胺环己酮可以诱导小鼠卵母细胞发生孤雌活化,但其机制尚未完全阐明。以前的研究提示亚胺环己酮可能是通过抑制蛋白激酶MOS的合成来发挥孤雌激活的作用的。本实验发现,CHX诱导的卵母细胞孤雌活化是Ca^2 依赖性,其效率可被钙离子载体A23187大大提高,免疫蛋白印迹结果表明,卵母细胞孤雌活化后MAPK发生去磷酸化。蛋白磷酸酶抑制剂冈田酸可以克服CHX＋A23187对小鼠放母细胞活化作用,并且部分阻止MAPK去磷酸化。以上结果表明,抑制MOS的合成并非CHX诱导的孤雌活化过程的惟一原因,并且蛋白磷酸酶抑制剂可以阻断这一激活事件。     

Oxytocin stimulates prostaglandin F(2alpha) secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C*

The mechanism for oxytocin's (OT) stimulation of PGF(2alpha) secretion from porcine endometrium is not clear, but is thought to involve mobilization of intracellular Ca(2+) and subsequent activation of protein kinase C (PKC). This study determined: (1) if mobilization of inositol trisphosphate-sensitive Ca(2+) by thapsigargin or activation of PKC by phorbol 12-myristate 13-acetate (PMA) could stimulate PGF(2alpha) release from luminal epithelial, glandular epithelial and stromal cells of porcine endometrium and (2) if inhibitors of various PKC isotypes could attenuate the ability of OT, thapsigargin and PMA to stimulate PGF(2alpha) secretion from these cells. Thapsigargin and PMA each stimulated (P < 0.01) PGF(2alpha) secretion from all three endometrial cell types examined. However, the effects of thapsigargin and PMA were synergistic (P < 0.05) only in stromal cells. Three protein kinase C inhibitors (i.e. G?6976, G?6983 and Ro-31-8220) differentially attenuated (P < 0.05) the ability of OT, thapsigargin and PMA to stimulate PGF(2alpha) release. These results are consistent with the hypothesis that OT mobilizes Ca(2+) to activate a Ca(2+)-dependent PKC pathway to promote PGF(2alpha) secretion from porcine endometrial cells. The differing pattern of response to isotype-specific inhibitors of PKC among cell types suggests that distinct PKC isoforms are differentially expressed in luminal epithelial, glandular epithelial and stromal cells.     

Multiple regulation of proenkephalin gene expression by protein kinase C

In the present study we investigated the role of protein kinase C (Ca2+/phospholipid-dependent enzyme)-mediated processes in the regulation of proenkephalin gene expression in primary cultures of bovine adrenal chromaffin cells. Activators of protein kinase C such as 1-oleoyl-2-acetylglycerol, mezerein, and the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate induced a time-dependent increase in proenkephalin mRNA levels, whereas the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. The increase in phorbol ester-induced proenkephalin mRNA was potentiated by low concentrations of the Ca2+ ionophore A23187, suggesting an interaction between protein kinase- and Ca2+-mediated processes in the regulation of proenkephalin mRNA. The phorbol ester-induced stimulation does not appear to be mediated by an interaction with the cAMP-generating system or increases in Ca2+ uptake. However, when proenkephalin mRNA levels were stimulated by KCl (10 mM) and the dihydropyridine BayK8644, PMA exhibited an inhibitory effect on proenkephalin mRNA, which was detectable at a 10-fold lower concentration of PMA than the stimulatory effect. This inhibitory effect appears to be mediated by an inhibition of Ca2+ entry through voltage-dependent Ca2+ channels, as suggested by 45Ca2+ uptake experiments. Thus, the net effect of PMA depends on and varies with the state of voltage-dependent Ca2+ channel activity. A third mode of action by protein kinase C to modulate proenkephalin gene expression is by interaction with the phosphatidylinositol second messenger system. Stimulation of phosphoinositide hydrolysis and proenkephalin mRNA by histaminic H1-receptor activation was inhibited by low concentrations of PMA. We suggest that protein kinase C may act as a positive and negative regulator of proenkephalin gene expression by interacting with at least three receptor-coupled second messenger systems.     

Phorbol ester induces both gene expression and phosphorylation of the plasma membrane Ca2+ pump.

Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.