Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.
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PMID:Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element. 1019 63

Mibefradil is a novel calcium channel blocker with activity at both L-type and T-type calcium channels. There are data suggesting that this compound can protect the ischemic/reperfused myocardium in spite of the fact that there is a very low abundance of T-type calcium channels within ventricular tissue. The aims of this study were two-fold. First, we wished to study the protective effect of mibefradil on ischemia/reperfusion injury in the isolated rat heart using infarct size as the endpoint of injury. In this respect, we compared mibefradil with amlodipine, a well-known and potent L-type calcium channel blocker, and with ischemic preconditioning, an intervention known to reduce infarct size consistently. Secondly, we investigated the possible mechanisms through which protection was achieved. For this second purpose, we examined the effects on protection of glibenclamide (an ATP-dependent K+ channel blocker) and chelerythrine (a protein kinase C inhibitor). Isolated rat hearts were perfused in the Langendorff mode at constant pressure. Control, mibefradil-treated (0.3 microM), mibefradil plus glibenclamide (50 microM), and mibefradil plus chelerythrine (10 microM) treated hearts underwent 35 minutes regional ischemia followed by 120 minutes reperfusion. At the end of the experiments, infarct size was determined with triphenyltetrazolium chloride and was expressed as a percentage of the ischemic risk zone (I/R%). A significant reduction in infarct size with mibefradil treatment was observed (I/R 11.1 +/- 2.1% vs. 35.5 +/- 3.1% in controls). This was comparable with the infarct reduction seen with two 5-minute cycles of ischemic preconditioning (17.7 +/- 2.5%). Amlodipine 0.1 microM, a concentration that caused equivalent coronary vasodilatation as that produced by mibefradil treatment, had no significant effect on infarct size (I/R 29.7 +/- 3.5%). The protective effect of mibefradil was not significantly modified by the presence of the PKC inhibitor chelerythrine 10 microM (I/R 19.1 +/- 4.9%) but was abolished when glibenclamide 50 microM was coadministered with mibefradil prior to ischemia (I/R 28.1 +/- 4.7%). Neither chlelerythrine nor glibenclamide alone had any influence on infarct size. We conclude from these data that mibefradil, unlike amlodipine, markedly reduces infarct size in the rat isolated heart. This protection is sensitive to inhibition by glibenclamide, suggesting that KATP channel opening may be an important additional and novel mechanism of mibefradil's action.
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PMID:Mibefradil, a T-type and L-type calcium channel blocker, limits infarct size through a glibenclamide-sensitive mechanism. 1037 26

To evaluate the role of protein kinase C (PKC) and intracellular calcium and particularly Ca(2+)-uptake in the initiation of lymphocyte mitogenesis, the proliferation of human peripheral blood mononuclear cells (PBMC) was investigated during calcium entry blockade with nifedipine (an L-type calcium channel blocker) and mibefradil (an L- and T-type calcium channel blocker with a higher selectivity for T-type channels). The rate of [3H]-thymidine, [3H]-uridine and [3H]-leucine incorporation into control and concanavalin A-stimulated PBMC cultured for 3 days in the presence or absence of the calcium channel blockers nifedipine or mibefradil (1, 10 or 50 microM) is assayed. Nifedipine and mibefradil concentration-dependently reduced cell number and [3H]-thymidine incorporation or de novo DNA synthesis in control and concanavalin A-stimulated PBMC, as well as de novo RNA and protein synthesis. The proliferative response of nifedipine- or mibefradil-treated cells was restored by addition of phorbol-12-myristate-13-acetate (PMA), an exogenous PKC activator. Our data show that PBMC treated with the Ca2+ channel blockers nifedipine or mibefradil are still capable of proliferating in response to PMA. However, in PKC-depleted cells, the proliferative response of PBMC was suppressed.
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PMID:Proliferation of human peripheral blood mononuclear cells during calcium entry blockade. Role of protein kinase C. 1039 31

Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca2+) in ET-1-induced prostacyclin (PGI2) release. ET-1 (10(-9) M) produced a significant increase in PGI2 release. Pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI2 release. Inhibition was first noted at 10(-9) M and was complete at 10(-6) M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI2 release. These results provide further support for the concept that PKC mediates ET-induced PGI2 release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.
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PMID:Endothelin-induced prostacyclin production in rat aortic endothelial cells: role of calcium. 1047 42

Chronic treatment with the immunosuppressive drug Cyclosporine A (CsA) is associated with increased intracellular calcium in vascular smooth muscle cells, which may activate phospholipase A2. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in CsA-induced prostacyclin (PGI2) release. CsA (10(-9) M) produced a significant increase in PGI2 release. CsA-induced PGI2 release were inhibited 80-85% by 10(-9) M, and 99-100% by 10(-6) M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), staurosporine or 1-(5-isoquinolinesulfonyl)piperazine. Pretreatment with (10(-9) M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced PGI2 release. Conversely, pretreatment with (10(-9) M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium, mediates CsA-induced PGI2 release in rat aortic endothelial cells.
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PMID:Cyclosporine A-induced prostacyclin release is maintained by extracellular calcium in rat aortic endothelial cells: role of protein kinase C. 1050 67

1 The present study examined effects of agonist enzymes and receptor-activating peptides for protease-activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. 2 Thrombin at 0.03-0.1 microM and the PAR-1-activating peptide SFLLR-NH2 at 3-100 microM or TFLLR-NH2 at 10-50 microM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH2 at 10-100 microM elicited only small contraction. Trypsin at 0.08 microM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH2 at 1000 microM exhibited no effect. 3 The contractile responses of the duodenal strips to TFLLR-NH2 and to SLIGRL-NH2 were partially attenuated by the L-type calcium channel blocker nifedipine (1 microM), the protein kinase C inhibitor GF109203X (1 microM) and the tyrosine kinase inhibitor genistein (15 microM), but were resistant to indomethacin (3 microM) and tetrodotoxin (1-10 microM). 4 The relaxation of the preparations exerted by TFLLR-NH2 was unaffected by indomethacin (3 microM), propranolol (5 microM), NG-nitro-L-arginine methyl ester (100 microM) and tetrodotoxin (1-10 microM). This relaxation was resistant to either GF109203X (1 microM) or genistein (15 microM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 microM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 microM), completely abolished the PAR-1-mediated duodenal relaxation, and significantly enhanced the PAR-1-mediated contraction. 6 These findings demonstrate that PAR-1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR-2 plays a minor excitatory role in the muscle, and that PAR-4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR-1 and PAR-2 activation are mediated, in part, by activation of L-type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR-1 activation occurs via activation of apamin-sensitive, but charybdotoxin-insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.
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PMID:Modulation by protease-activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation. 1055 20

A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.
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PMID:Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK. 1059 63

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.
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PMID:Modulation of N-type calcium channel activity by G-proteins and protein kinase C. 1069 56

The effect of estradiol-17beta-BSA (E(2)-BSA) on Ca(2+) uptake and its related signal pathways were examined in the primary cultured rabbit kidney proximal tubule cells. E(2)-BSA (10(-9) M) significantly stimulated Ca(2+) uptake from 2 h by 13% and at 8 h by 35% as compared to control, respectively. This stimulatory effect of E(2)-BSA was not inhibited by tamoxifen (10(-8) M, an intracellular estrogen receptor antagonist), actinomycin D (10(-7) M, a transcription inhibitor), and cycloheximide (4 x 10(-5) M, a protein synthesis inhibitor). However, E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by methoxyverapamil (10(-6) M, an L-type calcium channel blocker) and 5-(N-ethyl-N-isopropyl)-amiloride (10(-5) M, a Na(+)/H(+) antiporter blocker). These results suggest that E(2)-BSA stimulates Ca(2+) uptake through nongenomic pathways. Thus, we investigated which signal pathways were related to E(2)-BSA-induced stimulation of Ca(2+) uptake. 8-Br-cAMP (10(-6) M) alone increased Ca(2+) uptake by 22% compared to control. When E(2)-BSA combined with 8-Br-cAMP, Ca(2+) uptake was not significantly stimulated compared to E(2)-BSA. SQ 22536 (10(-6) M, an adenylate cyclase inhibitor) and myristoylated protein kinase A inhibitor amide 14-22 (10(-6) M, a protein kinase A inhibitor) blocked E(2)-BSA-induced stimulation of Ca(2+) uptake and E(2)-BSA also increased cAMP generation by 26% of that of control. In addition, TPA (0.02 ng/ml, an artificial PKC promoter) stimulated the Ca(2+) uptake by 14%, and the cotreatment of TPA and E(2)-BSA did not significantly stimulate Ca(2+) uptake compared to E(2)-BSA. E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by U 73122 (10(-6) M, a phospholipase C inhibitor) or bisindolylmaleimide I (10(-6) M, a protein kinase C inhibitor). Indeed, E(2)-BSA stimulated PKC activity by 26%. In conclusion, E(2)-BSA (10(-9) M) stimulated Ca(2+) uptake by nongenomic action, which is mediated by cAMP and PKC pathways.
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PMID:Estradiol-17beta-BSA stimulates Ca(2+) uptake through nongenomic pathways in primary rabbit kidney proximal tubule cells: involvement of cAMP and PKC. 1069 64

Catfish cone horizontal cells contain a voltage-gated L-type calcium channel that is modulated by activation of metabotropic glutamate receptors (mGluRs). Activation of group I mGluRs with the mGluR I agonist, (S)-3,5-dihydroxyphenylglycine [(S) 3,5-DHPG], potentiated peak calcium current amplitude, shifted the membrane potential corresponding to peak current activity, and widened the calcium current's activation range. In this study, we have examined the mechanisms linking activation of the mGluRs with "up-regulation" of calcium current activity. Under whole-cell voltage-clamp conditions favoring expression of the L-type calcium current, we provide evidence that activation of mGluRs initiate the diacylglyceral (DG) second messenger pathway to activate protein kinase C (PKC) and up-regulate calcium channel activity. This evidence was based on results using a number of PKC activators and inhibitors. PKC activators mimicked the effect of (S) 3,5-DHPG on calcium current activity. Up-regulation of the calcium channel by PKC activators or (S) 3,5-DHPG was eliminated if PKC inhibitors were present. These results also demonstrated that activation of group I mGluRs were linked to a pertussis toxin sensitive G-protein. When the GTP analog, guanosine 5-0-(3-thiotriphosphate (GTPgammaS), was allowed to diffuse into voltage-clamp cells, up-regulation of the calcium channel occurred and mimicked the effect of (S) 3,5-DHPG. However, when pertussis toxin (PTX) was allowed to diffuse into the cell along with GTPgammaS, GTPgammaS failed to modulate calcium current activity. IP3 (inositol 1,4,5 triphosphate) is a second product produced by activation of group I mGluRs. Once formed, IP3 can trigger calcium release from IP3-sensitive intracellular stores. To determine if the IP3 second messenger system was involved in up-regulation of calcium channel, (S) 3,5-DHPG was applied to voltage-clamped cone horizontal cells containing different concentrations of the calcium buffer, EGTA. Low concentrations of EGTA failed to buffer calcium released from intracellular stores. In the presence of low EGTA concentrations, (S) 3,5-DHPG's enhancement of the calcium current amplitude was reduced. Inhibition of the calcium current amplitude in low concentrations of EGTA was eliminated in the presence of the intracellular calcium store blocker, heparin. These results suggest that both the DG and IP3 second messenger pathways are involved in modulation of the voltage-gated calcium channel in catfish cone horizontal cells. The DG pathway up-regulates the voltage-gated calcium channel activity whereas calcium released from IP3 intracellular stores inhibits peak current amplitude.
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PMID:Second messenger pathways involved in up-regulation of an L-type calcium channel. 1091 Jan 13


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