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)

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.
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PMID:Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets. 184 26

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.
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PMID:A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers. 184 52

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation. 185 Jul 50

The influence of protein kinase C (PKC) activation on canine coronary vasoreactivity was assessed in vitro. Activation of PKC by phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused slow sustained constriction of isolated coronary artery rings. PDBu was a more potent and efficacious constrictor than PMA (169 +/- 21 vs. 81 +/- 7% of maximum KCl constriction). Constriction to PDBu was reduced slightly by deendothelialization and by meclofenamate. Pretreatment with threshold concentrations of PDBu increased constriction to serotonin from 3 +/- 1 to 48 +/- 4% of maximum KCl constriction whether or not the endothelium was present but had no effect on response to the thromboxane analogue U-46619. In addition, in arteries constricted with PDBu, dilations to ADP, thrombin, acetylcholine, and sodium nitroprusside were impaired when compared with arteries constricted with U-46619. These results suggest that activation of PKC in coronary arteries 1) produces potent constriction mediated only in small part by the endothelium and by cyclooxygenase products, 2) potentiates markedly the constrictor response to serotonin by an endothelium-independent mechanism, and 3) attenuates both endothelium-dependent and endothelium-independent vasodilation.
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PMID:Effects of phorbol esters on canine coronary artery constriction and dilation in vitro. 185 25

Ristocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 +/- 11% by 1.25 microM ADP, 41 +/- 14% by 1 microM A23187, and 26 +/- 7% by 0.1 microgram/ml platelet activating factor (PAF). The effect of 5-110 microM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 microM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 microM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A23187, but not phorbol myristate acetate (0.1 microM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 microM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of agonists and antagonists of platelet aggregation on von Willebrand factor-mediated platelet agglutination. 187 18

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.
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PMID:The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. 187 34

Effects of adenylates on the activity of mitochondrial NAD-malic enzyme from NAD-malic-enzyme (NAD-ME)-type and phosphoenolpyruvate-carboxykinase-(PKC)-type C4 plants are examined. At physiological concentrations, ATP, ADP, and AMP all inhibit the enzyme from Atriplex spongiosa and Panicum miliaceum (NAD-ME-type plants), with ATP the most inhibitory species. The degree of inhibition is greater with subsaturating levels of activator, malate, and Mn2+. NAD-malic enzyme from Urochloa panicoides (PCK-type) is activated by ATP (up to 10-fold) and inhibited by ADP and AMP. These effects are discussed in relation to regulation of C4 photosynthesis.
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PMID:Regulation of C4 photosynthesis: modulation of mitochondrial NAD-malic enzyme by adenylates. 189 77

Calcium fluxes were studied in fura-2-labeled rat platelets. Thrombin, ADP and ionomycin induced rapid mobilization of internally stored Ca2+, which resulted in only a moderate increase of cytosolic [Ca2+]i. Thrombin and ADP stimulated influx of extracellular Ca2+, which was monitored as uptake of 45Ca2+ and of Mn2+. With either agonist, the influx of Ca2+ magnified the initial increase of [Ca2+]i. Since responses of rat platelets were dependent on external [Ca2+], we conclude that Ca2+ influx complements the mobilization of internal stores to reach sufficiently high [Ca2+]i for full activation. A regulatory effect of protein kinase C modulators was observed on both agonist-induced elevation of [Ca2+]i and receptor-mediated Ca2+ entry.
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PMID:Rat platelets are deficient in internal Ca2+ release and require influx of extracellular Ca2+ for activation. 190 51

Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic. In vitro, ajoene reversibly inhibits platelet aggregation as well as the release reaction induced by all known agonists. In this paper we show that ajoene has a unique locus of action, that is not shared by any other known antiplatelet compound. For example, ajoene inhibits agonist-induced exposure of fibrinogen receptors, as well as intracellular responses such as activation of protein kinase C and the increase in cytoplasmic free calcium induced by receptor-dependent agonists (collagen, ADP, PAF, low-dose thrombin). On the other hand, with agonists that can by-pass (at least partially) the receptor-transductor-effector sequence, such as high-dose thrombin, PMA, NaF, only the exposure of fibrinogen receptors is blocked by ajoene. Binding of fibrinogen to chymotrypsin-treated platelets is only slightly inhibited by ajoene. The results reported here also show that: (a) ajoene does not act as a calcium chelator, does not impair the initial agonist-receptor interaction and does not influence the basal levels of intracellular inhibitors of platelet activation such as cyclic GMP; (b) the locus of action of ajoene is a yet unknown molecular step that links, in the case of physiological agonists, specific agonist-receptor complexes to the sequence of the signal transduction system on the plasma membrane of platelets. In the case of non-physiological, receptor-independent agonists (PMA, NaF), we can only speculate on the hypothesis that they somehow mimic the effect of the agonist-receptor complexes on the signal transduction system; and (c) the exposure of fibrinogen receptors is not a direct consequence of other intracellular processes. These observations clearly show, for the first time, that the exposure of fibrinogen receptors is a membrane event proximally and obligatorily coupled to the occupancy of other membrane receptors by their agonists without any intervention by the cytoplasmic biochemical processes. Additional results support the involvement of G-proteins in these early events of platelet activation. Furthermore, a role of the beta tau subunits of G-proteins in the exposure of fibrinogen receptors is proposed.
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PMID:Evidence for direct coupling of primary agonist-receptor interaction to the exposure of functional IIb-IIIa complexes in human blood platelets. Results from studies with the antiplatelet compound ajoene. 191 78

In the terminal stages of exocytosis from permeabilised mast cells, ATP has a number of modulatory actions, although its presence (and by implication, phosphorylation) is not obligatory for secretion to occur. These effects include (1) the enhancement of the sensitivity to both of the essential effectors (Ca2+ and guanine nucleotide); (2) the maintenance of the responsiveness of permeabilised cells; (3) restoration of responsiveness to cells rendered refractory by previous permeabilisation, and (4) induction of delays in the onset of exocytosis from permeabilised cells. We define the modulatory reactions induced by ATP by characterising their specificity to other potential phosphorylating nucleotides and their requirement for Mg2+. GTP and AppNHp are without effect in any of the modulatory actions. ATP, ATP-gamma-S, ITP, XTP, CTP and UTP all appear to support an enhancement of the sensitivity to GTP-gamma-S when applied immediately at the time of permeabilisation. However, the non-adenine nucleoside triphosphates appear to mediate their effect by transphosphorylation to ADP, and therefore the active species appears to be ATP. Only ATP is capable of maintaining and restoring responsiveness (2 and 3 above). Only ATP and ATP-gamma-S induce onset delays and do so moreover in the absence (less than 10(-8) M) of Mg2+. We conclude that three of the modulatory effects (1, 2 and 3 above) which all express a requirement for Mg2+, and can be prevented by inhibitors of protein kinase C are likely to result from phosphorylation reactions. The induction of delays by ATP is unlikely to incur phosphorylation.
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PMID:Modulation of the exocytotic reaction of permeabilised rat mast cells by ATP, other nucleotides and Mg2+. 191 82


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