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

In order to determine the mechanisms which could be responsible for the hypertrophy of vascular wall associated with primary hypertension, we have investigated the mechanisms involved in the proliferation of aortic cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) controls. In this study we have examined the role of phospholipase C which is responsible for the formation of second messengers, the function of protein kinase C and that of GTP-binding proteins (G proteins) which couple membrane receptors to phospholipase C. The adventitial fibroblasts from thoracic aorta were chosen as cell model in culture. The capacity of proliferation in response to 10% serum was analyzed by cell number determination and by measuring the incorporation of 3H-thymidine into newly synthesized DNA. Our results showed that SHR-derived fibroblasts proliferated more rapidly and incorporated more 3H-thymidine than WKY-derived fibroblasts. However, the phospholipase C activity, measured by the serum-stimulated production of 3H-inositol phosphates in cells prelabeled with 3H-inositol, did not differ between SHR- and WKY-derived cells. The desensitization of protein kinase C, by long term (2 d) treatment with high dose (300 nM) of phorbol 12-myristate, 13-acetate (TPA), markedly augmented, but to the same extent, the 3H-thymidine incorporation into DNA of SHR- and WKY-derived fibroblasts. Moreover, protein kinase C activation by TPA caused a parallel reduction (25-30%) of the incorporation of 3H-thymidine into DNA of SHR- and WKY-derived cells. Under the same experimental conditions, the growth of SHR- and WKY-derived fibroblasts was reduced by TPA in a dose-dependent manner (up to 20%) to the same extent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Study of mechanisms responsible for hyperproliferation of aortic cells in spontaneously hypertensive rats]. 195 50

To gain insight into the mechanisms that could account for the augmentation of cellular reactivity in primary hypertension, we have examined some of the biochemical events which are implicated in the transmission of mitogenic signal as well as in cell reactivity. This study focussed on phospholipase C, protein kinase C and GTP-binding proteins (G-proteins), in response to thrombin or arginin-vasopressin (AVP). Cultured fibroblasts prepared from the adventitia of thoracic aorta of spontaneously hypertensive rat (SHR) were used as cell models and were compared with fibroblasts prepared from controls Wistar-Kyoto (WKY) rats. The mitogenicity of each agonist was estimated by measuring the incorporation of 3H-thymidine into the newly synthesized DNA. The agonist-induced phospholipase C activity was evaluated by measuring the production of 3H-inositol phosphates in cells prelabeled with 3H-inositol. The influence of protein kinase C and that of G proteins on the mitogenesis in cells stimulated by thrombin or AVP was determined by pretreating cells with phorbol 12-myristate, 13-acetate (TPA) and pertussis toxin, respectively. Kinetics and dose response studies have demonstrated that in response to thrombin and AVP, the phospholipase C activity and the incorporation of 3H-thymidine were significantly higher in the fibroblasts derived from SHR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Activation mechanisms by thrombin and vasopressin of fibroblasts in spontaneously hypertensive rats]. 195 75

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.
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PMID:Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils. 195 59

Subcellular liver fractions from rats receiving a subcutaneous injection of turpentine, which causes a local inflammation, show an increased synthesis of Prostaglandin E2 and Prostaglandin F2 alpha which reaches a peak 90 minutes and 3 hours after treatment, respectively. Stimulation of phospholipase A2 activity of liver cell preparations seems to be responsible for the supply of arachidonic acid necessary to feed PG synthesis: this stimulation is accompanied by unchanged levels of diacylglycerol lipase, diacylglycerol kinase and protein kinase C activities and by an unchanged content of diacylglycerol in the liver tissue. This picture does not favour the hypothesis of an involvement of phospholipase C in the early stages after turpentine treatment. Determinations of GTP-ase activity in plasma membrane-rich liver preparations give ambiguous results, which do not allow any conclusion on the possible role of G-proteins in phospholipase A2 activation.
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PMID:Rat liver eicosanoid synthesis during turpentine-induced inflammation. 195 99

The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels.
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PMID:Regulation of the amiloride-blockable sodium channel from epithelial tissue. 196 46

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.
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PMID:Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S. 196 91

In AtT-20 cells somatostatin inhibits the secretion of adrenocorticotropic hormone (ACTH) through the activation of GTP binding proteins (G proteins) linked to second messengers such as calcium and cyclic AMP (cAMP). Recently, it has been proposed that there may be G proteins that regulate directly the exocytotic machinery. We have investigated whether somatostatin could inhibit secretion at a step distal to second messengers through a GTP binding protein. For these studies two experimental paradigms were used: (1) intact cells stimulated by calcium ionophores and (2) digitonin-permeabilized cells exposed to buffers of increasing Ca2+ concentrations. Somatostatin inhibited by 70% the ACTH release caused by the calcium ionophore ionomycin without modifying the ionophore-induced elevation in cytosolic [Ca2+]. This effect was cAMP independent because (1) it was observed in the presence of high concentrations of membrane-permeant cAMP analogues, and (2) it was not accompanied by a change in cAMP levels. The effect was also independent of the levels of activators of protein kinase C because it could be produced in the presence of high concentrations of phorbol esters. The action of somatostatin was prevented by pertussis toxin. In digitonin-permeabilized AtT-20 cells somatostatin inhibited release induced by calcium buffers in a GTP-dependent manner. These two observations indicate the involvement of a G protein. It is proposed that a G protein coupled to somatostatin receptors inhibits the intracellular machinery of secretion at a step distal to second messengers, perhaps at the exocytotic site.
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PMID:Evidence that receptor-linked G protein inhibits exocytosis by a post-second-messenger mechanism in AtT-20 cells. 196 44

Incubation of human platelets with the hemolysin-producing Escherichia coli strain K-12 (pANN5211) induced the activation of protein kinase C, aggregation of platelets, calcium influx, low amounts of 12-hydroxyeicosatetraenoic acid (12-HETE), and release of serotonin from dense granules. Nonhemolytic isogenic strains of E. coli 536/21 which differed only in their types of adhesins (MSH+ MS-Fim+; S-MRH+ S-Fim+; P-MRH+ P-Fim+) released neither serotonin nor 12-HETE from human platelets nor induced platelet aggregation. All hemolysin-negative bacteria except E. coli 536/21, without any adhesins, were able to activate protein kinase C reversibly but did not induce calcium influx. Activation of platelets with fluoride, an activator of the GTP-binding protein, was associated with protein kinase C activation, calcium influx, platelet aggregation, serotonin release, and 12-HETE formation. The simultaneous stimulation of platelets with NaF and the nonhemolytic E. coli strains suppressed several of the NaF-induced platelet responses. Membrane preparations isolated from stimulated platelets with hemolysin-negative and hemolysin-positive E. coli showed increased binding of guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and enhanced GTPase activity.
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PMID:Signal transduction in human platelets and inflammatory mediator release induced by genetically cloned hemolysin-positive and -negative Escherichia coli strains. 197 Dec 56

The nef gene product of the human immunodeficiency virus (HIV) is suggested to be a negative factor involved in down-regulating viral expression by a mechanism in which the correct conformation of the nef protein is essential. The nef protein expressed by vaccinia virus recombinants is phosphorylated by protein kinase C. We investigated the synthesis of the nef protein and its state of phosphorylation during HIV-1 infection of a T4 cell line (CEM cells). Maximum synthesis of viral proteins occurred 3 days after infection, when more than 90% of cells were producing viral proteins. The synthesis of the nef protein was detected in parallel with the env and gag proteins. As expected, the nef protein was myristylated but not phosphorylated, and its half-life was less than 1 h. By the use of the polymerase chain reaction technique, we isolated and sequenced the nef gene of this HIV-1 stock. Two significant mutations were observed. Firstly, threonine, at amino acid number 15, the site of phosphorylation by protein kinase C, was mutated into an alanine, and secondly aspartic acid of the tetrapeptide WRFD, which is probably involved in GTP binding, was mutated into an asparagine. The mutated nef gene was expressed in a vaccinia virus system, in which it was not phosphorylated and its half-life was dramatically reduced compared to the wild-type nef gene product. Furthermore, down-regulation of CD4 cell surface expression was no longer affected by the mutated nef gene. These results emphasize that phosphorylation of the nef protein provides an efficient test to monitor its biological activity.
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PMID:Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells. 197 71

Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers.
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PMID:Regulation of the formation and water permeability of endosomes from toad bladder granular cells. 197 9


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