Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have used the bradykinin analog, [DArg0-Hyp3-DPhe7]-bradykinin, as a model stimulus with which to examine peptide-induced mediator release from human skin mast cells (SMC) and to compare it with IgE-mediated release from the same cells. The bradykinin analog was an effective histamine secretagogue, inducing a comparable maximal level of release to that observed for anti-IgE. By contrast to anti-IgE, however, [DArg0-Hyp3-DPhe7]-bradykinin did not stimulate marked release of prostaglandin D2 (PGD2) from these cells. In experiments where cells were exposed to both stimuli simultaneously, histamine release was additive, while PGD2 release was the same as that observed for anti-IgE alone. The kinetics of [DArg0-Hyp3-DPhe7]-bradykinin-stimulated histamine release were rapid, with 50% of maximal release being achieved within 30 sec, compared to 2-3 min for anti-IgE. Interestingly, when both stimuli were applied simultaneously, the kinetics of release were intermediate between those of either stimulus alone. Studies of the signal transduction pathways that may be involved in [DArg0-Hyp3-DPhe7]-bradykinin-induced histamine release revealed striking differences to results obtained with anti-IgE. While agents that increase intracellular cyclic AMP have a pronounced inhibitory effect on IgE-mediated release, forskolin, isobutylmethylxanthine and isoproterenol were all totally ineffective at inhibiting histamine release induced by the bradykinin analog. Similarly, staurosporine, a relatively selective inhibitor of protein kinase C, and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) an activator of this enzyme, both have pronounced effects on IgE-mediated histamine release from SMC but were completely inactive with regard to [DArg0-Hyp3-DPhe7]-bradykinin-stimulated release. SMC stimulated with this peptide showed characteristic changes in intracellular free calcium levels, as assessed by digital video microscopy. This response differs from that induced by anti-IgE in that it had a more rapd onset, achieved a lower peak, and decayed much more rapidly. Analysis at the single cell level showed that cells that responded in this fashion upon exposure to the bradykinin analog were capable of showing an additional response upon subsequent exposure to anti-IgE. We conclude that histamine release from SMC in response to [DArg0-Hyp3-DPhe7]-bradykinin occurs via a completely different mechanism from that in response to IgE-mediated stimuli. Peptide-induced release is rapid and is not susceptible to pharmacologic manipulation of intracellular cyclic AMP or protein kinase C but utilizes a rapid transient shift in intracellular calcium concentrations as part of its signal transduction pathway.
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PMID:Mechanisms of mediator release from human skin mast cells upon stimulation by the bradykinin analog, [DArg0-Hyp3-DPhe7]bradykinin. 170 13

The goal of this study was to determine whether protein kinase C mediates bradykinin-induced increases in microvascular permeability. Permeability of the hamster cheek pouch was evaluated using intravital fluorescent microscopy and fluorescein isothiocyanate (FITC)-dextran (MW 70,000). We examined effects of sphingosine, a protein kinase C inhibitor, on bradykinin-induced increases in permeability. Increases in permeability were quantitated by counting the number of leaky sites and calculating the clearance of FITC-dextran. During bradykinin (10(-6) M), leaky sites increased from 0 to 40 +/- 4 (mean +/- SEM) sites/0.11 cm2, and clearance increased from 1.7 +/- 1.0 to 22 +/- 9 ml/sec x 10(-6). The bradykinin type-2 receptor antagonist D-Arg,[Hyp3,Thi5,8,D-Phe7]-bradykinin virtually abolished formation of leaky sites in response to bradykinin. To determine whether changes in microvascular pressure contribute to the increase in leaky sites, venular pressure was measured using a micropipette and survo-null device. Increases in cheek pouch venular pressure were similar during application of bradykinin and adenosine, which increased permeability, and isoproterenol, which did not increase permeability in the cheek pouch. Thus, increases in permeability were not linked to changes in microvascular pressure. The protein kinase C inhibitor, sphingosine (10(-6) M), markedly attenuated responses to bradykinin. Leaky sites increased from 0 to only 2 +/- 1 sites/0.11 cm2, and clearance increased from 3.9 +/- 1.4 to only 6.7 +/- 2.2 ml/sec x 10(-6). To test the specificity of sphingosine, we examined effects of adenosine (10(-6) M). Sphingosine did not significantly alter increases in microvascular permeability in responses to adenosine. We also examined effects of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), another protein kinase C inhibitor, on responses to bradykinin and adenosine. H-7 greatly attenuated formation of leaky sites during stimulation with bradykinin and did not alter the number of leaky sites produced during adenosine. The findings suggest that protein kinase C may mediate increases in vascular permeability in response to bradykinin.
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PMID:Role of protein kinase C in bradykinin-induced increases in microvascular permeability. 170 11

C6-2B rat glioma cells were stably transfected with substance K receptor cDNA and used to study interactions between cAMP and Ca2+ signaling pathways. Activation of the newly expressed receptors by substance K increased the intracellular free Ca2+ concentration, as monitored by single-cell fura-2 imaging, and markedly inhibited agonist-stimulated cAMP accumulation. Blockade of intracellular Ca2+ mobilization abolished the substance K receptor-mediated inhibition of isoproterenol-induced cAMP production. Phosphodiesterase inhibitors, down-regulation or inhibition of protein kinase C, and pertussis toxin failed to prevent substance K-induced inhibition of agonist-stimulated cAMP accumulation. An increased intracellular Ca2+ concentration caused by either calcium ionophores or activation of endogenous bradykinin receptors was found to markedly reduce cAMP production in wild-type cells. These results demonstrate that elevated intracellular Ca2+ concentration can negatively modulate agonist-stimulated adenylate cyclase activity in C6-2B glioma cells.
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PMID:Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA. 171 1

Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells.
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PMID:Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells. 172 Oct 65

The actions of tumor-promoting phorbol esters in smooth muscle excitation-contraction coupling were studied in isolated guinea pig ileum in the presence of various contractile agents. Muscarinic agonists, histamine and bradykinin elicited an initial transient phasic contraction and a subsequent sustained tonic contraction in guinea pig ileum. The Ca2+ channel antagonist nifedipine selectively inhibited the tonic contraction. Phorbol esters, protein kinase C activators, induced immediate muscle relaxation followed by oscillatory contractions when added during the tonic phase of contraction. Phorbol esters, when added in advance, slightly altered the ligand-induced phasic contraction but converted tonic contractions into oscillatory spikes. The amplitude, frequency and shape of the oscillation induced by phorbol esters were dependent upon the dose of phorbol ester: amplitude was increased and frequency was decreased by increasing the doses of phorbol ester. In contrast, the phorbol ester potentiated the tonic contraction induced by high potassium chloride with little effect on the phasic component. It also sensitized the muscles to Bay K 8644. Bay K 8644, which was ineffective in stimulating muscle contraction at 1 nM, became a very effective stimulator in the presence of the phorbol ester. All of these phorbol ester-induced potentiations and oscillations were sensitive to inhibition by staurosporine or nifedipine. These data suggest that in guinea pig ileum, protein kinase C plays a positive regulatory role in Ca2+ channel activation and promotes a complex regulatory effect on Ca(2+)-mobilizing ligand-stimulated Ca2+ channel activity, which results in oscillatory contractile responses to carbachol, methacholine, histamine and bradykinin.
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PMID:Phorbol esters induce oscillatory contractions of intestinal smooth muscles. 172 50

In rings of rat portal vein, endothelin-1, endothelin-2, and endothelin-3 caused graded slow contractions and potentiated spontaneous contractions. The apparent EC50 values and maximal responses to 30 nM endothelin were 1.4 nM and 0.96 g for endothelin-1, 5.2 nM and 0.65 g for endothelin-2, and 1.7 nM and 0.62 g for endothelin-3 (n = 4-12). At concentrations producing half the contraction triggered by 80 mM KCl, the order of potencies was endothelin-1 greater than U46619 = angiotensin II greater than bradykinin greater than substance P greater than phenylephrine. Longitudinal portal-mesenteric vein preparations developed very modest contractions to endothelin-1 (0.13 g at 30 nM; n = 5), but their responses to 80 mM KCl and phenylephrine were greater than those of rings. Responses of rings to endothelin-1 were profoundly reduced in Ca(2+)-free medium, but less inhibition was obtained after incubation with nicardipine (up to 1 microM) and/or nickel (up to 0.5 mM), phorbol (up to 0.3 microM), staurosporine (up to 10 nM), or cromakalim (3 microM). Indomethacin (5.6 microM) did not affect responses to endothelin-1. Cromakalim (0.1-3 microM) also relaxed rings constricted with 0.3 nM endothelin-1, and this effect was partially reversed by glibenclamide (3 microM). Thus, endothelins, especially endothelin-1, are potent constrictors of portal vein rings but not of portal-mesenteric vein strips. Their action appears to rely largely on Ca2+ influx from the external medium (only in part via L- and T-type Ca2+ channels) and activation of protein kinase C but not on eicosanoid generation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Potent constrictor actions of endothelin-1, endothelin-2, and endothelin-3 in rat isolated portal vein. 173 99

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.
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PMID:Regulation of arachidonic acid release in vascular endothelium. Ca(2+)-dependent and -independent pathways. 174 1

Basic Fibroblast Growth Factor (bFGF) is shown to be a potent mitogen for cultured glomerular mesangial cells. bFGF induces an increase in cell number and stimulates DNA synthesis measured by [3H]thymidine incorporation in normal as well as in protein kinase C-depleted cells. The ED50 observed in both cases are nearly identical (approximately 0.04 nM) and maximal responses are obtained at 1 nM. Staurosporine, a potent protein kinase C inhibitor, does not prevent bFGF from inducing mitogenesis. On the contrary, the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and the bradykinin derivative Des-Arg9bradykinin that we have previously shown as mitogens for mesangial cells, fail to trigger DNA synthesis or cell proliferation upon staurosporine treatment or in protein kinase C-depleted cells. bFGF is unable to induce the association of the enzyme to membranes, the so-called translocation process, although the growth factor induces a slight production of diacylglycerol. Using a highly resolutive two-dimensional electrophoresis, we show that bFGF, in contrast to TPA, is unable to stimulate the phosphorylation of a Mr 80,000/pI 4.5 protein, a major and specific protein kinase C substrate. By contrast, bFGF stimulates the phosphorylation of a Mr 28,000/pI 5.7-5.9 protein in normal as well as in protein kinase C-depleted cells while TPA induces this protein phosphorylation only in normal cells. Our results suggest that bFGF exerts its proliferative action on mesangial cells through a protein kinase C-independent pathway and that the growth factor does not activate anyway the enzyme in this cell type.
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PMID:Basic fibroblast growth factor stimulates glomerular mesangial cell proliferation through a protein kinase C-independent pathway. 177 35

Alpha 1-Adrenergic receptors and bradykinin receptors are two distinct membrane receptors that stimulate phospholipid breakdown and arachidonic acid and arachidonic acid metabolite release. In the current studies, we have examined several mechanisms to assess their possible contribution to arachidonic acid release in the Madin-Darby canine kidney cell line by agonist stimulation of these receptors: 1) activation of phospholipase A2 (PLA2); 2) sequential activation of phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase; and 3) inhibition of the sequential action of fatty acyl-CoA synthetase and lysophosphatide acyltransferase. Experiments were conducted to measure the stimulation of lysophospholipid production by epinephrine and bradykinin, the rate of incorporation of [3H]arachidonic acid into stimulated and unstimulated cells, and the effect on [3H]arachidonic acid release of treating cells with exogenous phospholipase C. The data indicate that stimulation of PLA2 activity is regulated by alpha 1-adrenergic and bradykinin receptors and that this stimulation is mediated, at least in part, by the activation of protein kinase C. We find that the role of diacylglycerol in arachidonic acid release is as an activator of protein kinase C and not as a substrate for a lipase. Moreover, the hormonal agonists do not appear to inhibit fatty acid reacylation. Experiments using the Ca2(+)-sensitive dye fura-2 and the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid suggest that bradykinin activates PLA2 by a transient elevation of intracellular Ca2+. This action appears to be less important for activation of PLA2 by epinephrine. Taken together, these data are consistent with the following conclusions. 1) Hormone-stimulated arachidonic acid release in Madin-Darby canine kidney-D1 cells occurs as a consequence of PLA2 activation. 2) The ability of an agonist both to mobilize Ca2+ and to activate protein kinase C contributes to its efficacy as a stimulator of PLA2-mediated arachidonic acid release.
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PMID:Intracellular Ca2+ and protein kinase C interact to regulate alpha 1-adrenergic- and bradykinin receptor-stimulated phospholipase A2 activation in Madin-Darby canine kidney cells. 184 14

[3H]Bradykinin (BDK) binds to two distinct binding sites (P less than .01, N = 12) in NG108-15 cell membranes; (site 1: Kd1 = 3.09 x 10(-10) M, Bmax1 = 242 +/- 24 fmol/mg protein) and (site 2: Kd2 = 1.94 x 10(-8) M, Bmax2 = 491 +/- 75 fmol/mg protein). Although site 1 comprises only 33 +/- 4% (N = 12) of the total binding site population, comparison of the binding affinity and functional potency for BDK agonist analogs exhibiting differential selectivity for the two sites reveals that this high affinity site is the receptor mediating inositol monophosphate (IP) production in this cell line. BDK-stimulated IP production undergoes a very rapid (5 min) desensitization that is characterized by both a loss in agonist potency (EC50 = 3.57 x 10(-9) M vs. 1.94 x 10(-10) M in controls; P less than .001, N = 12) and a decrease in amplitude of response (fold stimulation = 1.45 +/- 0.06 vs. 1.80 +/- 0.09 in controls; P less than .01, N = 12). Only the decrease in response amplitude is attenuated by down-regulation of protein kinase C by prior long term treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating an involvement of protein kinase C activation in the desensitization process. Desensitization is accompanied by down-regulation of site 1 only (Bmax1 = 71 +/- 8 fmol/mg (N = 10; P less than .001 vs. controls)); Bmax2 and the Kd for BDK at both sites remain unchanged, further supporting the contention that site 1 is the functionally relevant receptor. In contrast to the functional data, long term TPA treatment does not attenuate the receptor down-regulation, indicating that the rapid desensitization involves both receptor-related and postreceptor mechanisms. The implications of this property of the BDK receptor for analog design and receptor classification are discussed.
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PMID:Bradykinin-stimulated inositol phosphate production in NG108-15 cells is mediated by a small population of binding sites which rapidly desensitize. 185 31


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