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 studied the extent of Ca2+ influx, myoplasmic free Ca2+ concentration changes, and phosphorylation of the regulatory 20-kDa myosin light chain (LC20) associated with the potentiation of stretch-induced myogenic tone in the rabbit facial vein. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was used to augment Ca(2+)-dependent stretch-induced myogenic tone. Veins stretched to an optimal resting tension in physiological salt solution (PSS) containing 0.4 mM of Ca2+ developed stretch-induced myogenic tone. Tissues incubated in Ca(2+)-free PSS, either with or without PMA (0.1 microM) did not develop myogenic tone. Readmission of Ca2+ (0.4 mM) caused a three-fold increase in the contraction in PMA-treated segments (710 +/- 60 mg, n = 29 v control: 188 +/- 10 mg, n = 24). This increased contraction was not associated with additional increases in either Ca2+ influx (73.5 +/- 6.9 pmol/mg of tissue/min, n = 29 v control: 61.1 +/- 5.7 pmol/mg of tissue/min, n = 24), myoplasmic free Ca2+ concentration or LC20, (0.44 +/- 0.02 mol PO4/mol LC20, n = 9 v control: 0.43 +/- 0.03 mol PO4/mol LC20, n = 7). Our results suggest that PKC activation amplifies stretch-induced myogenic tone in the rabbit facial vein through target proteins that are not associated with regulation of Ca2+ influx, myoplasmic free Ca2+ concentration, or LC20 phosphorylation. We conclude that the PKC-mediated potentiation of stretch-induced myogenic tone is due to an increased sensitivity of the contractile apparatus to Ca2+.
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PMID:Phorbol ester-induced potentiation of myogenic tone is not associated with increases in Ca2+ influx, myoplasmic free Ca2+ concentration, or 20-kDa myosin light chain phosphorylation. 802 13

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca2+ ([Ca2+]i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca2+]i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca2+]i in a dose-dependent manner. [Ca2+]i maximally increased from 85 +/- 5 nmol/l to 608 +/- 99 nmol/l with PTH, 10(-6) M, and to 766 +/- 162 nmol/l with AVP, 10(-7) M. The increment in [Ca2+]i by both hormones was by intracellular Ca2+ release and entry through plasma membrane Ca2+ channels. 8-Bromo-adenosine-3',5'-cyclic monophosphate (8-BrcAMP), 10(-4) M, increased [Ca2+]i (basal 83 +/- 3 to 427 +/- 121 nmol/l) but only from internal sources as nifedipine (10 mumol), ([Ca2+]i changes: 86 +/- 4 to 390 +/- 29 nmol/l) and removal of bath Ca/+o, ([Ca2+]i changes: 84 +/- 6 to 517 +/- 142 nmol/l), were without effect on agonist-induced [Ca2+]i. Thapsigargin, 1.5 mumol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca2+ transients, and partially inhibited PTH-mediated Ca2+ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca2+ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca2+]i transients.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells. 805 57

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na(+)-K(+)-2Cl- cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3- (3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a approximately 180-kDa peptide, prominently labeled, and diffuse bands at approximately 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of approximately 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of approximately 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI approximately 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+/calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphorylation-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a approximately 175-kDa BBM protein that can be regulated by phosphorylation.
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PMID:Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter. 807 74

The vasoconstrictor responses to alpha-1 adrenoceptor agonists, binding behavior of alpha-1 adrenoceptors and postreceptor events in the mesenteric vasculature from deoxycorticosterone acetate-salt hypertensive rats were studied. The reactivity of perfused mesenteric artery to norepinephrine (NE) and phenylephrine, but not KCl, was enhanced significantly in the hypertensive rats compared with control rats. Prazosin antagonized the pressor response to NE more effectively in the hypertension than in the control. [3H]Prazosin binding was saturable and a single class of specific sites. Scatchard analysis revealed that the dissociation constant for [3H]prazosin was lower and the maximum binding capacity was greater in the hypertensive rats than in the control rats. The NE-stimulated phosphatidylinositol hydrolysis, estimated by measuring inositol 1,4,5-triphosphate (IP3) accumulation, was greater in the hypertensive rat artery compared with the control one. The IP3-induced contraction in the beta-escin-treated mesenteric large resistance vessel was smaller in the hypertensive rats. The vasoconstrictor response to phorbol 12,13-dibutyrate of the perfused mesenteric artery was larger in the hypertensive animals than in the control. Staurosporine antagonized the phorbol 12,13-dibutyrate-induced vasoconstriction in preparations from both rats. These results suggest that the increases in the number and affinity of alpha-1 adrenoceptors may result in an enhanced phosphatidylinositol hydrolysis accounting for an increased vascular reactivity to alpha-1 adrenoceptor agonists in deoxycorticosterone acetate-salt hypertensive rats. Furthermore, the enhancement of vasoconstrictor mechanism mediated by protein kinase C pathway may also contribute to vascular hyper-reactivity to NE, whereas the decreased IP3-induced contraction may function to minimize the hyper-reactivity observed in this model of experimental hypertension.
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PMID:Characterization of the alpha-1 adrenoceptors in the mesenteric vasculature from deoxycorticosterone-salt hypertensive rats: studies on vasoconstriction, radioligand binding and postreceptor events. 811 68

Esophageal circular muscle cells isolated by enzymatic digestion contracted in response to acetylcholine (ACh) and in response to the protein kinase C (PKC) agonist 1,2-dioctanoylglycerol (1,2 DAG). Both responses were blocked by PKC antagonists but not by calmodulin antagonists. Furthermore, specific PKC activity, measured in the particulate fraction of the muscle, increased in response to cholinergic stimulation, suggesting that ACh-induced contraction is mediated by a PKC-dependent pathway. ACh-induced contraction decreased with decreasing extracellular Ca2+ and was blocked in Ca(2+)-free physiological salt solution (PSS). Similarly, contraction by the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) was blocked by removal of Ca2+ from the PSS. Diacylglycerol production in response to ACh was reduced when extracellular Ca2+ was reduced from 2 to 0.5 mM and was abolished in Ca(2+)-free PSS. The response to 1,2-DAG, however, did not significantly change as extracellular Ca2+ or cytosolic Ca2+ was reduced to zero. Heparin (10 micrograms/ml), thapsigargin (3 microM), or the Ca2+ ionophore A-23187 (3 microM) had no effect on 1,2-DAG or ACh-induced contraction in permeable cells. The data suggest that contraction in response to ACh is mediated by influx of extracellular Ca2+ and a PKC-dependent pathway. Ca2+ may be required mainly to activate the phospholipases responsible for production of diacylglycerol, since contraction of esophageal muscle cells in response to 1,2-DAG is Ca2+ independent.
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PMID:Calcium requirements for acetylcholine-induced contraction of cat esophageal circular muscle cells. 814 7

Activation of neutrophils may contribute to lung injury in the adult respiratory distress syndrome. We added rabbit neutrophils to the pulmonary circulation of salt-perfused and ventilated isolated rabbit lungs. These neutrophils were activated by adding synthetically pure melittin to the perfusate. This led to lung injury as measured by filtration coefficient under no-flow conditions. We also activated neutrophils in vitro before addition to the pulmonary circulation. These preactivated neutrophils also produced lung injury, indicating a primary action of melittin on neutrophils rather than on lung. The injury was prevented by aristolochic acid, which is an inhibitor of phospholipase A2 (PLA2), and independently by catalase, which is scavenger of hydrogen peroxide (H2O2). Aristolochic acid also appeared to act primarily on neutrophils since addition to neutrophils in vitro prevented injury from in vitro activation by melittin. Aristolochic acid did not appear to act as a free radical scavenger since it did not prevent injury from neutrophils activated by phorbol myristate acetate (PMA). PMA is a direct activator of protein kinase C in neutrophils and leads to formation of H2O2 with consequent lung injury. We conclude that activation of neutrophils by melittin leads to oxidant lung injury possibly from activation of PLA2. Since PLA2 does not directly produce a second messenger, such as diacylglycerol or inositol triphosphate, it is likely that other actions of PLA2 produce an intermediary mediator. We previously showed that an inhibitor of eicosanoid synthesis prevents lung injury from exogenous PLA2. This suggests that the formation of leukotriene B4 (LTB4), a 5-lipoxygenase product of arachidonic acid, may contribute to the oxidant lung injury from melittin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Increase in filtration coefficient from actions of melittin on neutrophils in isolated rabbit lungs. 814 48

Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.
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PMID:Protein kinase C in rat cerebral microvessels. 817 27

This study examined the contribution of phosphatidylinositol metabolism and the efficacy of protein kinase C-mediated desensitization in the exaggerated alpha 1b-adrenergic receptor-mediated inositol phosphate response in the aorta of the deoxycorticosterone acetate (DOCA)-salt rat model of hypertension. The basal accumulation of inositol phosphates and the basal incorporation of [3H]myo-inositol in the phosphatidylinositol lipid pool were significantly higher in the aorta of these hypertensive rats. A positive correlation (r = .88, P < .01) was demonstrated between basal inositol phosphate levels and the [3H]myo-inositol-labeled phosphatidylinositol lipid pool. In hypertensive rats, alpha 1b-adrenergic receptor-mediated inositol phosphate production in response to phenylephrine was significantly higher compared with normotensive rats. Despite the normalization of phenylephrine-mediated inositol phosphate production to the [3H]myo-inositol-labeled phosphatidylinositol lipid pool, the alpha 1b-adrenergic response remained significantly higher in the hypertensive rats. Phorbol ester activation of protein kinase C attenuated to a lesser extent phenylephrine-mediated inositol phosphate production (40%) in the aorta of hypertensive rats compared with the 80% attenuation observed in the aorta of normotensive rats. This desensitization was inhibited in both groups by the protein kinase C inhibitor staurosporine. The blunted desensitization of the alpha 1b-adrenergic receptor by protein kinase C activation was not associated with a decrease in protein kinase C activity in the hypertensive rats, because aortic strips from these animals were more responsive to phorbol ester activation than aortic strips from normotensive animals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Altered protein kinase C regulation of phosphoinositide-coupled receptors in deoxycorticosterone acetate-salt hypertensive rats. 820 69

The Saccharomyces cerevisiae PKC1 gene encodes a homolog of mammalian protein kinase C (Levin, D. E., Fields, F.O., Kunisawa, R., Bishop, J.M., and Thorner, J. (1990) Cell 62, 213-224). A protein of 150 kDa is recognized by a polyclonal antiserum raised against a trpE-Pkc1 fusion protein. In subcellular fractionations, Pkc1p associates with the 100,000 x g particulate fraction. This association is resistant to extraction with high salt concentrations, alkali buffer, or nonionic detergents, suggesting that Pkc1p may be associated with a large protein complex. Pkc1p modified at its COOH terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ sequence tag) was able to fully restore the growth defects of a pkc1ts strain at restrictive temperature. ZZ-tagged Pkc1p was partially purified by chromatography on DEAE-Sepharose, followed by IgG-Sepharose. In vitro, Pkc1p phosphorylates the pseudosubstrate peptide and myelin basic protein, but not histones. Replacing an isoleucine with an arginine 2 amino acids COOH-terminal of the acceptor serine in the substrate peptide resulted in a 10-fold decrease of Km. Pkc1p activity was independent of cofactors such as phospholipids, diacylglycerol, and Ca2+, known to activate several mammalian protein kinase C isoenzymes, making it a rather distantly related member of the protein kinase C superfamily.
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PMID:Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product. 820 4

This study determined if phorbol ester-induced contraction of vascular smooth muscle requires calcium-dependent myosin light chain (MLC) phosphorylation and, if not, whether the mechanical characteristics of the contraction in terms of stiffness and crossbridge cycling are similar to those during a calcium- and MLC phosphorylation-dependent contraction. Carotid arterial strips were exposed to 1.0 microM phorbol 12,13-dibutyrate (PDBu) in the presence of normal physiological salt solution (PSS) or after calcium depletion in calcium-free PSS and compared with contraction elicited by calcium-containing 110 mM KCl-PSS. PDBu induced maximal stress in both the presence and absence of calcium. While there was a temporal correlation between MLC phosphorylation and shortening velocity during KCl depolarization, shortening velocity was dissociated from MLC phosphorylation during PDBu stimulation. The stress-stiffness relationship was not different during KCl and PDBu stimulation, suggesting similar crossbridge interactions even though MLC phosphorylation levels were significantly different. These results demonstrate that PDBu-induced contraction of the swine carotid artery is not dependent on calcium or MLC phosphorylation. We suggest the possibility that activation of a calcium-independent PKC isoform may result in the expression of an inherent level of actin-activated myosin ATPase activity resulting in the slow development of stress.
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PMID:Phorbol ester-induced contractions of swine carotid artery are supported by slowly cycling crossbridges which are not dependent on calcium or myosin light chain phosphorylation. 824 64


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