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)

Phagocytic cells can be primed for enhanced stimulated release of superoxide anion (O2-) by exposure to a variety of biologic agents, including gamma-interferon and lipopolysaccharide. We examined the role of calcium ion in this priming, using the calcium ionophore ionomycin. Preincubation with ionomycin, 1 to 10 nM, primed human neutrophils to release up to 7-fold more O2- during stimulation with 1 microM formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). With 160 nM phorbol myristate acetate as stimulus, ionomycin caused a doubling of O2- production in mouse peritoneal macrophages. Incubation of phagocytes with ionomycin at priming concentrations did not directly stimulate O2- release. Priming of neutrophils occurred in 1-2 min and was associated with a marked reduction in the lag time for O2- release after f-Met-Leu-Phe stimulation and with an increase in the rate of O2- production. Kinetic analysis of NADPH-dependent O2(-)-producing activity in sonicates of resting human neutrophils incubated with sodium dodecyl sulfate suggested that modification of the enzyme responsible for the respiratory burst was not responsible for priming. Priming of neutrophils with ionomycin had no apparent effect on either the activity or subcellular distribution of protein kinase C. The effect of ionomycin on the cytosolic free calcium concentration ([Ca2+]c) was assessed in neutrophils using the calcium-sensitive fluorescent dye fura-2. Ionomycin at priming concentrations caused an approximate doubling of the base-line [Ca2+]c. When neutrophils were exposed to various concentrations of ionomycin, a parallel rise in [Ca2+]c and priming was observed. A rise in [Ca2+]c of approximately 0.8 microM caused half-maximal priming. These results suggest that an increase in [Ca2+]c is not sufficient to initiate release of O2-, but they support the concept that Ca2+ can serve as a second messenger in this event.
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PMID:Priming of neutrophils and macrophages for enhanced release of superoxide anion by the calcium ionophore ionomycin. Implications for regulation of the respiratory burst. 304 Jul 59

The phorbol myristate acetate (PMA) stimulation of the human neutrophil NADPH-oxidase has been demonstrated through the activation of protein kinase C (PK-C), using light membrane fractions from nitrogen-cavitated cells. Both arachidonic acid (AA) and sodium dodecyl sulfate (SDS) can also generate an active oxidase in cellfree systems. That the source of O2- with AA and SDS activation is the same NADPH-oxidase as previously studied was confirmed by the similar pH optima and Km values for NADPH as those previously described for the O2- -generating activity harvested from pre-stimulated human neutrophils. In contrast to the stimulation by PMA, however, the stimulation of the NADPH-oxidase by AA and SDS does not appear to require protein kinase C activation: the action of AA and SDS is independent of the addition of PK-C cofactors to the system, and the inhibitor of PK-C activity, H-7, had no effect on the stimulation by AA or SDS. AA and SDS activation are comparable, but the level of NADPH-oxidase expression is sixfold greater with each of these agents than that obtained with a reconstituted PK-C system. The basis of this difference in oxidase expression is unclear, but these findings suggest strongly that although activated PK-C is capable of stimulating a dormant NADPH-oxidase in a cellfree system, this is not the sole pathway for oxidase activation.
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PMID:Comparison of subcellular activation of the human neutrophil NADPH-oxidase by arachidonic acid, sodium dodecyl sulfate (SDS), and phorbol myristate acetate (PMA). 310 4

Inhibitors of pancreatic islet lipoxygenase (LPX) impair nutrient-induced insulin (I) release. To define the mechanism of action of these inhibitors, studies were carried out at subthreshold glucose concentrations (0-1.7 mM) in order to minimize any effects of LPX blockade on the potentiating effect of extracellular fuels. Barium chloride (Ba2+; 2 mM) increased 45Ca2+ release from prelabeled islets in Ca2+-free medium and, thus, is a model for the mobilization of intracellular Ca2+ stores. Inhibition of LPX (using nordihydroguaiaretic acid, BW755c [3-amino-1-(trifluomethyl-phenyl)2-pyrazoline] or butylated hydroxytoluene) did not have any consistent effect on the influx of Ba2+ (as assessed by 133Ba uptake) or on the consequent release of cellular Ca2+ stores; however, each LPX inhibitor vitiated Ba2+-induced I release. The LPX inhibitors were not merely acting as nonspecific antioxidants, since two inhibitors which do not act by scavenging hydroperoxides (5,8,11,14-eicosatetraynoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid) also impeded the effect of Ba2+ on I secretion; furthermore, a series of hydroxyl radical scavengers, reducing agents, or agents that block nonenzymatic and/or NADPH-activated lipid peroxidation did not inhibit I secretion. LPX inhibitors also blocked the residual I response to 16.7 mM glucose in Ca2+-free medium. Additionally, they reduced secretion induced by 46 mM K+ or 1 mM isobutylmethylxanthine (provided in the presence of extracellular Ca2+), without inhibiting K+- or isobutylmethylxanthine-induced Ca2+ fluxes. Stimuli sensitive to LPX blockade were also antagonized by antimycin A (an inhibitor of energy flux) or TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; which appeared to deplete critical intracellular Ca2+ stores]. In contrast, the effects of exogenous phospholipase C (and several other Ca2+-dependent membrane-active agonists) were resistant to the LPX inhibitors, TMB-8, and antimycin A; thus, LPX inhibitors are not nonspecific global poisons of all Ca2+-dependent exocytotic hormone release. We conclude that LPX (or a very similar enzyme) may modulate the effects (or redistribution) of an ATP-dependent trigger pool of Ca2+ at a site distal to and independent of its mobilization by primary islet agonists. LPX inhibitors also blocked secretion induced by 12-O-tetradecanoyl-phorbol-13-acetate; this effect may reflect an effect of LPX on the activation of protein kinase C or a modulation of its synergism with the same trigger Ca2+ pool(s).
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PMID:Lipoxygenase inhibitors reduce insulin secretion without impairing calcium mobilization. 310 21

We have reported previously that calcium ions and phospholipid activate the heme-stabilized proinhibitor form (pro-HCI) of the heme-controlled translational inhibitor (HCI) in reticulocyte lysates and promote the first step of the reaction pro-HCI in equilibrium reversible HCI----irreversible HCI. This suggested the possible involvement of a Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the activation. However, further investigation revealed, among other things, that polyunsaturated fatty acids (e.g., arachidonic acid) were as effective as Ca2+/phospholipid in promoting translational inhibition and phosphorylation of the alpha subunit of the chain-initiation factor eIF-2 and, moreover, HCI activation could be prevented or reversed in either case by NADPH-generating systems or by dithiols. Our results suggest that pro-HCI is activated by lipoperoxides produced in reticulocyte lysates from either phospholipid or polyunsaturated fatty acids; the presence of Ca2+ is required in the former but not in the latter case. The reversible activation of HCI by Ca2+ and phospholipid might suggest a possible modulatory role of Ca2+ in translational control.
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PMID:Mechanism of activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid. 315 12

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.
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PMID:Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway. 333 94

It has been reported that respiratory bursts with N-formylmethionylleucylphenylalanine, A23187, phorbol ester and fatty acids are switched off and on by modulating the net charges of plasma membranes in guinea-pig neutrophils (Miyahara, M. et al. (1987), Biochim. Biophys. Acta, 929, 253-262). In the present study, this was further extended in cells treated with protein kinase C inhibitors which completely suppressed the phorbol ester-dependent respiratory burst. This suggested that the initiation of the respiratory burst, which is generally accepted as linked to protein kinase C activation, might also be implicated in the net charge changes of plasma membranes. The above results were also supported by data obtained with a cell-free system reconstituted with plasma membranes and cytosolic fractions from unstimulated neutrophils, guanosine 5'-[gamma-thio]triphosphate and NADPH. Arachidonate stimulated NADPH oxidase activity accompanied by a marked phosphorylation of membrane proteins. The phosphorylation was sensitive to H-7, but it did not appear to be essential for the respiratory burst, because the oxidase activation was insensitive to H-7. Pretreating the plasma membranes with positively charged cetylamine inhibited the oxidase activation by arachidonate. These results suggest that a charge-dependent process, which does not use protein kinase C, may play an important role in the reaction leading to NADPH oxidase activation, and this may be related to the interaction of plasma membranes with the cytosolic activation factor.
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PMID:Charge-dependent regulation of NADPH oxidase activities in intact and subcellular systems of polymorphonuclear leukocytes. 340 44

We have demonstrated that the purified guanine nine nucleotide exchange factor (GEF) may be isolated as a complex with NADPH. Complete inhibition of the GEF-catalyzed exchange of eukaryotic initiation factor 2-bound GDP for GTP was observed in the presence of either 0.5-0.75 mM NAD+ or NADP+. Incubation of GEF with ATP results in the phosphorylation of its Mr 82,000 polypeptide. This phosphorylation is strongly inhibited by heparin but is not affected by heme or H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP- and cGMP-dependent protein kinases and protein kinase C. The purification of GEF was modified to eliminate any contaminating kinase activity and the isolated protein appears to be homogeneous as judged by NaDodSO4/polyacrylamide gel electrophoresis and silver staining. The Mr 82,000 subunit of GEF is phosphorylated only upon addition of ATP and casein kinase II. The extent of phosphorylation is approximately equal to 0.55 mol of phosphate per mol of GEF, and this results in a 2.3-fold increase in the guanine nucleotide exchange activity. Following treatment of the phosphorylated GEF with alkaline phosphatase, the activity of the protein is reduced by a factor of 5. Rephosphorylation of GEF increases its specific activity to that of the phosphorylated protein. The results of this study suggest that phosphorylation/dephosphorylation of GEF plays a role in regulating polypeptide chain initiation.
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PMID:Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in polypeptide chain initiation. 342 26

Plasma membranes isolated from human neutrophils after brief exposure to phorbol 12-myristate 13-acetate contain a large portion (30-40%) of the total cellular protein kinase C (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1986) Biochem. Biophys. Res. Commun. 136, 228-234) and also retain almost 90% of their content of neutral serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G., and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1685-1689). When ATP is added to the isolated membranes, a substantial amount (approximately 25%) of the intrinsic proteinase is released into the incubation medium. The addition of ATP in the presence of NADPH also caused a significant enhancement of the production of O2 radicals. These effects of ATP were not observed with membranes isolated from untreated neutrophils. The release of the serine proteinase is almost fully dependent on the addition of ATP and is correlated with the phosphorylation of membrane proteins. It is also markedly inhibited by the addition of retinal or trifluoperazine inhibitors of native protein kinase C. The results represent the first direct demonstration of a role for membrane-bound protein kinase C activity in the release of neutral proteinase and the production of O2 radicals, responses related to the cytotoxic effects of activated neutrophils.
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PMID:ATP induces the release of a neutral serine proteinase and enhances the production of superoxide anion in membranes from phorbol ester-activated neutrophils. 352 41

It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.
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PMID:Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels. 623 73

The dynamics and mechanisms of extracellular release of hydrogen peroxide (H2O2) from bovine pulmonary artery endothelial cells (EC) subjected to anoxia, hypoxia, and hypoxia followed by reoxygenation were examined using various inhibitors of enzymatic systems in intact cells and by direct measurement of H2O2 production from isolated EC plasma membranes. Extracellular H2O2 was measured with a fluorometric assay. EC exposed to hypoxia (3% O2) and anoxia (0% O2) released less H2O2 (29.6 +/- 1.3% and 4.2 +/- 0.7%, respectively) compared with EC exposed to normoxia (20% O2). The extracellular release of H2O2 from EC previously exposed to hypoxia for 24 h increased immediately after reoxygenation (20% O2) to 272 +/- 48%, as compared with EC exposed continuously to normoxia (100% release). Inhibition of xanthine oxidase (XO) by allopurinol did not reduce the release of H2O2 from cells exposed to normoxia or hypoxia followed by reoxygenation. Furthermore, inhibitors of cyclooxygenase (indomethacin), phospholipase A2 (quinacrine and chlorpromazine), nitric oxide synthase (L-arginine analogs), the mitochondrial electron transport chain (rotenone and cyanide), and cytochrome P-450 (methoxypsoralen) had no or minimal effect on this release. On the other hand, inhibitors of protein kinase C (calphostin and staurosporine) and NADPH oxidase (diphenyliodonium) reduced the release of H2O2 from EC in a dose-dependent manner in both exposure groups. In separate experiments, plasma membranes isolated from EC were found to produce H2O2 in the presence of NADH or NADPH as electron donors. This was inhibited by diphenyliodonium but not by allopurinol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. 752 30


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