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)

Alterations of glucose metabolism and the oxidation of glutamine and palmitate were studied, by using specifically labelled substrates, in freshly isolated Kupffer cells and hepatic endothelial cells after infusion in vivo of human recombinant tumour necrosis factor-alpha (TNF; 7.5 x 10(5) IU/30 min per kg body wt., intravenously). Cells were incubated in a medium containing 5 mM-glucose, 0.4 mM-palmitate, 1 mM-lactate and 0.5 mM-glutamine. Administration of TNF in vivo increased glucose use in Kupffer cells by 70%. Glucose oxidation in the tricarboxylic acid cycle and flux in the Embden-Meyerhof (EM) pathway were elevated by 40 and 80% respectively. Treatment in vitro with 1 microM-phorbol 12-myristate 13-acetate (PMA) resulted in a similar percentage increase in glucose use by Kupffer cells prepared from either saline- or TNF-treated rats. However, PMA increased the activity of the hexose monophosphate shunt (HMS) by 3- and 10-fold in cells isolated from saline- or TNF-infused animals respectively. A phagocyte stimulus in vitro, opsonized zymosan, increased glucose use by 30% and doubled the flux through the HMS in Kupffer cells from saline-infused animals. The activity of the HMS in response to zymosan was increased by 400% after TNF treatment. In endothelial cells, basal glucose utilization was not altered by TNF treatment. PMA increased HMS activity in endothelial cells to a similar degree after saline or TNF infusion. Zymosan, however, increased HMS activity only in endothelial cells from TNF-treated rats. Oxidation of palmitate or glutamine was not affected by TNF treatment either under basal conditions or after challenge in vitro. Our data indicate that, after phagocytosis in vitro or protein kinase C activation, glucose use and flux through the HMS increase in Kupffer cells. This is accompanied by increased glycolytic flux, with no changes in glucose oxidation in the tricarboxylic acid cycle. After TNF exposure, followed by a secondary stimulus, the enhanced glucose use by Kupffer cells is primarily channelled through the HMS pathway. These data suggest that the increased glucose use in vivo by Kupffer cells found after immune-stimulated conditions may subserve primarily the increased need for NADPH and HMS intermediates.
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PMID:Up-regulation of glucose metabolism in Kupffer cells following infusion of tumour necrosis factor. 189 44

The cell free activation of NADPH-Oxidase in membranes of mouse peritoneal macrophages by purified PKC-isotypes was investigated. Unstimulated intrinsic activity of PKC-isotypes showed little dependence on Ca2+ for activation of the oxidase. In the presence of TPA, the activation of the oxidase was greatly enhanced, and alpha-, and gamma-subtypes were strongly Ca2+ dependent in this system. Beta-, delta- and epsilon-subtypes were active both in the presence and absence of free Ca2+ ions. The results suggest that at resting Ca2+ levels certain PKC-isotypes can activate NADPH-oxidase.
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PMID:NADPH-oxidase activation by protein kinase C-isotypes. 205 95

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.
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PMID:Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate. 215 19

Neutrophils (PMN) from newborn calves generate significantly less superoxide anion (O2-) than do their adult counterparts after stimulation with direct protein kinase C agonists. To better understand this observation, we compared the activity and kinetics of NADPH oxidase in membrane fractions from phorbol 12-myristate 13-acetate-stimulated adult and newborn PMN. After phorbol 12-myristate 13-acetate stimulation, PMN were sonicated and the membranes assayed for O2- production with increasing concentrations of NADPH. O2- production was calculated 1 and 2 min after the beginning of the reaction. At all concentrations of NADPH used, there was no difference (p greater than 0.05) in O2- production between adult (n = 8) and newborn (n = 9) PMN membrane preparations. Enzyme kinetics calculations revealed no differences (p greater than 0.05) between age groups in Km and Vmax or in the velocity of the reactions. Determination of the protein content in the membrane pellet, however, showed that adult PMN yielded significantly (p less than 0.01) higher amounts of protein (2.82 +/- 0.14 mg/mL) than did newborn PMN (1.78 +/- 0.07 mg/mL). This difference could be partly attributed to cell size; flow cytometric analysis showed that newborn PMN had a significantly (p less than 0.01) smaller diameter (10.94 +/- 0.07 microns) than did adult PMN (11.65 +/- 0.06 microns), and calculated cell volume and surface area were also both significantly less (p less than 0.01) in newborn PMN.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Membrane NADPH oxidase activity and cell size in bovine neonatal and adult neutrophils. 217

In a "respiratory burst", fertilized sea urchin eggs consume oxygen to produce H2O2 as an extracellular oxidant to crosslink their protective surface envelopes. The egg generates H2O2 via a NADPH-specific oxidase that requires protein kinase C for activation. To further study the physiological regulation of the respiratory burst and cortical granule exocytosis, we have measured azide-insensitive oxygen uptake and fertilization envelope assembly in ionophore-stimulated eggs. Procaine, trifluoperazine, staurosporine, and H-7, which inhibit protein kinase C by different mechanisms, suppressed egg oxygen consumption without affecting fertilization envelope assembly. In contrast, both exocytosis and oxygen uptake were blocked in N-ethylmaleimide-treated eggs. When the eggs were stimulated with ionophore in Na-free artificial seawater, which prevents the increase in pHi, oxidase activity was inhibited. This effect was reversed by elevation of cytoplasmic pH with the membrane-permeant base NH4Cl. We conclude that protein kinase C was not involved in the events downstream from the ionophore-dependent elevation of Ca2+ which induced cortical granule exocytosis. However, the respiratory burst was inhibited despite the increase in Ca2+ that triggered exocytosis. The likely target for inhibition of the burst was protein kinase C. Cytoplasmic alkalinization was necessary for optimal rates of H2O2 synthesis, further implicating pHi as a regulator of the egg oxidase.
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PMID:Protein kinase C activates the respiratory burst of fertilization, but not cortical granule exocytosis, in ionophore-stimulated sea urchin eggs. 222 97

We have investigated the possible role of plasma membrane oxidoreductases in the Ca2+ export mechanisms in rat brain synaptic membranes. Ca2+ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca2+ pump and by a low affinity/high capacity ATP-independent Na(+)-Ca2+ exchanger. Both Ca2+ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP greater than NAD greater than NADPH greater than NADH with IC50 values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca2+ pump and with IC50 values between 8 and 10 mM for the Na(+)-Ca2+ exchanger. Oxidizing agents such as DCIP3 and ferricyanide inhibited the ATP-driven Ca2+ efflux mechanism but not the Na(+)-Ca2+ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca2+ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na(+)-Ca2+ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by either cAMP-dependent protein kinase, Ca2+ calmodulin-dependent protein kinases, or protein kinase C.
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PMID:Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes. 224 77

The non-tumour promoting irritant, resiniferatoxin, was capable of activating the NADPH-oxidase respiratory burst of starch-elicited, but not resident mouse peritoneal macrophages in vitro. Unlike TPA, the response was synergised by incubation with zymosan. The Rx-stimulated NADPH-oxidase activity in a cell-free assay was selectively enhanced in the presence of exogenous Rx-kinase rather than PKC and in the absence of Ca2+. Since resiniferatoxin is a poor activator of PKC, it is probable that the Ca2(+)-independent Rx-kinase plays a role in activation of the macrophage respiratory burst following stimulation by zymosan.
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PMID:TPA and resiniferatoxin-mediated activation of NADPH-oxidase. A possible role for Rx-kinase augmentation of PKC. 237 85

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction. 242 6

Lipopolysaccharide (LPS) pretreatment "primes" neutrophils to release increased amounts of superoxide anion (O2-) when stimulated. We investigated the molecular basis of this enhanced activity. Comparison of kinetic parameters of the respiratory burst NADPH oxidase in unstimulated LPS-primed and control neutrophils disclosed a similar Km for NADPH and no difference was seen in the content of cytochrome b. Pertussis toxin, which inhibits some G proteins, did not prevent priming. Change in membrane potential (delta psi) was five-fold greater in LPS-primed cells and paralleled the increased O2- release. Cytofluorographic analysis indicated that the increased change in delta psi was due to the creation of a new population of active cells. Changes in the concentration of intracellular free Ca2+ ([Ca2+]i) are believed to antecede changes in delta psi. There was a consistent increment (67 +/- 8%, n = 12) in resting [Ca2+]i in cells preincubated with LPS compared with control. When stimulated, the peak [Ca2+]i was significantly higher in LPS-primed cells. Ca2+-dependent protein kinase C activity was unaltered in resting and FMLP-stimulated neutrophils preexposed to LPS. Addition to cells of the intracellular Ca2+ chelator MAPTAM before preincubation with LPS blocked the changes in [Ca2+]i and the enhanced respiratory burst that characterize LPS priming. The increased resting [Ca2+]i in LPS-primed cells may enhance stimulus-induced cellular activity by modifying a Ca2+-dependent step in signal transduction.
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PMID:Lipopolysaccharide priming of human neutrophils for an enhanced respiratory burst. Role of intracellular free calcium. 253 46

Ebselen (PZ51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) was shown to be an inhibitor of human granulocyte oxidative burst stimulated by phorbol myristate acetate (IC50 25 microM). Estimation of the primary oxygen metabolites of the burst was complicated by the redox chemistry of Ebselen. Ebselen inhibited NADPH-stimulated superoxide generation by a partially purified NADPH oxidase preparation with an IC50 of 0.5-1.0 microM. Ebselen was also shown to inhibit the activity of partially purified Ca2+- and phospholipid-dependent protein kinase C (IC50 ca. 0.5 microM). Phorbol ester-stimulated phosphorylation of protein in intact cells was inhibited by Ebselen (IC50 ca. 50 microM). These pharmacodynamic properties of Ebselen are discussed in terms of its anti-inflammatory activity in vivo. The findings are also discussed in terms of Ebselen's known ability to interact with sulfhydryl components of cells, particularly critical thiol components of the enzymes studied.
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PMID:Studies on the anti-inflammatory activity of ebselen. Ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C? 253 84


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