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)

Proton accumulation and efflux associated specifically with NADPH oxidation in neutrophils remains to be elucidated. Using confocal fluorescence and patch-clamp recordings from single human neutrophils, in the presence of protein kinase C inhibitors, we studied the transient cytosolic acidification and whole-cell H+ current induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and recombinant human tumor necrosis factor alpha (rhTNF alpha). Intracellular pH changes were monitored utilizing the ratiometric imaging of the dual emission fluoroprobe, carboxyseminaphthorhodafluor-1, AM acetate. Bath application of 1000 units/ml rhTNF alpha or 0.1 microM fMLP changed the fluorescence of fluoroprobe-loaded cells, indicating generation of cytosolic H+ ions. In the absence of Ca2+ in the pipette solution, exposure of cells to rhTNF alpha or fMLP for 10 s activated voltage-dependent H+ currents. From tail current analysis, the threshold voltage for H+ current activation was approximately -50 mV. These fMLP- or rhTNF alpha-activated voltage-dependent H+ currents were augmented further in the presence of 0.1 mM of NADPH in the pipette solution, and they were inhibited by bath application of 50 microM of apocynin, an NADPH oxidase inhibitor. These results indicate that rhTNF alpha- or fMLP-induced NADPH oxidase in human neutrophils gives rise to the activation of voltage-dependent H+ currents.
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PMID:Activation of NADPH-oxidase and its associated whole-cell H+ current in human neutrophils by recombinant human tumor necrosis factor alpha and formyl-methionyl-leucyl-phenylalanine. 753 23

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases.
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PMID:Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects? 757 84

1. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a non-toxic seleno-organic drug with antiinflammatory, antiatherosclerotic and cytoprotective properties. 2. Ebselen and some of its metabolites are effective reductants of hydroperoxides including those arising in biomembranes and lipoproteins. 3. By reactions with hydroperoxides and thiols several interconversion cycles are formed which include ebselen metabolites with varying oxidation number of the selenium. 4. In the presence of thiols ebselen mimics the catalytic activities of phospholipid hydroperoxide glutathione peroxidase. 5. Ebselen inhibits at low concentrations a number of enzymes involved in inflammation such as lipoxygenases, NO synthases, NADPH, oxidase, protein kinase C and H+/K(+)-ATPase. The inhibitions are manifested on the cellular level and may contribute to the antiinflammatory potential of ebselen.
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PMID:Molecular actions of ebselen--an antiinflammatory antioxidant. 759 Jan 3

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate- and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurin-mediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme's catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.
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PMID:Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. 823 9

The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca2+]i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca2+]i, it restored the burst response to agonists in Ca(2+)-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca(2+)-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G(i)-proteins, but that in the presence of PMA portions of the agonist response could be recovered.
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PMID:Activation of the respiratory burst in eosinophil leucocytes--a transduction sequence decoupled from cytosolic Ca2+ rise. 770 83

Human peripheral blood monocytes (PBM) produce superoxide anions (O2-.) by a process involving electron transfer from NADPH to O2, catalyzed by the respiratory burst enzyme NADPH oxidase. We have previously shown that phagocytosis, while activating NADPH oxidase, induced in PBM the synthesis of heat shock (HS) proteins (HSP). The present study was undertaken to establish whether this increase in HSP expression was related to O2-. and/or to classical second messengers such as protein kinase C (PKC). Thus, the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) were compared with those of heat shock on the expression, in PBM, of the major HSP, hsp70 and hsp90, using biometabolic labeling, Western and Northern blotting, and gel mobility shift assays. PMA induced the accumulation of mRNA and an increased expression of hsp90 and, to a lesser extent, hsp/hsc70 (hsc is the cognate, constitutive form). This induction was also observed in PBM from patients with chronic granulomatous disease, a genetic defect in NADPH oxidase, and was abolished by the PKC inhibitors staurosporine and H-7. PMA did not cause activation of the HS factor, and the PMA-induced overexpression expression of HSP was not blocked by the transcriptional inhibitor actinomycin D. HSP-specific mRNA stability was increased after PMA exposure as compared with heat shock. These results suggest that O2-. is not involved in the PMA-mediated induction of hsp70 and hsp90 and that, in contrast to HS, PMA increases the expression of HSP as a result of PKC-induced mRNA stabilization rather than of transcriptional activation of HS genes.
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PMID:Differential expression and regulation of hsp70 and hsp90 by phorbol esters and heat shock. 777 71

Superoxide is produced by a NADPH oxidase of phagocytic cells and contributes to their microbicidal activities. The oxidase is activated when receptors in the neutrophil plasma membrane bind to the target microbe. These receptors recognise antibodies and complement fragments which coat the target cell. The oxidase electron transport chain, located in the plasma membrane, comprises a low potential cytochrome b heterodimer (gp 91-phox and p22-phox) associated with FAD. It is non-functional until at least three proteins, p67-phox, p47-phox and p21rac (and possibly others), move from the cytosol to dock on the cytochrome b. The docking involves the interaction of SH3 domains on p47-phox or p67-phox with a proline-rich sequence on the small subunit of the cytochrome b. These SH3 domains may become exposed following phosphorylation of p47-phox by protein kinase C or, in model systems, by addition of arachidonic acid to reconstitution mixtures. Following the docking process the electron-transporting component is able to transfer electrons from NADPH to oxygen. This electrogenic event is charge-compensated by the opening of a proton channel. Components of the oxidase are expressed in non-phagocytes, where their function is uncertain but could be related to some signal function of superoxide.
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PMID:The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells. 784 Jul 72

f-Met-Leu-Phe-stimulated luminol-enhanced chemiluminescence was found to be repeatedly defective in some MDS patients. This defect was not attributed to myeloperoxidase deficiency, nor to a defect in NADPH oxidase function, because PMA chemiluminescence was found to be normal in these individuals. An arbitrary value of 7 mV (half the mean control value) was chosen to subdivide the group: MDS patients with values < 7 mV had a mean f-Met-Leu-Phe chemiluminescence response of 2.5 +/- 0.5 compared to MDS patients with values > 7 mV who had a mean response of 15.6 +/- 1.6 mV, P < 0.01 (healthy controls 14 +/- 2 mV). The characteristics of the f-Met-Leu-Phe receptor and initial calcium flux results suggested that the receptor itself was normal in number and function in low f-Met-Leu-Phe responders. The rate of superoxide generation, which is calcium-dependent, was also found to be in the normal range in low f-Met-Leu-Phe responders, although total superoxide production was reduced in some of these patients. When MDS neutrophils with a low f-Met-Leu-Phe response were stimulated with PMA, chemiluminescence was normal, suggesting normal activity of the NADPH-oxidase complex. Furthermore, myeloperoxidase activity was reduced in only three out of the 11 low f-Met-Leu-Phe responders. Following priming with GM-CSF, f-Met-Leu-Phe chemiluminescence was 27 +/- 1.6 mV in low f-Met-Leu-Phe responders compared to controls (87.7 +/- 11 mV, P < 0.005). Thus, although responses were improved, they were not as marked as in control neutrophils. These data suggest that a subgroup of MDS patients have a low f-Met-Leu-Phe chemiluminescence response which is not due to a defect in the f-Met-Leu-Phe receptor or oxidase activity, and in the majority of cases MPO activity is normal. Initial patient survival data suggest that these patients may have an increased risk of infective mortality. It is proposed that defective f-Met-Leu-Phe chemiluminescence results from a putative defect in cell-signalling mechanism upstream of PKC, and GM-CSF priming only partially improves responsiveness.
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PMID:Identification of a subgroup of myelodysplastic patients with a neutrophil stimulation-signalling defect. 791 69

Human antioxidant-response element (hARE) containing two copies of the AP1/AP1-like elements arranged as inverse repeat is known to mediate basal and beta-naphthoflavone-induced transcription of the type 1 NAD(P)H:quinone oxidoreductase (NQO1) gene. Band-shift assays revealed that beta-naphthoflavone increased binding of nuclear proteins at the hARE. Super shift assays identified Jun-D and c-Fos proteins in the band-shift complexes observed with control and beta-naphthoflavone-treated Hepa-1 nuclear extracts. Hepa-1 cells stably transformed with hARE-tk-chloramphenicol acetyl transferase (CAT) recombinant plasmid were used to demonstrate that, in addition to beta-naphthoflavone, a variety of antioxidants, tumor promoters and hydrogen peroxide (H2O2) also increased expression of hARE-mediated CAT gene. beta-naphthoflavone induction of the CAT gene expression in Hepa-1 cells was found insensitive to inhibitors of protein kinase C and tyrosine kinases. However, binding of regulatory proteins at the hARE and the CAT gene expression in Hepa-1 cells were increased by dithiothreitol, 2-mercaptoethanol and diamide. Treatment of the Hepa-1 cells with N-ethylmaleimide reduced binding of proteins at the hARE and interfered with expression and beta-naphthoflavone induction of the CAT gene. These results suggested a role of sulfhydryl modification of hARE binding (Jun and Fos) proteins which mediate basal and induced expression of the NQO1 gene. We also report that in-vitro-translated products of the proto-oncogenes, Jun and Fos, bind to the hARE in band-shift assays. The incubation of Jun and Fos proteins with small amounts of nuclear extract from dimethylsulfoxide-treated (control) or beta-naphthoflavone treated Hepa-1 cells prior to band-shift assays increased the binding of Jun and Fos proteins to the hARE. Interestingly, the increase in binding of Jun and Fos proteins to the hARE was more prominent with beta-naphthoflavone-treated nuclear extract as compared to the control nuclear extract. In addition, incubation of control nuclear extract with beta-naphthoflavone, microsomes and NADPH increased the binding of Jun and Fos proteins to the hARE. Evidence from in vitro studies indicate the presence of unknown nuclear factor(s) that receive signals from metabolites of beta-naphthoflavone and modulate Jun and Fos binding to the AP1 site contained within the hARE.
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PMID:Human antioxidant-response-element-mediated regulation of type 1 NAD(P)H:quinone oxidoreductase gene expression. Effect of sulfhydryl modifying agents. 795 57

Thioredoxin (Trx) catalyzes thiol-disulfide oxidoreductions. We and others recently showed that human Trx could function as an autocrine growth factor for human lymphoid cells immortalized by the human T-lymphotrophic virus type I or the Epstein-Barr virus. Here we report that reduced Trx from Escherichia coli generated by NADPH and thioredoxin reductase increases the proliferation of an Epstein-barr virus(+)-B cell line 1G8, which constitutively produces low amounts of human Trx. This proliferative effect involved the activation of protein kinase C through its translocation to the membrane. Staurosporin and calphostin C, two inhibitors of protein kinase C, but not of H8, a protein kinase A inhibitor, were able to block Trx-dependent proliferation. The addition of Trx to 1G8 cells resulted in the formation of inositol 1,4,5-triphosphate and sn-1,2-diacylglycerol by a phosphoinositide-specific phospholipase C, as well as increased free calcium concentration. Diacylglycerol showed a biphasic increase; the first phase, corresponding to an early peak (30 s) of inositol 1,4,5-triphosphate and a second larger, prolonged phase. The second phase was inhibited by propranolol, a specific inhibitor of phosphohydrolase, indicating that it is most likely derived from phosphatidylcholine hydrolysis by the sequential action of phospholipase D and phosphatidic acid phosphohydrolase. Our data suggest that enhanced phosphoinositide-specific phospholipase C activity induced by the dithiol form of Trx in 1G8 cells is associated to protein kinase C activation, and thus plays a role in the permanent growth of Epstein-Barr virus-infected B cells.
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PMID:Thioredoxin increases the proliferation of human B-cell lines through a protein kinase C-dependent mechanism. 796 46


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