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:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein kinase C inhibitor, staurosporine, inhibited NADPH oxidase activity of human neutrophils activated by phorbol myristate acetate. However, this inhibitor had no effect on either the initiation or the maximal rate of O2- secretion activated by the chemotactic peptide, fMet-Leu-Phe, but resulted in a more rapid termination of oxidant production. Similarly, staurosporine had no effect on the rapid (1 min) increase in luminol-dependent chemiluminescence activated by fMet-Leu-Phe, but the second (intracellular) phase of oxidant production was inhibited. The initial burst of oxidant production during phagocytosis was similarly protein kinase C-independent, but again the later phases of oxidase activity were staurosporine-sensitive. Neutrophils loaded with Quin-2 at concentrations sufficient to act as a Ca2+ buffer could not secrete O2- in response to fMet-Leu-Phe; although the initial (protein kinase C-independent) burst of luminol chemiluminescence was not observed in fMet-Leu-Phe-stimulated Ca2(+)-buffered cells, the second phase of (protein kinase C-dependent) oxidant production was largely unaffected. Hence, the initial burst of oxidant production activated by fMet-Leu-Phe, opsonized zymosan, and latex beads is independent of the activity of protein kinase C-dependent intracellular activation processes, but the activity of this kinase is required to extend or sustain the duration of oxidant production.
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PMID:Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils. 185 Apr 7

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.
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PMID:Staurosporine inhibits the respiratory burst and induces exocytosis in human neutrophils. 255 21

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 approximately 16 nM) and AA (EC50 approximately 6 microM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5-6 nmol/min/10(6) cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 approximately 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4beta-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.
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PMID:Leukotriene B4 activates the NADPH oxidase in eosinophils by a pertussis toxin-sensitive mechanism that is largely independent of arachidonic acid mobilization. 957 59

We studied differences between the NADPH oxidase activation pathways triggered by pervanadate, a protein tyrosine phosphatase inhibitor, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in guinea pig neutrophils. Previously, pervanadate has been shown to activate NADPH oxidase via the tyrosine kinase-dependent pathway (Yamaguchi et al. Arch. Biochem. Biophys. 323, 382-386, 1995). Both pervanadate and PMA induced superoxide anion (O-2) production, translocation of the 47-kDa protein component of the phagocyte oxidase (p47-phox) to the plasma membrane, and phosphorylation of p47-phox in the membrane. A selective protein kinase C inhibitor, GF 109203X, markedly inhibited PMA-induced O-2 production, p47-phox translocation, and p47-phox phosphorylation, but did not inhibit pervanadate-induced O-2 production and only slightly suppressed pervanadate-induced translocation and phosphorylation. These results demonstrate that pervanadate activates NADPH oxidase independently of protein kinase C. Phosphopeptide mapping of p47-phox revealed differences in the mechanism between pervanadate-induced and PMA-induced phosphorylation. Furthermore, some protein kinases which phosphorylate p47-phox-derived peptide are activated by pervanadate. These results suggest the existence of novel protein kinases responsible for the phosphorylation of p47-phox and the activation of these protein kinases by tyrosine kinase.
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PMID:Pervanadate activates NADPH oxidase via protein kinase C-independent phosphorylation of p47-phox. 988 22

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces respiratory burst (O-2 generation) in permeabilized human neutrophils. The signal pathway from GTPgammaS to the enzyme responsible for O-2 generation (NADPH oxidase) is not well defined. To elucidate the signaling pathway activated by GTPgammaS, we used selective inhibitors to test for the involvement of several enzymes, comparing the effects of these inhibitors on fMet-Leu-Phe (fMLP) activation. GTPgammaS-induced respiratory burst was not influenced by genistein, a selective inhibitor of tyrosine kinase, while fMLP-induced response was completely abolished. The respiratory burst by GTPgammaS was efficiently inhibited by the protein kinase C inhibitor GF109203X even more than fMLP activation. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 showed a partial inhibition of both GTPgammaS and fMLP activation. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, completely blocked fMLP activation, but had no effect on the GTPgammaS-induced respiratory burst. Using U73122, phospholipase C is shown to be essential in GTPgammaS signaling as well as fMLP signaling. Butanol blocked fMLP signaling but not GTPgammaS signaling, indicating that only fMLP activation involves phospholipase D. These results suggest that there are several differences between GTPgammaS- and fMLP-induced activation, but both activators share a common pathway including phospholipase C, protein kinase C, and MAPK kinase.
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PMID:Guanosine 5'-O-(3-thiotriphosphate)-induced O-2 generation in permeabilized neutrophils requires protein kinase C and phospholipase C but not tyrosine kinase or phospholipase D. 988 54

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.
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PMID:Thrombin stimulated reactive oxygen species production in cultured human endothelial cells. 993 Jun 45

Neutrophil superoxide production can be potentiated by prior exposure to "priming" agents such as granulocyte/macrophage colony stimulating factor (GM-CSF). Because the mechanism underlying GM-CSF-dependent priming is not understood, we investigated the effects of GM-CSF on the phosphorylation of the cytosolic NADPH oxidase components p47(phox) and p67(phox). Preincubation of neutrophils with GM-CSF alone increased the phosphorylation of p47(phox) but not that of p67(phox). Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosphorylation of p47(phox) than with GM-CSF alone and fMLP alone. GM-CSF-induced p47(phox) phosphorylation was time- and concentration-dependent and ran parallel to the priming effect of GM-CSF on superoxide production. Two-dimensional tryptic peptide mapping of p47(phox) showed that GM-CSF induced phosphorylation of one major peptide. fMLP alone induced phosphorylation of several peptides, an effect enhanced by GM-CSF pretreatment. In contrast to fMLP and phorbol 12-myristate 13-acetate, GM-CSF-induced phosphorylation of p47(phox) was not inhibited by the protein kinase C inhibitor GF109203X. The protein-tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitor wortmannin inhibited the phosphorylation of p47(phox) induced by GM-CSF and by fMLP but not that induced by phorbol 12-myristate 13-acetate. GM-CSF alone did not induce p47(phox) or p67(phox) translocation to the membrane, but neutrophils treated consecutively with GM-CSF and fMLP showed an increase (compared with fMLP alone) in membrane translocation of p47(phox) and p67(phox). Taken together, these results show that the priming action of GM-CSF on the neutrophil respiratory burst involves partial phosphorylation of p47(phox) on specific serines and suggest the involvement of a priming pathway regulated by protein-tyrosine kinase and phosphatidylinositol 3-kinase.
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PMID:Priming of human neutrophil respiratory burst by granulocyte/macrophage colony-stimulating factor (GM-CSF) involves partial phosphorylation of p47(phox). 1040 Jul 4

NADPH oxidase, a superoxide-producing enzyme in phagocytic cells, consists of membrane-associated cytochrome b558 and cytosolic components (p47-phox, p67-phox, p40-phox, rac 1/2). Activation of NADPH oxidase is accompanied by the phosphorylation of cytosolic components (p47-phox and p67-phox). In this study, we have examined whether p40-phox, a newly identified cytosolic component, is phosphorylated during neutrophil activation, and the relationship between p40-phox phosphorylation and NADPH oxidase activation. When 32P-labeled guinea pig neutrophils were stimulated by phorbol 12-myristate 13-acetate, p40-phox was phosphorylated as p47-phox. It is interesting that phosphorylation of p40-phox was markedly inhibited by protein kinase C inhibitor, H-7, but not by casein kinase II inhibitor, A-3, and H-7 inhibited translocation of p40-phox and activation of NADPH oxidase. Furthermore, purified protein kinase C but not casein kinase II directly phosphorylated p40-phox of p40-phox/p47-phox/p67-phox complex. Together these observations suggest that p40-phox is phosphorylated by protein kinase C during neutrophil activation, and phosphorylation of p40-phox may be important for the activation of NADPH oxidase.
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PMID:Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase. 1057 19

This study investigates the effects of aliphatic (n-heptane, n-nonane), naphtenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)), and aromatic (methylbenzene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on respiratory burst in human granulocytes. The free radical formation was measured as 2,7-dichlorofluorescein diacetate-amplified (DCF) fluorescence, by electron paramagnetic resonance (EPR) spectroscopy and by hydroxylation of 4-hydroxybenzoate. The chemotactic peptide N-formyl-met-leu-phe (fMLP) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analogue, were included as positive controls. DCF fluorescence was elevated in a concentration-dependent manner by C9 hydrocarbons. The C7 hydrocarbons did not stimulate respiratory burst in the concentration range examined. The naphtenic hydrocarbon TMCH showed the strongest effect on respiratory burst and was therefore selected for mechanistic studies of this free radical formation. In the absence of extracellular Ca(2+), fluorescence in response to TMCH and fMLP was reduced by 77 and 90%, respectively. Preincubation of the granulocytes with the protein kinase C inhibitor bisindolylmaleimide reduced the DCF fluorescence stimulated with TMCH, fMLP, and PMA by 82, 56, and 90%, respectively. The phospholipase C inhibitor U73122 lowered the TMCH- and fMLP-activated DCF fluorescence by 87 and 76%. In addition, the TMCH- and fMLP-induced DCF fluorescence, after the preincubation with the phospholipase D modulator n-butanol, was lowered by 83 and 52%, respectively. The importance of protein kinase C, phospholipase C, and phospholipase D for elevation of respiratory burst was also demonstrated by the EPR experiments using the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Preincubation with the NADPH oxidase inhibitor diphenyleneiodonium and diethyldithiocarbamate, which inhibits superoxide dismutase, led to an almost complete reduction of DCF fluorescence in response to TMCH, fMLP, and PMA. Preincubation with diethyldithiocarbamate led to the elevation of superoxide adducts of DEPMPO. The hydrocarbons stimulated formation of mainly the superoxide (O(*-)(2)) adduct of DEPMPO (DEPMPO-OOH) but also small amounts of the hydroxyl adduct ((*)OH) (DEPMPO-OH). Using 4-hydroxybenzoate as a hydroxyl radical trap confirmed formation of (*)OH after stimulation with the hydrocarbons. In conclusion, our findings indicate that TMCH-activated respiratory burst is dependent on the Ca(2+)-dependent phospholipase C, phospholipase D, and protein kinase C prior to activation of the NADPH oxidase.
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PMID:The effects of aliphatic (n-nonane), naphtenic (1,2, 4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neutrophil granulocytes. 1098 13

Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.
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PMID:The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils. 1104 57


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