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)

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.
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PMID:Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions. 42 8

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.
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PMID:Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols. 203 43

The early signalling events that may ultimately contribute to the assembly and subsequent activation of the NADPH oxidase in guinea-pig peritoneal eosinophils were investigated in response to leukotriene B4 (LTB4). LTB4 promoted a rapid, transient and receptor-mediated increase in the rate of H2O2 generation that was potentiated by R 59 022, a diradylglycerol (DRG) kinase inhibitor, implicating protein kinase C (PKC) in the genesis of this response. This conclusion was supported by the finding that the PKC inhibitor, Ro 31-8220, attenuated (by about 30%) the peak rate of LTB4-induced H2O2 generation under conditions where the same response evoked by 4 beta-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90%. Paradoxically, Ro 31-8220 doubled the amount of H2O2 produced by LTB4 which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB4-induced Ins(1,4,5)P3 accumulation and the duration of the Ca2+ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB4-induced oxidase activation indicated that phospholipase D (PLD) did not play a major role. Thus, although H2O2 generation was abolished by butan-1-ol, this was apparently unrelated to the inhibition of PLD, as LTB4 failed to stimulate the formation of Ptd[3H]BuOH in [3H]butan-1-ol-treated eosinophils. Rather, the inhibition was probably due to the ability of butan-1-ol to increase the eosinophil cyclic AMP content. In contrast, Ca(2+)- and PLC-driven mechanisms were implicated in H2O2 generation, as LTB4 elevated the Ins(1,4,5)P3 content and intracellular free Ca2+ concentration in intact cells, and cochelation of extracellular and intracellular Ca2+ significantly attenuated LTB4-induced H2O2 generation. Pretreatment of eosinophils with wortmannin did not affect LTB4-induced H2O2 production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB4 activates the NADPH oxidase in eosinophils by PLD- and PtdIns 3-kinase-independent mechanisms that involve Ca2+, PLC and PKC. Furthermore, the activation of additional pathways that do not require Ca2+ is also suggested by the finding that LTB4 evoked a significant respiratory burst in Ca(2+)-depleted cells.
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PMID:Early signalling events implicated in leukotriene B4-induced activation of the NADPH oxidase in eosinophils: role of Ca2+, protein kinase C and phospholipases C and D. 757 12

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 activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.
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PMID:A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase. 1093 44

Immunoglobulin G (IgG) receptors (FcgammaRs) on myeloid cells are responsible for the internalization of immune complexes. Activation of the oxidase burst is an important component of the integrated cellular response mediated by Fc receptors. Previous work has demonstrated that, in interferon-gamma-primed U937 cells, the high-affinity receptor for IgG, FcgammaRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D (PLD), sphingosine kinase, and calcium transients. Here, it is shown that both known PLD isozymes, PLD1 and PLD2, were present in these cells. With the use of antisense oligonucleotides to specifically reduce the expression of either isozyme, PLD1, but not PLD2, was found to be coupled to FcgammaRI activation and be required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, coupling of FcgammaRI to activation of the nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase burst was inhibited by pretreating cells with 0.3% butan-1-ol, indicating an absolute requirement for PLD. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required to couple FcgammaRI to the activation of NADPH oxidase and trafficking of internalized immune complexes for degradation. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by FcgammaRI and its functional role in coordinating the response to antigen-antibody complexes.
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PMID:Functional coupling of FcgammaRI to nicotinamide adenine dinucleotide phosphate (reduced form) oxidative burst and immune complex trafficking requires the activation of phospholipase D1. 1171 83

Cyclic AMP affects microvascular smooth muscle contraction and growth. Therefore, it is important to elucidate mechanisms regulating cyclic AMP production in microvascular smooth muscle. In this study, we determined whether several signal transduction pathways regulate receptor-induced cyclic AMP in isolated preglomerular microvessels and microvascular smooth muscle cells. Preglomerular microvessels were incubated with isoproterenol (beta-adrenoceptor agonist) and with and without U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), 1-butanol (phospholipase D inhibitor), CGP77675 (c-src inhibitor), HA1077 (Rho kinase inhibitor), Y27632 (Rho kinase inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), dipenyleneiodonium (NADPH oxidase inhibitor), or Tempol (superoxide dismutase mimetic). Cultured preglomerular microvascular smooth muscle cells were incubated with isoproterenol or forskolin (direct activator of adenylyl cyclase) and with or without U73122, C(2)-ceramide (phospholipase D inhibitor), or PP1 [src family inhibitor, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine]. All studies were conducted with 3-isobutyl-1-methylxanthine (broad-spectrum phosphodiesterase inhibitor) to eliminate changes in cyclic AMP degradation. In microvessels isoproterenol-induced cyclic AMP was not affected by Y27632, HA1007, LY294002, dipenylene-iodonium, or Tempol; was increased by U73122 and GF109203X; and was decreased by 1-butanol and CGP77675. In cells, U73122 increased and C(2)-ceramide and PP1 decreased isoproterenol-induced cyclic AMP. Forskolin-induced cyclic AMP was not altered. These results indicate that receptor-mediated activation of adenylyl cyclase is 1) not modulated by Rho kinase, phosphatidylinositol-3-kinase, NADPH oxidase, or superoxide; 2) is attenuated by phospholipase C and protein kinase C; and 3) is augmented by phospholipase D and src. Phospholipase C, phospholipase D, and src modulate receptor-induced cyclic AMP by affecting beta-adrenoreceptor/G protein/adenylyl cyclase coupling rather than by directly affecting adenylyl cyclase activity.
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PMID:Modulation of cyclic AMP production by signal transduction pathways in preglomerular microvessels and microvascular smooth muscle cells. 1508 74

Wheat seedlings (Triticum durum Desf.) were incubated in 100muM Cu(2+) for different periods of time ranging from 1min up to 16h. Following metal addition a rapid intake of copper ions into the roots was observed. Cu(2+) induced an accumulation of both phosphatidic acid and phosphatidylbutanol within 1min of incubation, the latter indicating a very rapid induction of phospholipase D (PLD) activity. The highest PLD stimulation was detected after 2h from copper addition and decreased almost to the initial value at increasing times. Cycloheximide treatment of roots lowered phosphatidylbutanol accumulation because of a reduced PLD activity. The expression profile of a T. durum putative PLD-encoding gene showed a peak after 1h of treatment as well, indicating that enhanced gene expression contributed to the increase in PLD activity. In the absence of copper ions, roots treated with the G protein activator mastoparan showed increases in phosphatidic acid and phosphatidylbutanol similar to those detected with the metal. PLD activity was also stimulated by cholera toxin. Two putatively G protein alpha subunit encoding sequences were isolated and no significant differences in transcription activity following Cu(2+) addition were observed. In copper-treated roots an early production of superoxide generated both by total and membrane-bound NADPH oxidase occurred. The G protein inhibitor suramin as well as the PLD antagonist 1-butanol abolished copper-induced superoxide production.
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PMID:Copper excess triggers phospholipase D activity in wheat roots. 1676 89

Tobacco BY-2 suspension cells were used to study the chemical damage and its associated mechanisms caused by Cu2+. Treatment with 100 micromol/L Cu2+ generated a large amount of H2O2 and thiobarbituric acid-reactive substances (TBARS) in cells. Using phospholipase D (PLD) specific inhibitor (1-butanol) or phosphatidic acid (PA), we demonstrated that PLD plays an important role in the generation of H2O2 and TBARS. Semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme activity assays with wild type and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-overexpressing BY-2 cells revealed that PLD and PA are the key factors leading to NADPH oxidase activation, which is responsible for H2O2 and TBARS production induced by Cu2+. Moreover, the content of ascorbic acid (AsA), an effective antioxidant, was sharply reduced in BY-2 cells exposed to excessive Cu2+. Furthermore, a significant downregulation of the enzymes of AsA biosynthesis and the antioxidant system was found. This evidence suggests that excessive Cu2+-elevated reactive oxygen species (ROS) production is caused by upregulated PLD that elevates the activity of NADPH oxidase and its collapsed antioxidant systems that scavenges ROS.
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PMID:Excessive copper induces the production of reactive oxygen species, which is mediated by phospholipase D, nicotinamide adenine dinucleotide phosphate oxidase and antioxidant systems. 1871 37

To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelial cells, A549 cells were exposed to H(2)O(2) (500 microM) which resulted in time-dependent injury and bi-phasic increase of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation, attenuated cell injury at 5 min of H(2)O(2) exposure, but enhanced injury at 3h of exposure. This activation was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimicked the effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in cell viability induced by H(2)O(2) exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) and quinacrine (phospholipase A2 inhibitor) had weak effects on H(2)O(2)-induced PLD activation but reversed H(2)O(2)-induced injury. We speculate that PLD activation at the initiation of H(2)O(2) exposure predominantly results in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survival as the H(2)O(2) attack continues, which might be mainly due to the accumulation of intracellular phosphatidic acid.
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PMID:Activation of phospholipase D involved in both injury and survival in A549 alveolar epithelial cells exposed to H2O2. 2041 98


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