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)

Cell adhesion to endothelial cells stimulated by tumor necrosis factor-alpha (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antioxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-kappa B (NF-kappa B). Since kappa B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or N-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 mumol/L PDTC) in HUVECs as assessed by flow cytometry and polymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-kappa B mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-kappa B. Since HUVECs released superoxide anions in response to TNF, and H2O2 induces VCAM-1, PDTC may act as a radical scavenger. Although ICAM-1 induction was unaffected, inhibitors of NADPH oxidase (apocynin) or cytochrome P-450 (SKF525a) suppressed VCAM-1 induction by TNF, revealing that several radical-generating systems are involved in its regulation. PDTC, apocynin, or SKF525a decreased adhesion of monocytic U937 cells to TNF-treated HUVECs (by 75% at 100 mumol/L PDTC). Inhibition by anti-VCAM-1 monoclonal antibody 1G11 indicated that U937 adhesion was VCAM-1 dependent and suppression by antioxidants was due to reduced VCAM-1 induction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. 752 48

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

The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved.
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PMID:Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation. 754 22

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

Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.
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PMID:Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain. 764 35

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.
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PMID:Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate. 769 72

This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O2.-, H2O2, and the H2O2-myeloperoxidase (MPO)-halide system. The H2O2-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3'-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H2O2, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H2O2 to yield oxidized DAB, H2O2 was localized to phagolysosomes. The subcellular sites of H2O2 generation were shown using cerium ions which react with H2O2 and precipitate electron opaque cerium perhydroxides (Ce(OH)2OOH and Ce(OH)3OOH). The results suggested that NADPH oxidase is associated with the plasma lemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H2O2 in the phagosome. As O2.- is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O2.- oxidizes Mn2+ to Mn3+, and Mn3+ oxidizes DAB, a medium containing DAB-Mn2+ was used to localize sites of O2.- production in stimulated PMN. The localizations were, as expected, similar to those for H2O2. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.
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PMID:Robert Feulgen Lecture 1994. Cytochemistry and reactive oxygen species: a retrospective. 781 66

Chronic granulomatous disease (CGD) is a rare recessive disorder caused by defects in the NADPH oxidase enzyme complex of phagocytes (neutrophils, eosinophils and monocytes). CGD phagocytes fail to produce superoxide and other reactive oxygen species following cell activation (Malech, 1993). The products of oxidase activation can be measured in individual cells by flow cytometry using specific fluorescent probes that increase fluorescence upon oxidation (Trinkle et al., 1987). This approach can be used to confirm a diagnosis of CGD, and to detect the normal/abnormal phagocyte mixture that characterizes the X-linked CGD carrier state. Three fluorescent probes have been described as useful for this purpose: 2'7'-dichlorofluorescin diacetate (DCF) (Bass et al., 1983), 5,6-carboxy-2'7'-dichlorofluorescein diacetate, bis(acetoxymethyl) ester (C-DCF) (Hockenbery et al., 1993) and dihydrorhodamine 123 (DHR) (Rothe et al., 1988; Kinsey et al., 1987). A direct comparison between these three probes has not been reported. In this study we performed a direct comparison between these three probes, evaluating their ability in flow cytometric analysis to maximize fluorescent separation between activated CGD patient and normal granulocytes. Using a whole blood technique with phorbol myristate acetate (PMA) as an activator, it was found that DHR loaded normal granulocytes had a fluorescence intensity which, upon activation, was 48-fold higher than that of C-DCF loaded granulocytes and seven-fold higher than DCF loaded granulocytes (P < 0.001). Use of sodium azide to decrease the catabolism of H2O2 enhanced the fluorescence of DCF by 140%, C-DCF by 45% and DHR by 25%, suggesting that DCF is primarily sensitive to H2O2. DCF and DHR were then evaluated for sensitivity in the detection of small percentages of normal cells in a CGD/normal granulocyte mixture. Normal sub-populations as small as 0.1% could clearly be distinguished using DHR, while DCF was insensitive at this level. Based on these findings, we used DHR in an effort to detect normal granulocytes in a CGD patient following therapeutic granulocyte transfusion. We were able to detect normal granulocytes in the circulation for up to 18 h after transfusion. With these data we show that DHR is the most sensitive flow cytometric indicator for the detection of oxygen reactive species in activated granulocytes and is the best probe for evaluating CGD patients and carriers. In addition, our data suggest that DHR is a useful tool for monitoring circulating normal granulocytes in CGD patients following transfusion, and potentially will be a sensitive probe for assessing the success of such future technologies as gene therapy for CGD.
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PMID:Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. 782 69

The production of H2O2 by cells in cold paraformaldehyde-fixed frozen sections of inflammatory lesions was histochemically demonstrated by incubating them with diaminobenzidine (DAB) for 2 to 6 h. Catalase (150 micrograms/ml, about 1400 U/ml) inhibited the reaction, indicating that H2O2 was required to produce the chromogenic DAB product. Granulocytes (PMNs and eosinophils) were the main types of cells stained by the DAB reaction. Positive staining of macrophages was less frequent. The H2O2 was produced by metabolic enzymes that were still active after cell death and mild fixation. An atmosphere of 95 to 100% oxygen enhanced the specific DAB reaction, and an atmosphere of 100% nitrogen eliminated it. The DAB histochemical reaction to detect H2O2 requires the presence of peroxidases to produce the colored reaction product. Within our tissue sections, such peroxidases were evidently present in excess, because addition of low concentrations of H2O2 significantly increased the reaction product. Although some of the H2O2 produced by the granulocytes may have been derived from the dismutation of superoxide (O2-), the NADPH oxidase pathway for O2- formation did not seem to be involved: NADPH oxidase, a rather labile enzyme, should not be active after mild fixation, and diphenyleneiodonium (100 microM), an inhibitor of flavine-requiring NADPH oxidase, did not inhibit the reaction. Reactive nitrogen intermediates were also not involved, because NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthetase, did not appreciably inhibit the reaction. We conclude that stable, non-flavine-requiring oxidases, possibly cyclooxygenases or lipoxygenases, produced the H2O2 measured histochemically by our DAB reaction. These studies were made on tissue sections of acute dermal inflammatory lesions produced in rabbits by the topical application of 1% sulfur mustard [bis(2-chloroethyl) sulfide] in methylene chloride. Both intact PMNs and disintegrating PMNs in the base of the crust produced H2O2. Despite the production of H2O2 and the presence of peroxidase activity, no tissue damage was seen microscopically near the H2O2-producing cells, which indicates that the tissues are well protected by the antioxidants present in this self-limiting inflammatory reaction.
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PMID:Histochemical demonstration of hydrogen peroxide production by leukocytes in fixed-frozen tissue sections of inflammatory lesions. 793 Sep 39

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O2-) and H2O2. The production of O2 was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H2O2 production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O2 and H2O2 release in a dose-dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O2 production in zymosan and phorbol ester stimulated cells were found to be 250 microM and 300 microM, respectively and the amounts required to cause one-half optimum levels of H2O2 production in these cells were found to be 50 microM and 100 microM, respectively. The effect of these drugs on O2 producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p < 0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient 'differential phagocytosis' assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O2 and H2O2 by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.
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PMID:Impairment of raw 264.7 macrophage function by antiarrhythmic drugs. 796 98


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