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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescence probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.
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PMID:Evaluation of oxidative processes in human pigment epithelial cells associated with retinal outer segment phagocytosis. 808 27

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.
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PMID:Role of hydroxyl radicals in the activation of human platelets. 817 49

The respiratory burst reaction, estimated as O2.- production, has been studied in rat peritoneal macrophages of different age (3, 12 and 24 months). To stimulate NADPH oxidase, the enzyme responsible for the respiratory burst, various stimuli that act in different ways have been used: PMA (phorbol myristate acetate), Con-A (concanavalin A) and N-FMLP (N-formyl-methionyl-leucyl-phenylalanine). All produced a decrease in response with age, with that from PMA being the greatest. The PMA-induced decrease in the O2.- production may be related to the inactivation of NADPH-producing enzymes such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase that we have found with age. Glutathione reductase, an enzyme that participates in the maintenance of the redox status in the cell, also showed an age-related decrease. Enzymes that participate in oxygen species scavenging, such as glutathione peroxidase and Cu/Zn superoxide dismutase, did not change with age, although an age-related decrease in catalase activity was found.
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PMID:Respiratory burst reaction changes with age in rat peritoneal macrophages. 821 68

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.
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PMID:Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2. 825 82

Primary cultures of rat Kupffer cells liberated significant amounts of prostaglandin (PG) D2, PGE2, and thromboxane (measured as thromboxane B2) when exposed to reoxygenation after 4 h of hypoxia. After a delayed onset, prostanoids were released at high rates for at least 8 h and after that time 700 pmol PGD2, 280 pmol PGE2, and 200 pmol thromboxane per 10(6) cells had been liberated. Unlike prostanoid release, leukotriene B4 production in reoxygenated cell cultures was only twice as much as in aerobic controls. Superoxide dismutase and catalase had no effect on PGD2, PGE2, and thromboxane production, indicating that prostanoid formation was independent of reactive oxygen species generated extracellularly and of cell injury. On the other hand, diphenyliodonium, as well as amiloride, blocked hypoxia-reoxygenation-induced PGD2, PGE2, and thromboxane release. The elevated prostanoid synthesis was preceded by increases in intracellular pH (from 7.23 to 7.38) and in intracellular Ca2+ (from 55 nM to a maximum level of 807 nM). These observations suggest a participation of NADPH oxidase and a related Na(+)-H+ exchange in the enhanced prostanoid synthesis, probably through the induction of an increased intracellular Ca2+ concentration.
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PMID:Prostanoid release by Kupffer cells upon hypoxia-reoxygenation: role of pHi and Cai2+. 838 98

The results presented in this paper demonstrate that the potentiation of phagocytosis of erythrocyte (E) IgG by TNF-alpha or PMA is not due to an oxygen-dependent mechanism. In fact, the potentiation of phagocytosis occurs normally in human neutrophils 1) when the respiratory burst is inhibited by diphenyleneiodonium, 2) in conditions where the reactive oxygen metabolites produced by the activation of NADPH oxidase, that accompanies the phagocytosis, were removed by catalase or superoxide dismutase, 3) of a patient lacking NADPH oxidase activity due to a genetic defect of p67-phox, 4) treated with staurosporine which allowed PMA to potentiate the ingestion of E-IgG at concentrations which inhibited the activation of the respiratory burst. Evidence is also presented that staurosporine not only did not inhibit, but amplified the potentiation of phagocytosis by PMA and TNF-alpha. This last finding suggests that the activation of protein kinase C plays a modulatory rather than a positive role in the mechanism of potentiation of phagocytosis.
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PMID:The potentiation by TNF-alpha and PMA of Fc receptor-mediated phagocytosis in neutrophils is independent of reactive oxygen metabolites produced by NADPH oxidase and of protein kinase C. 839 10

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the result of the interaction among H2O2, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H2O2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn2+, Fe2+, Cu2+, and Co2+ very markedly enhanced CL induced by mixtures of H2O2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by superoxide dismutase (SOD) and by deferoxamine LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H2O2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.
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PMID:Chemiluminescence in activated human neutrophils: role of buffers and scavengers. 839 91

The activation of human platelets by polymorphonuclear leukocytes (PMN) was investigated in human whole blood challenged with "priming" concentrations of arachidonic acid or collagen in the presence or absence of N-formyl-Met-Leu-Phe (FMLP), a selective activator of PMN. With the use of arachidonic acid or collagen alone at priming concentrations or FMLP alone, no platelet response was observed. In contrast, FMLP in combination with arachidonic acid or collagen caused irreversible platelet aggregation with thromboxane A2 production. Platelet response to FMLP-activated PMN was enhanced by superoxide dismutase and blocked by catalase or the NADPH oxidase inhibitor diphenyliodonium, suggesting a role for the O2-.-H2O2 system in this cellular interaction. This was corroborated by experiments with exogenously added H2O2, which mimicked FMLP effects in the activation of primed platelets in whole blood. The present investigation indicates that platelets primed with minute amounts of arachidonic acid or collagen can be activated, in human whole blood, by oxygen-reactive species released by PMN.
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PMID:Polymorphonuclear leukocyte-derived O2-reactive species activate primed platelets in human whole blood. 849 72

Mice with chronic granulomatous disease (X-CGD mice) generated by mutating the X-linked gene for a subunit of NADPH oxidase have been analyzed for their ability to respond to intravenous injection of purified cobra venom factor (CVF). This agent in wild-type mice produces a neutrophil-dependent and catalase-sensitive form of lung injury. Lung injury was evaluated by measuring the accumulation of extravascular albumin. Quite unexpectedly, the lungs of X-CGD mice showed no difference in the increased accumulation of extravascular albumin after injection of CVF when compared to wild-type mice. In both X-CGD and wild-type mice, full development of injury required neutrophils. While catalase was highly protective in wild-type mice, its protective effects were completely lost in the X-CGD mice. Furthermore, a competitive antagonist of L-arginine, N(G)-methyl-L-arginine, was protective in X-CGD mice but not in wild-type mice. Allopurinol was protective in both types of mice. Both the basal and the CVF-inducible lung mRNA for inducible nitric oxide synthase and IL-1beta was similar in X-CGD and wild-type mice. These data indicate that oxygen radical production and lung injury in response to injection of CVF occurs through alternative pathways in mice with genetic deletion of NADPH oxidase.
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PMID:Preservation of complement-induced lung injury in mice with deficiency of NADPH oxidase. 864 63

Src family tyrosine kinases have been implicated in the adhesion-dependent activation of neutrophil functions (Yan, S. R., Fumagalli, L., and Berton, G. (1995) J. Inflamm. 45, 297-312; Lowell, C. A., Fumagalli, L., and Berton, G. (1996) J. Cell Biol. 133, 895-910). Because the activity of tyrosine kinases can be affected by oxidants, we investigated whether reactive oxygen intermediates (ROI) produced by adherent neutrophils regulate Src family kinase activities. Inhibition of ROI production by diphenylene iodonium, an inhibitor of NADPH oxidase, or degradation of H2O2 by exogenously added catalase inhibited the adhesion-stimulated activities of p58(c-fgr) and p53/56(lyn). In addition, adhesion-stimulated p58(c-fgr) and p53/56(lyn) activities were greatly reduced in neutrophils from patients with chronic granulomatous disease (CGD) that are deficient in the production of ROI. Exogenously added H2O2 increased p58(c-fgr) and p53/56(lyn) activities in nonadherent neutrophils. Although ROI regulated the activities of p58(c-fgr) and p53/56(lyn), they did not affect the redistribution of the two kinases to a Triton X-100-insoluble, cytoskeletal fraction that occurs in adherent neutrophils. Tyrosine phosphorylation of proteins in adherent, CGD neutrophils was only partially inhibited, suggesting that the full activation of p58(c-fgr) and p53/56(lyn), which depends on endogenously produced ROI, does not represent an absolute requirement for protein tyrosine phosphorylation. The adhesion-stimulated activity of the tyrosine kinase p72(syk) was not affected by catalase in normal neutrophils, and it was comparable in normal and CGD neutrophils. These findings suggest that ROI endogenously produced by adherent neutrophils regulate Src family kinases activity selectively and establish the existence of a cross-talk between reorganization of the cytoskeleton, production of ROI, and Src family tyrosine kinase activities in signaling by adhesion.
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PMID:Regulation of Src family tyrosine kinase activities in adherent human neutrophils. Evidence that reactive oxygen intermediates produced by adherent neutrophils increase the activity of the p58c-fgr and p53/56lyn tyrosine kinases. 879 54


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