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)

In this article, the evidence for the involvement of free radicals in some of the gastrointestinal disorders is reviewed. Oxygen radicals are partially reduced oxygen species that include superoxide, and hydroxyl radicals, and hypophthalous acids. Most cells possess numerous antioxidant enzymes and scavengers to protect themselves from these injurious agents; the rate of production of reactive oxygen metabolites may exceed the capacity of the antioxidant defenses thus resulting in tissue damage. The gastrointestinal tract is particularly well endowed with the enzymatic machinery necessary to form large amounts of oxygen radicals. Sources of radicals in the gastrointestinal tract include mucosal xanthine oxidase and NADPH oxidase found in the resident phagocytotic leukocytes (macrophages, neutrophils, eosinophils) of the lamina propria. Other sources of oxygen radicals in the gastrointestinal tract involve ischemia and reperfusion, drug ingestion, diet and radiation therapy. Recent studies have demonstrated the involvement of oxygen radicals following active episodes of small-intestinal ischemia, ulcerative colitis, pancreatitis and gastric ulcer. In contrast to cell antioxidants, control of tissue free radical levels is now pharmacologically feasible and perhaps justified for specific diseases. However, carefully designed and controlled clinical trials are needed.
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PMID:Oxygen radicals: their role in selected gastrointestinal disorders. 186 20

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to approximately 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.
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PMID:Metabolic and biosynthetic alterations in cultured astrocytes exposed to hypoxia/reoxygenation. 790 52

The effect of an inhibitor of protein kinase, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine HCl], and its hydroxylated metabolite, HA1100, on the activation of NADPH oxidase in human neutrophils were studied. Cells were preincubated with each drug for 10 min and then activated by treatment with phorbol myristate acetate (PMA) or formylmethionyl leucyl phenylalanine (FMLP). After activation, the rate of superoxide dismutase-inhibitable reduction of cytochrome c was estimated. HA1077 and HA1100 inhibited the PMA-induced production of O2- by neutrophil NADPH oxidase in a concentration-dependent manner (IC50 = 15 and 24 microM, respectively). The sensitivity of the FMLP-induced production of O2- to these drugs was similar. The production of O2- in 1,25-dihydroxyvitamin D3-treated HL-60 cells, which differentiated to macrophage-like cells, was also inhibited by the drugs. The extent of inhibition by HA1077 was almost the same as that by a calmodulin inhibitor (W-7) and by inhibitors of protein kinase (H-7 and H-8). In a cell-free lysate of neutrophils, the NADPH-dependent production of O2- can be induced by sodium dodecyl sulfate (SDS). HA1077 at 100 microM had only a weak inhibitory effect on the cell-free, SDS-induced production of O2-, an indication that HA1077 inhibits the activation of NADPH oxidase, not the actual activity. The effects of H-7 and H-8 were similar to that of HA1077, whereas W-7 inhibited the production of O2- by the cell-free extract of HL-60 cells. This action of HA1077 could explain, in part, its ability to protect neuronal cells from death after ischemia.
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PMID:Inhibition by the protein kinase inhibitor HA1077 of the activation of NADPH oxidase in human neutrophils. 824 Apr

Lazaroids (21-aminosteroids and 2-methylaminochromans) are a new series of drugs designed and demonstrated to protect against tissue damage after trauma and/or ischemia. It has been suggested that the protective effects of lazaroids are derived from their potent actions to inhibit iron-dependent lipid peroxidation, but whether this is sufficient to explain their therapeutic effects is unknown. In an effort to better understand their mechanism of action, these drugs were tested for other modes of antioxidant activity such as scavenging superoxide and hydroxyl radicals and inhibition of production of oxygen free radicals by human neutrophils stimulated with phorbol myristate acetate. Using an ESR spin-trapping technique, with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for superoxide and hydroxyl radicals, we found that the lazaroids U74500A and U78518F are, at best, weak scavengers of superoxide radicals whereas U78518F is a strong scavenger of hydroxyl radicals. In addition, lazaroids were found to be strong inhibitors (60-80% inhibition at 50 microM) of the superoxide-generating NADPH oxidase of human neutrophils. Inhibition of NADPH oxidase by lazaroids in cell-free systems suggested the action to be on the activated enzyme rather than on the process of activation. This may represent an important mode action of lazaroids and suggests their potential use in ischemic/inflammatory conditions involving oxygen free radical production by activated phagocytic cells.
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PMID:Inhibition of superoxide-generating NADPH oxidase of human neutrophils by lazaroids (21-aminosteroids and 2-methylaminochromans). 838 Oct 5

The continuing study of multiple organ failure (MOF) has led to the development of inflammatory models of tissue injury in contrast to earlier infectious models. This change of focus is in response to more recent clinical observations suggesting that postinjury MOF frequently occurs in the absence of infection. In the alternative "two-hit" inflammatory model that has been proposed, the initial traumatic insult "primes" the inflammatory response such that a delayed, otherwise innocuous, inflammatory insult triggers an exaggerated response. The neutrophil (PMN), being uniquely equipped to cause oxidative tissue injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, has been implicated as an early pivotal player in this model of postinjury MOF. Similar to the "two-hit" inflammatory model, circulating PMNs respond to proinflammatory mediators by becoming primed for enhanced superoxide anion (O2.) production and by increasing adherence to endothelium of organs that are susceptible to PMN-mediated injury. Subsequent proinflammatory insults promote further neutrophil sequestration and activate them for enhanced release of O2.-. The resulting tissue injury can be perpetuated and lead to eventual end-organ damage and failure. In terms of the NADPH oxidase system, PMN priming and activation by various agonists have been well documented in vitro and lead to increased endothelial damage. PMN priming and activation are also operable in an in vivo model of gut ischemia/reperfusion, a surrogate of shock and trauma resuscitation, leading to distant organ damage. Finally, in clinical studies of severely injured trauma patients, PMN priming and activation sequences identify patients at risk for developing MOF with its associated high mortality. Further characterization of the mechanisms that regulate PMN priming and activation in the trauma patient is necessary for the development of new therapeutic interventions designed to block deleterious PMN responses which lead to MOF while not compromising beneficial PMN functions of host defense and tissue repair.
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PMID:Neutrophil priming and activation in the pathogenesis of postinjury multiple organ failure. 877 96

Reactive oxygen species (ROS) play an important role in the pathogenesis of ischemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to anoxia-reoxygenation (A-R). To determine potential sources of intracellular ROS formation in EC in response to A-R, a fluorometric assay based on the oxidation of 2',7'-dichlorofluorescin was used. Intracellular ROS production declined 40% during 6 h of anoxia (P < 0.05). After A-R, the rates of intracellular ROS formation increased to 148 +/- 9% (P < 0.001) that of normoxic EC (100 +/- 3%). In EC exposed to A-R, allopurinol and NG-methyl-L-arginine (L-NMMA), inhibitors of xanthine oxidase (XO) and nitric oxide synthase (NOS), respectively, reduced intracellular ROS formation by 25 +/- 1% (P < 0.001) and 36 +/- 4% (P < 0.01). Furthermore, at low doses (i.e., 20 microM), deferoxamine and diethylenetriaminepentaacetic acid (DTPA) significantly inhibited intracellular ROS formation. However, at 100 microM, only deferoxamine caused further reduction in DCF fluorescence. In summary, EC respond to A-R by generating increased amounts of XO- and NOS-derived intracellular ROS. The inhibition, to a similar extent, caused by allopurinol and L-NMMA, as well as the effect of deferoxamine and DTPA suggest that the ROS detected is peroxynitrite. Based on these findings and previous work, we conclude that EC generate ROS in response to A-R from at least two different sources: a plasma membrane-bound NADPH oxidase-like enzyme that releases H2O2 extracellularly and XO, which generates intracellular O2-, which in turn may react with nitric oxide to form peroxynitrite.
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PMID:Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation. 917 54

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of approximately 70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with these in vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes.
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PMID:Cloning of a putative vesicle transport-related protein, RA410, from cultured rat astrocytes and its expression in ischemic rat brain. 919 52

Extracellular oxygen radicals produced by H9c2 rat heart cells in monolayer cultures during ischemia and subsequent reoxygenation were monitored using the luminol-horseradish peroxidase-enhanced chemiluminescence technique. As expected, the photon count diminishes during ischemia but again rapidly attains normal values following reoxygenation. In the presence of superoxide dismutase, this photon emission is repressed, as is also the case in the presence of diphenylene iodonium, a specific inhibitor of NADPH-oxidase activity. Thus, the conclusion seems justified that H9c2 rat heart cells in monolayer cultures produce superoxide radicals extracellularly due to an NADPH oxidase-like action. In order to characterize this extracellular superoxide-generating system, we determined its sensitivity to increased temperatures, inhibition of protein synthesis and perturbations of cytoskeletal structures. Heat shocks result in a delayed inactivation of the NADPH oxidase activity followed by recovery, the kinetics of which depend on the imposed heat shock temperature. This inactivation is independent of protein synthesis and actin cytoskeletal structures, but the recovery of the enzyme's activity is dependent on these entities.
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PMID:NADPH-oxidase-dependent superoxide production by myocyte-derived H9c2 cells: influence of ischemia, heat shock, cycloheximide and cytochalasin D. 934 74

Respiratory burst activity of murine microglial cells was investigated in vitro under normoxic and hypoxic conditions with a chemoluminometric assay. Hypoxia for 24 hours reduced the release of extracellular reactive oxygen intermediates (ROIs), whereas reoxygenation increased the chemoluminescence more than sevenfold. Blockade of potassium channels inhibited the increase of oxidative burst after reoxygenation, indicating that potassium ions, which were increased in the supernatant of hypoxic microglial cells, were involved in this activation process. Also, blockade of voltage-gated calcium channels with nifedipine attenuated the increased release of ROIs. With fura-2 analysis, it was shown that the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by potassium ions was mediated by calcium influx via voltage-gated calcium channels. Thus, influx of calcium ions through voltage-gated channels activates the NADPH oxidase in microglial cells during reoxygenation. By the increased production of ROIs, microglial cells may add to the reperfusion injury after ischemia in vivo.
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PMID:Reoxygenation increases the release of reactive oxygen intermediates in murine microglia. 962 91

We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.
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PMID:Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. 975 43


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