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:1.17.3.2 (xanthine oxidase)
8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (.NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the NG-methyl-L-arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of L-[3H]arginine to L-[3H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 microgram/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 microgram/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O2.-) scavenger superoxide dismutase. Incubation of the cells with both the .NO donor S-nitroso-N-acetylpenicillamine and the O2.(-)-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between .NO and O2.-, to form the peroxynitrite anion (ONOO-), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO- derivative hydroxyl radical (.OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO- caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO-). The pathophysiological relevance of ONOO(-)-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.
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PMID:Induction of nitric oxide synthase inhibits gap junction permeability in cultured rat astrocytes. 878 40

We investigated the role of nitric oxide (NO) in the development of gastric mucosal lesions induced by serotonine (5-HT) in rats. Repeated subcutaneous administration of 5-HT (20 mg kg-1) produced damage in the stomach with severe edema in the submucosa. Gastric lesions induced by 5-HT were prevented by simultaneous administration of aminoguanidine, a selective inducible NO synthase (iNOS) inhibitor, as well as by methysergide, a 5-HT antagonist. In addition, the lesions were inhibited by pretreatment with the antioxidative drugs, such as allopurinol (a xanthine oxidase inhibitor) and hydroxyurea (a neutrophil reducing agent). Following 5-HT treatment, the Ca(2+)-independent NOS activity in the gastric mucosa was significantly increased within 6 h and remained elevated for 2 days thereafter. The serum NOx levels increased 12 h after the administration of 5-HT, reaching a peak 24 h later. Gastric mucosal thiobarbituric acid (TBA) reactants and myeloperoxidase (MPO) activity were also significantly increased after 2 days treatment with 5-HT. Our results suggest that: (1) the repeated administration of 5-HT induced inflammatory gastric lesions in the rat stomach; (2) iNOS is upreguated during 5-HT treatment, and NO produced by iNOS contributes to development of gastric lesions in response to 5-HT, in addition to the oxyradical formation, and (3) the deleterious role of NO in this model may be accounted for by a cytotoxic action of peroxynitrite that is formed in the presence of NO and superoxide radicals.
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PMID:Role of nitric oxide in pathogenesis of serotonine-induced gastric lesions in rats. 942 24

Intracellularly generated reactive species of both oxygen (ROS) and nitrogen (RNS) have been implicated in signaling responses in airway epithelial cells, but these radicals have not been measured directly in such cells. In this study, intracellular production of both ROS and RNS were measured in the same cell lysates of guinea pig tracheal epithelial (GPTE) cells maintained in primary culture. ROS and RNS were quantified under basal (constitutive) conditions and in response to different stimuli: LPS and TNFalpha [activators of inducible nitric oxide synthase (iNOS)]; several activators of calcium-dependent cNOS (ATP, bradykinin, ionophore A23187, and thapsigargin); and exogenous oxidant stress generated by addition of xanthine oxidase to purine (p + XO). Studies with LPS and TNFalpha also were performed using the murine macrophage cell line, RAW 264.7, as a positive control. Intracellular oxidant production was detected from oxidation of dihydrorhodamine to rhodamine. NOx was quantified by either chemiluminescent or fluorescent detection. NOS activity was measured as citrulline production from arginine. Basal production of oxidants by GPTE cells (0.08 + 0.00 nmol rhodamine) was less than 10% that of RAW.267 cells (0.91 + 0.03 nmol rhodamine). TNFalpha and LPS significantly increased intracellular oxidant production in GPTE cells, as did p + XO, but none of the cNOS activators affected production of oxidants in these cells. Concentrations of NO2 after 4 h in unstimulated RAW 264.7 and GPTE cells were similar and comprised 63% of total NOx in GPTE and 62% in RAW cells. TNFalpha and LPS both increased NO2 in GPTE cells, but none of the Ca++-mobilizing agents nor p + XO significantly affected intracellular RNS. The results suggest both ROS and RNS can be measured in the same lysates from airway epithelial cells, and that both ROS and RNS are produced in these cells in response to different stimuli.
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PMID:Concurrent production of reactive oxygen and nitrogen species by airway epithelial cells in vitro. 958 18

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy for pulmonary hypertension, suggesting that inhaled NO may downregulate basal NO production. However, the exact mechanism of this rebound pulmonary hypertension remains unclear. The objectives of these studies were to determine the effect of NO exposure on endothelial NO synthase (eNOS) gene expression, enzyme activity, and posttranslational modification in cultured pulmonary arterial endothelial cells. Sodium nitroprusside (SNP) treatment had no effect on eNOS mRNA or protein levels but did produce a significant decrease in enzyme activity. Furthermore, although SNP treatment induced protein kinase C (PKC)-dependent eNOS phosphorylation, blockade of PKC activity did not protect against the effects of SNP. When the xanthine oxidase inhibitor allopurinol or the superoxide scavenger 4,5-dihydroxy-1-benzene-disulfonic acid were co-incubated with SNP, the inhibitory effects on eNOS activity could be partially alleviated. Also, the levels of superoxide were found to be elevated 4.5-fold when cultured pulmonary arterial endothelial cells were exposed to the NO donor spermine/NO. This suggests that NO can stimulate xanthine oxidase to cause an increase in cellular superoxide generation. A reaction between NO and superoxide would produce peroxynitrite, which could then react with the eNOS protein, resulting in enzyme inactivation. This mechanism may explain, at least in part, how NO produces NOS inhibition in vivo and may delineate, in part, the mechanism of rebound pulmonary hypertension after withdrawal of inhaled NO.
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PMID:Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide. 961

We have reported that peroxynitrite (PON) selectively inactivated prostacyclin synthase (PGIS) by a mechanism of tyrosine nitration at the active site [Zou, Martin and Ullrich (1997) Biol. Chem. Hoppe-Seyler 378, 707-713]. We have now extended our studies on rat mesangial cells (RMC) and show that nitration can occur under the influence of cytokines. Pretreatment of RMC with interleukin 1beta (IL-1beta), which up-regulated cyclo-oxygenase 2 and inducible nitric oxide synthase (NOS-2), significantly attenuated the conversion of [14C]prostaglandin H2 (PGH2) into the stable prostacyclin (PGI2) metabolite 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha). The presence of superoxide dismutase (SOD, 100 units/ml) or the NOS synthase inhibitor Nomega-monomethyl-l-arginine (100 microM) as well as cycloheximide (10 microM) plus actinomycin (10 microM) abolished IL-1beta-mediated down-regulation of 6-oxo-PGF1alpha from PGH2. At the same time, 6-oxo-PGF1alpha production from arachidonate (AA) increased at the expense of prostaglandin E2 (PGE2). Neither NO alone generated from different NO donors nor superoxide from xanthine/xanthine oxidase (1-100 m-units/ml) inhibited PGI2 synthesis, either from PGH2 or from AA. Bolus additions of chemically synthesized PON or the PON generator 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) exhibited a potent inhibition of 6-oxo-PGF1alpha release from both PGH2 and AA. In addition, immunoprecipitation of nitrotyrosine-containing proteins from PON- and SIN-1-treated RMC yielded distinct nitrated PGIS bands but also from IL-1beta-pretreated cells alone, compared with a lack of nitrated PGIS in control cells. Taken together, our results strongly suggest that IL-1beta pretreatment of RMC via NOS-2 leads to the production of PON with the consequence of a partial nitration and inhibition of PGIS.
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PMID:Interleukin 1beta decreases prostacyclin synthase activity in rat mesangial cells via endogenous peroxynitrite formation. 982 Aug 30

Brain ischemia initiates a complex cascade of metabolic events, several of which involve the generation of nitrogen and oxygen free radicals. These free radicals and related reactive chemical species mediate much of damage that occurs after transient brain ischemia, and in the penumbral region of infarcts caused by permanent ischemia. Nitric oxide, a water- and lipid-soluble free radical, is generated by the action of nitric oxide synthases. Ischemia causes a surge in nitric oxide synthase 1 (NOS 1) activity in neurons and, possibly, glia, increased NOS 3 activity in vascular endothelium, and later an increase in NOS 2 activity in a range of cells including infiltrating neutrophils and macrophages, activated microglia and astrocytes. The effects of ischemia on the activity of NOS 1, a Ca2+-dependent enzyme, are thought to be secondary to reversal of glutamate reuptake at synapses, activation of NMDA receptors, and resulting elevation of intracellular Ca2+. The up-regulation of NOS 2 activity is mediated by transcriptional inducers. In the context of brain ischemia, the activity of NOS 1 and NOS 2 is broadly deleterious, and their inhibition or inactivation is neuroprotective. However, the production of nitric oxide in blood vessels by NOS 3, which, like NOS 1, is Ca2+-dependent, causes vasodilatation and improves blood flow in the penumbral region of brain infarcts. In addition to causing the synthesis of nitric oxide, brain ischemia leads to the generation of superoxide, through the action of nitric oxide synthases, xanthine oxidase, leakage from the mitochondrial electron transport chain, and other mechanisms. Nitric oxide and superoxide are themselves highly reactive but can also combine to form a highly toxic anion, peroxynitrite. The toxicity of the free radicals and peroxynitrite results from their modification of macromolecules, especially DNA, and from the resulting induction of apoptotic and necrotic pathways. The mode of cell death that prevails probably depends on the severity and precise nature of the ischemic injury. Recent studies have emphasized the role of peroxynitrite in causing single-strand breaks in DNA, which activate the DNA repair protein poly(ADP-ribose) polymerase (PARP). This catalyzes the cleavage and thereby the consumption of NAD+, the source of energy for many vital cellular processes. Over-activation of PARP, with resulting depletion of NAD+, has been shown to make a major contribution to brain damage after transient focal ischemia in experimental animals. Neuronal accumulation of poly(ADP-ribose), the end-product of PARP activity has been demonstrated after brain ischemia in man. Several therapeutic strategies have been used to try to prevent oxidative damage and its consequences after brain ischemia in man. Although some of the drugs used in early studies were ineffective or had unacceptable side effects, other trials with antioxidant drugs have proven highly encouraging. The findings in recent animal studies are likely to lead to a range of further pharmacological strategies to limit brain injury in stroke patients.
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PMID:Oxidative stress in brain ischemia. 998 55

The effect of the induction of i-NOS in primary glial cultures was studied with respect to the protein levels of reactive oxygen species (ROS) scavenging enzymes and the cytotoxicity of nitric oxide (.NO) formation at different levels of artificially generated superoxide. Stimulation of the cultures by bacterial lipopolysaccharides and gamma-interferon resulted in an induction of i-NOS exclusively in microglial cells. Among the ROS scavenging enzymes superoxide dismutase (Cu/Zn- and Mn-isoform), glutathione peroxidase and catalase only mitochondrial Mn-SOD was found to be upregulated in the course of i-NOS induction (Western blots). Although .NO formation did not affect cell viability at physiological levels of superoxide over a time period of 4 days, it caused an oxidative load particularly in microglial cells as observed by monitoring the oxidation of dichloro-dihydrofluorescein, an indicator for the formation of peroxynitrite and ROS. Elevated levels of superoxide, generated either intracellularly by paraquat or extracellularly via xanthine oxidase and hypoxanthine, resulted dose-dependently in a larger decline of cell viability in the .NO forming cultures compared to controls (release of lactate dehydrogenase, citrate synthase, stainability by propidium iodide, and tetramethylrhodamine). NOS-inhibitors reduced the degree of cell damage to that seen for control cultures, indicating an ONOO--/.NO mediated mechanism of cell damage. Our data support the concept that i-NOS catalyzed .NO-formation leads to an ONOO--mediated increased oxidative load. At physiological levels of superoxide and within a wide range of higher superoxide levels this nitrosative stress is well balanced in cultured glial cells by protective mechanisms.
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PMID:Peroxynitrite mediated damage and lowered superoxide tolerance in primary cortical glial cultures after induction of the inducible isoform of NOS. 1049 18

Although endothelium-derived hyperpolarizing factor (EDHF) is thought to be a cytochrome P-450 product (arachidonic acid metabolite) in some tissues, in porcine coronary arteries (PCAs) its nature remains unclear. Because phospholipase A2 and C are involved in the synthesis and/or release of EDHF in the PCA, the arachidonic acid (AA) pathway may be involved. In the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) and the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), both bradykinin (BK; 10(-9)-10(-6) M) and AA (10(-7)-10(-4) M) induced dose-dependent relaxation of PGF2alpha-contracted PCA rings, which was blocked by a high extracellular concentration of KCl (30 mM) or pretreatment with ouabain, a Na+/K+-adenosine triphosphatase (ATPase) inhibitor (5 x 10(-7) M). Eicosatetraynoic acid (ETYA; 20 microM), which inhibits all AA pathways, slightly affected the response to BK and AA; however, lipoxygenase or cytochrome P-450 inhibitors had no effect, suggesting that relaxation is independent of these enzymatic pathways. Because endothelial cells can generate reactive oxygen species (ROS) via metabolism of AA and independent of cyclooxygenase activity, we also studied (a) whether ROS can relax the PCA, as well as the mechanism(s) involved, and (b) the role of ROS in BK- and AA-induced relaxation. Xanthine (X; 100 microM) plus xanthine oxidase (XO; 0.02 U/ml) induced time-dependent relaxation of PGF2alpha-contracted PCA rings in the presence of indomethacin and L-NAME. Dilatation was not affected by superoxide dismutase (SOD; 500 U/ml) but was abolished by catalase (300 U/ml), suggesting that hydrogen peroxide (H2O2) is involved. When rings were contracted by depolarizing them with 30 mM KCl, X/XO failed to elicit relaxation. Ouabain abolished the response to X/XO, suggesting that X/XO may induce relaxation by hyperpolarizing vascular smooth muscle cells via stimulation of the Na+/K+-ATPase pump. We therefore questioned whether ROS might be involved in BK- and AA-induced relaxation. Because catalase combined with SOD had little or no effect, we concluded that in the PCA, the relaxation induced by BK via EDHF involves some mechanism independent of NO, AA metabolism, or ROS.
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PMID:Reactive oxygen species: role in the relaxation induced by bradykinin or arachidonic acid via EDHF in isolated porcine coronary arteries. 1051 Nov 33

1 In order to understand mechanisms that limit the safe ischaemic time of donor hearts, this study evaluated NO/cyclic GMP biosignalling in the recovery of function after cardioplegia and hypothermic storage. 2 Hearts removed from anaesthetized rats were either perfused in working mode (Fresh) or arrested (St. Thomas' II cardioplegia) and stored at 3 degrees C for 8 h (CPL) prior to working mode perfusion. LV work and indices of the production of NO (Ca2+-dependent and Ca2+-independent NOS), cyclic GMP (soluble guanylyl cyclase (sGC) and GTP) and superoxide (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)) were measured. 3 Relative to Fresh hearts, CPL hearts were deficient in cyclic GMP and had poor function. Correction of cyclic GMP deficiency (SNP, 200 microM) improved LV work and LV compliance. SNP effects were prevented by inhibition of sGC (ODQ, 3 microM), and potentiated by inhibition of cyclic GMP-dependent phosphodiesterase (zaprinast, 20 microM). SNP (200 microM) had no effect on function of Fresh hearts. 4 NOS activities (pH = 7.2) were similar in CPL and Fresh hearts, but at end-ischaemic pH (6.3), Ca2+-dependent NOS activity was reduced. The sensitivity of sGC to SNP was greater, and activities of XO and XDH were higher, in CPL than in Fresh hearts. 5 The deficiency in NO biosignalling in CPL hearts may arise due to acidosis-induced inhibition of NOS activity, reduced availability of GTP and/or enhanced inactivation of NO by superoxide. These findings provide rationales for novel strategies to prevent the deficiency in NO biosignalling and so improve the function of the transplanted heart.
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PMID:Deficiency in myocardial NO biosignalling after cardioplegic arrest: mechanisms and contribution to post-storage mechanical dysfunction. 1055 23

Iodonium compounds, especially diphenylene iodonium and iodonium diphenyl are used extensively as inhibitors of NADH-ubiquinone reductase and NADPH oxidase activity. Here, the use of a new iodonium compound, phenoxaiodonium is reported. The IC(50) of neutrophil superoxide production, measured using the superoxide dismutase inhibitable rate of cytochrome c reduction, was approximately 0.75 microM, while 50% inhibition of mitochondrial respiration, measured by the rate of oxygen uptake using a Clark type oxygen electrode, was at approximately 20 microM. The inhibition of oxidation of xanthine to urate by xanthine oxidase was also studied, giving a K(i) of 0.2 microM. Inhibition of nitric oxidase synthase (NOS: from rat brain) by 0.2 microM phenoxaiodonium was equivalent to 1 mM N(G)-nitro-L-arginine methyl ester HCl (L-NAME), that is total abolition of activity. We conclude that phenoxaiodonium is an extremely good inhibitor of flavo-enzymes, but like diphenylene iodonium and iodonium diphenyl, will be of limited use as a pharmacological tool for the elucidation of the involvement of such enzymes in specific cellular functions.
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PMID:The inhibition of flavoproteins by phenoxaiodonium, a new iodonium analogue. 1092 15


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