Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes.

The oxygen-derived free radical superoxide anion (.O2-) plays an important role in the pathogenesis of various diseases. Recent demonstrations that .O2- inactivates the potent vasodilator endothelium-derived relaxing factor (EDRF) and that EDRF is probably nitric oxide (NO) suggest that EDRF(NO) may act as an endogenous free radical scavenger. This hypothesis was tested in an in vitro system by analyzing the effect of authentic NO (dilutions of a saturated aqueous solution) on .O2- production (detected spectrophotometrically as reduction of cytochrome c) by fMet-Leu-Phe-activated human leukocytes (PMN). NO depressed the rate of reduction of cytochrome c by .O2- released from PMN's or generated from the oxidation of hypoxanthine by xanthine oxidase. This effect was concentration-dependent and occurred at dilutions of the saturated NO solution (1:250 to 1:10) which inhibited platelet aggregation. NO had no direct effect on cytochrome c or on xanthine oxidase. These observations indicate that NO(EDRF) can be regarded as a scavenger of superoxide anion and they suggest that EDRF(NO) may provide a chemical barrier to cytotoxic free radicals (.O2-).     

Concomitant production of nitric oxide and superoxide in human macrophages

Many harmful effects of nitric oxide are caused by the reaction of NO with superoxide anion. The present study was carried out to find out the concomitant production of superoxide and to investigate a suitable inhibitor of NO, which is produced by iNOS. THP-1 cells were differentiated into macrophages by PMA and cytokine. Addition of L-NAME showed decrement in superoxide production. Addition of apocynin, aminoguanidine or ONO 1714 brought about a significant reduction in superoxide production. The expressions of p67 and p47(phox) were reduced by the addition of apocynin, aminoguanidine or ONO 1714 whereas xanthine oxidase and cyclooxygenase did not have a major role in superoxide production. The results of the present study show that iNOS and NADPH oxidase play an important role in superoxide release. It suggests that addition of iNOS inhibitor together with apocynin may be more effective in case of therapeutic application in disease conditions like atherosclerosis.     

Mechanism of Kainate Toxicity to Cerebellar Neurons In Vitro Is Analogous to Reperfusion Tissue Injury

The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate-induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine oxidase, a cellular source of cytotoxic superoxide radicals (O2-.). Moreover, neurons are also protected from excitotoxin-induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin-induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.     

The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties

Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (−)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation.     

Sickle cell disease vasculopathy: a state of nitric oxide resistance

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by microvascular vaso-occlusion with erythrocytes containing polymerized sickle (S) hemoglobin, erythrocyte hemolysis, vasculopathy, and both acute and chronic multiorgan injury. It is associated with steady state increases in plasma cell-free hemoglobin and overproduction of reactive oxygen species (ROS). Hereditary and acquired hemolytic conditions release into plasma hemoglobin and other erythrocyte components that scavenge endothelium-derived NO and metabolize its precursor arginine, impairing NO homeostasis. Overproduction of ROS, such as superoxide, by enzymatic (xanthine oxidase, NADPH oxidase, uncoupled eNOS) and nonenzymatic pathways (Fenton chemistry), promotes intravascular oxidant stress that can likewise disrupt NO homeostasis. The synergistic bioinactivation of NO by dioxygenation and oxidation reactions with cell-free plasma hemoglobin and ROS, respectively, is discussed as a mechanism for NO resistance in SCD vasculopathy. Human physiological and transgenic animal studies provide experimental evidence of cardiovascular and pulmonary resistance to NO donors and reduced NO bioavailability that is associated with vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression, and end-organ injury. Emerging epidemiological data now suggest that chronic intravascular hemolysis is associated with certain clinical complications: pulmonary hypertension, cutaneous leg ulcerations, priapism, and possibly stroke. New therapeutic strategies to limit intravascular hemolysis and ROS generation and increase NO bioavailability are discussed.     

Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-mediated oxidation reactions in vivo

Peroxynitrite (ONOO(-)/ONOOH), the product of the diffusion-limited reaction of nitric oxide (*NO) with superoxide (O(-*)(2)), has been implicated as an important mediator of tissue injury during conditions associated with enhanced *NO and O(-*)(2) production. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized peroxynitrite, others have proposed that the relative rates of *NO and production may be critical in determining the reactivity of peroxynitrite formed in situ (Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., and Grisham, M. B. (1996) J. Biol. Chem. 271, 40-47). In the present study, we examined the mechanisms by which excess O(-*)(2) or *NO production inhibits peroxynitrite-mediated oxidation reactions. Peroxynitrite was generated in situ by the co-addition of a chemical source of *NO, spermineNONOate, and an enzymatic source of O(-*)(2), xanthine oxidase, with either hypoxanthine or lumazine as a substrate. We found that the oxidation of the model compound dihydrorhodamine by peroxynitrite occurred via the free radical intermediates OH and NO(2), formed during the spontaneous decomposition of peroxynitrite and not via direct reaction with peroxynitrite. The inhibitory effect of excess O(-*)(2) on the oxidation of dihydrorhodamine could not be ascribed to the accumulation of the peroxynitrite scavenger urate produced from the oxidation of hypoxanthine by xanthine oxidase. A biphasic oxidation profile was also observed upon oxidation of NADH by the simultaneous generation of *NO and O(-*)(2). Conversely, the oxidation of glutathione, which occurs via direct reaction with peroxynitrite, was not affected by excess production of *NO. We conclude that the oxidative processes initiated by the free radical intermediates formed from the decomposition of peroxynitrite are inhibited by excess production of *NO or O(-*)(2), whereas oxidative pathways involving a direct reaction with peroxynitrite are not altered. The physiological implications of these findings are discussed.     

Cognac polyphenolic compounds increase bradykinin-induced nitric oxide production in endothelial cells

We recently reported that in vitro Cognac polyphenolic compounds (CPC) induce NO-dependent vasorelaxant effects and stimulate cardiac function. In the present study, we aim to investigate the effect of CPC on both nitric oxide (NO) and superoxide anions (O(2)(-)) production in cultured human endothelial cells. In addition, its effect on the bradykinin (BK)-induced NO production was also tested. The role and sources of O(2)(-) in the concomitant effect of BK plus CPC were pharmacologically determined. NO and O(2)(-) signals were measured using electron paramagnetic resonance technique using specific spin trappings. Both, CPC and BK induced an increase in NO production in human endothelial cells. The combination of both further enhanced NO release. The capacity of CPC plus BK to increase NO signal was blunted by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and was enhanced in the presence either of superoxide dismutase or catalase. Moreover, CPC plus BK response was greater after inhibition of either NADPH oxidase by apocynin or xanthine oxidase by allopurinol but it was not affected by rotenone. CPC did not affect O(2)(-) level either alone or after its increase upon lipopolysaccharide treatment. Finally, the capacity of BK alone to increase NO was enhanced either by apocynin or allopurinol. Altogether, these data demonstrate that CPC is able to directly increase NO production without affecting O(2)(-) and enhances the BK-induced NO production in human endothelial cells. The data highlight the ability of BK to stimulate not only NADPH oxidase- but also xanthine oxidase-inhibitor sensitive mechanisms that reduce its efficiency in increasing NO either alone or in the presence of CPC. These results bring pharmacological evidence for vascular protection by CPC via its potentiating effect of BK response in terms of endothelial NO release.     

Mitigation of peroxynitrite-mediated nitric oxide (NO) toxicity as a mechanism of induced adaptive NO resistance in the CNS

During CNS injury and diseases, nitric oxide (NO) is released at a high flux rate leading to formation of peroxynitrite (ONOO^•) and other reactive nitrogenous species, which nitrate tyrosines of proteins to form 3-nitrotyrosine (3NY), leading to cell death. Previously, we have found that motor neurons exposed to low levels of NO become resistant to subsequent cytotoxic NO challenge; an effect dubbed induced adaptive resistance (IAR). Here, we report IAR mitigates, not only cell death, but 3NY formation in response to cytotoxic NO. Addition of an NO scavenger before NO challenge duplicates IAR, implicating reactive nitrogenous species in cell death. Addition of uric acid (a peroxynitrite scavenger) before cytotoxic NO challenge, duplicates IAR, implicating peroxynitrite, with subsequent 3NY formation, in cell death, and abrogation of this pathway as a mechanism of IAR. IAR is dependent on the heme-metabolizing enzyme, heme oxygenase-1 (HO1), as indicated by the elimination of IAR by a specific HO1 inhibitor, and by the finding that neurons isolated from HO1 null mice have increased NO sensitivity with concomitant increased 3NY formation. This data indicate that IAR is an HO1-dependent mechanism that prevents peroxynitrite-mediated NO toxicity in motor neurons, thereby elucidating therapeutic targets for the mitigation of CNS disease and injury.     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions.     

Hyperthermia enhances the cytotoxic effects of reactive oxygen species to Chinese hamster cells and bovine endothelial cells in vitro.

Hyperthermia is under intensive investigation as a treatment for tumors both alone and in combination with other therapeutic agents. Hyperthermia has a profound effect on the function and structural integrity of tumor microvasculature; this has often been cited as a reason for its effectiveness in treatment of tumors. To test the role of hyperthermia in cytotoxic effects of active oxygen species, Chinese hamster, V79, and bovine endothelial cells were treated by the active oxygens, O not equal to 2 and H2O2, generated from the hypoxanthine/purine and xanthine oxidase reactions. It was found that cytotoxicity to V79 cells depends on the concentrations of purine and xanthine oxidase. A high level of cytotoxicity may be initiated in hyperthermia-treated tumors because high xanthine oxidase activity is known to be associated with tumors and endothelial cells, and degradation processes produce high concentrations of xanthine oxidase substrates in tumors. Since the cytotoxic effect can be reduced by the xanthine oxidase inhibitor, allopurinol, and the H2O2 removal enzyme, catalase, the cytotoxic effect in this experimental system is dependent on xanthine oxidase and H2O2. Adding erythrocytes at the same time as purine and xanthine oxidase could also prevent the cytotoxicity. Elevated temperatures stimulated the reaction of purine and xanthine oxidase and resulted in an increased cytotoxic effect. A similar effect is observed in growth inhibition and colony formation in endothelial cells without adding xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and 相似文献   

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Role of nitric oxide and superoxide in acute cardiac allograft rejection in rats

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.     

Phosphatidylinositol 3-kinase and xanthine oxidase regulate nitric oxide and reactive oxygen species productions by apoptotic lymphocyte microparticles in endothelial cells

Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. We have previously shown that MPs from apoptotic T cells induce endothelial dysfunction, but the mechanisms implicated are not completely elucidated. In this study, we dissect the pathways involved in endothelial cells with respect to both NO and reactive oxygen species (ROS). Incubation of endothelial cells with MPs decreased NO production that was associated with overexpression and phosphorylation of endothelial NO synthase (eNOS). Also, MPs enhanced expression of caveolin-1 and decreased its phosphorylation. Microparticles enhanced ROS by a mechanism sensitive to xanthine oxidase and P-IkappaBalpha inhibitors. PI3K inhibition reduced the effects of MPs on eNOS, but not on caveolin-1, whereas it enhanced the effects of MPs on ROS production. Microparticles stimulated ERK1/2 phosphorylation via a PI3K-depedent mechanism. Inhibition of MEK reversed eNOS phosphorylation but had no effect on ROS production induced by MPs. In vivo injection of MPs in mice impaired endothelial function. In summary, MPs activate pathways related to NO and ROS productions through PI3K, xanthine oxidase, and NF-kappaB pathways. These data underscore the pleiotropic effects of MPs on NO and ROS, leading to an increase oxidative stress that may account for the deleterious effects of MPs on endothelial function.     

Mitochondrial nitric oxide synthase, oxidative stress and apoptosis

Nitric oxide (NO) exerts a wide range of its biological properties via its interaction with mitochondria. By competing with O(2), physiologically relevant concentrations of NO reversibly inhibit cytochrome oxidase and decrease O(2) consumption, in a manner resembling a pharmacological competitive antagonism. The inhibition regulates many cellular functions, by e.g., regulating the synthesis of ATP and the formation of mitochondrial transmembrane potential (Delta Psi). NO regulates the oxygen consumption of both the NO-producing and the neighboring cells; thus, it can serve as autoregulator and paracrine modulator of the respiration. On the other hand, NO reacts avidly with superoxide anion (O(2)(-)) to produce the powerful oxidizing agent, peroxynitrite (ONOO(-)) which affects mitochondrial functions mostly in an irreversible manner. How mitochondria and cells harmonize the reversible effects of NO versus the irreversible effects of ONOO(-) will be discussed in this review article. The exciting recent finding of mitochondrial NO synthase will also be discussed.     

Regulation of xanthine oxidase by nitric oxide and peroxynitrite

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.     

Excitatory Amino Acid Release from Rat Hippocampal Slices as a Consequence of Free-Radical Formation

The release of D-[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hydroxyl-radical scavenger D-mannitol, or the superoxide-radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.     

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: evaluation of its role in nitrite and nitric oxide generation in anoxic tissues

In addition to nitric oxide (NO) generation from specific NO synthases, NO is also formed during anoxia from nitrite reduction, and xanthine oxidase (XO) catalyzes this process. While in tissues and blood high nitrate levels are present, questions remain regarding whether nitrate is also a source of NO and if XO-mediated nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-electrode studies were performed. Typical XO reducing substrates, xanthine, NADH, and 2,3-dihydroxybenz-aldehyde, triggered nitrate reduction to nitrite and NO. The rate of nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of nitrite, suggesting stepwise-reduction of nitrate to nitrite then to NO. The molybdenum-binding XO inhibitor, oxypurinol, inhibited both nitrite and NO production, indicating that nitrate reduction occurs at the molybdenum site. At higher xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. The pH dependence of nitrite and NO formation indicate that XO-mediated nitrate reduction occurs via an acid-catalyzed mechanism. With conditions occurring during ischemia, myocardial xanthine oxidoreductase and nitrate levels were determined to generate up to 20 microM nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed nitrate reduction to nitrite and NO occurs and can be an important source of NO production in ischemic tissues.