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H Ischiropoulos

Publications and source records attributed to H Ischiropoulos.

At least 73 records · Page 4Linked to original sources
PubMed1995

Peroxynitrite stimulates vascular smooth muscle cell cyclic GMP synthesis.

Peroxynitrite stimulated the synthesis of cyclic GMP by rat aortic smooth muscle in a time- and dose-dependent manner. Peak formation of cyclic GMP occurred at 1 min with 100 microM peroxynitrite and was inhibited by oxyhemoglobin. Peroxynitrite was less potent than nitric oxide in stimulating cyclic GMP synthesis. Peroxynitrite also enhanced endothelial-dependent cyclic GMP synthesis, via generation of a long-lived substance, which was prevented by inhibition of glutathione synthesis. These data show that peroxynitrite stimulates cyclic GMP synthesis, inferring production of low yields of nitric oxide or associated derivatives. Additionally, vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase.

Animals↗
PubMed1995

Endotoxin triggers the expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in the rat aorta in vivo.

The free radicals nitric oxide (.NO) and superoxide (O2-) are known to react to form peroxynitrite (ONOO-), a highly reactive species. Peroxynitrite has been suggested to play an important role in the cellular damage associated with the overproduction of .NO, but there are very limited data regarding its in vivo formation. Here we demonstrate that injection of endotoxin into rats leads to the expression of an inducible isoform of .NO synthase (iNOS) in the thoracic aorta at 6 h and an increase in the circulating levels of nitrite/nitrate. Moreover, at the same time point, there is a marked increase in the immunoreactivity of nitrotyrosine, a marker of peroxynitrite in the aorta. The formation of nitrotyrosine was prevented by inhibiting the activity of NOS by NG-methyl-L-arginine in vivo. Our data suggest that during endotoxin shock, part of .NO, produced following the induction of iNOS, is converted into peroxynitrite in the vicinity of large blood vessels. The demonstration of the in vivo formation of peroxynitrite at sites of .NO overproduction may necessitate the development of novel and additional approaches for limiting or preventing .NO-related cytotoxic or vasodilatory actions during circulatory shock.

Amino Acid Oxidoreductases↗
PubMed1995

Endotoxin-stimulated nitric oxide production increases injury and reduces rat liver chemiluminescence during reperfusion.

BACKGROUND/AIMS: Nitric oxide has many physiological functions and may play an important role in modulating tissue injury. However, the mechanism of NO action in ischemia/reperfusion injury is completely unknown. This report investigates the role of NO in hepatic reperfusion injury. METHODS: Rat liver was oxygenated for 30 minutes, followed by 30 minutes of ischemia, and then reperfused for 30 minutes. Perfusate was sampled for aspartate aminotransferase content, as an indication of hepatic injury, and for nitrite, an index of NO production. Spontaneous organ chemiluminescence was continuously monitored as a measure of oxyradical production. RESULTS: NO production by the perfused rat liver was induced in vivo by pretreatment with Escherichia coli lipopolysaccharide. This induction led to an increase in hepatic injury during reperfusion that was partially ameliorated by the NO synthase inhibitor NG-monomethyl-L-arginine. Chemiluminescence during reperfusion, a measure of superoxide production in this system, was also decreased in the lipopolysaccharide-treated animals, and this effect was blunted by NG-monomethyl-L-arginine. CONCLUSIONS: These data suggest that NO may combine with superoxide formed during reperfusion to directly cause hepatocellular injury. In vitro work shows that this chemical product is the highly toxic species peroxynitrite.

Animals↗
PubMed1995

Peroxynitrite-mediated inhibition of DOPA synthesis in PC12 cells.

Experimental evidence has implicated oxidative stress in the development of Parkinson's disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion-limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant-induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinson's disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3-morpholinosydnonimine (SIN-1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN-1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN-1-mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN-1 persisted even after removal of SIN-1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.

Animals↗
PubMed1995

Reactive species in ischemic rat lung injury: contribution of peroxynitrite.

Lung ischemia-reperfusion represents a potentially important mechanism for diverse forms of tissue injury associated with decreased pulmonary flow. Previous studies demonstrated oxidative injury in ischemic-reperfused lungs. The present study was designed to evaluate the contribution of nitric oxide and peroxynitrite in tissue injury. The levels of the stable decomposition products of nitric oxide and peroxynitrite, nitrite plus nitrate, were twofold greater than control during reperfusion after 60 min of ischemia. Inhibition of nitric oxide synthesis by endotracheal insufflation of 5 mM NG-nitro-L-arginine methyl ester, 30 min before the induction of ischemia, decreased the production of lung thiobarbituric acid reactive substances (TBARS) by 67% (P < 0.05, n = 5), TBARS released into the lung perfusate by 55% (P < 0.05, n = 5), lung-conjugated dienes by 61% (P < 0.05, n = 5), and dinitrophenylhydrazine-reactive protein carbonyl levels by 86% (P < 0.05, n = 5). Amino acid analysis of tissue homogenates from lungs exposed to 60 min of ischemia and 60 min of reperfusion revealed a 1.8-fold (P < 0.05, n = 5) increase in nitrotyrosine concentration compared with 2 h continuously perfused lungs. Inhibition of nitric oxide synthesis abolished the increase in nitrotyrosine levels. Furthermore, lungs exposed to 60 min of reperfusion after 60 min of ischemia showed specific binding of an anti-nitrotyrosine antibody. In reperfused tissues, antibody binding was observed throughout the lung. The binding was blocked with excess of nitrotyrosine, and minimal binding was observed in nonperfused blood-free control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Nitric oxide released by platelets inhibits neutrophil B2 integrin function following acute carbon monoxide poisoning.

Carbon monoxide (CO) poisoning has been reported to temporarily inhibit B2 integrin adherence molecules on leukocytes in previous studies in a rat model. The aim of this study was to investigate the mechanism for this effect. Studies were conducted using blood obtained from rats after they were exposed to CO and also with blood cells exposed to CO in vitro. Initial investigations indicated that inhibition of neutrophil (polymorphonuclear leukocyte, PMN) B2 integrin function was linked to the platelets in blood, as the effect was resolved by decreasing platelet number before PMN adherence was tested. The platelet effect could also be shown by incubating either platelet-rich plasma or whole blood with CO in vitro. The effect of platelets was blocked by superoxide radicals and by NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase. These observations suggested that CO caused platelets to release NO, an agent known to inhibit the function of B2 integrins. The concentration of NO measured in suspensions of platelets from rats poisoned with CO according to the established model (exposure to 1000 ppm CO for 40 min and 3000 ppm CO for 20 min) was 47 nmol/10(8) platelets, in contrast to only 0.3 nmol NO/10(8) platelets from control rats. Enhanced NO release occurred despite a 60% inhibition of NO synthase activity, assessed by measuring conversion of [14C]L-arginine to citrulline. Exposure to only 1000 ppm CO for 1 hr caused platelets to release 74 nmol NO/10(8) platelets, and no inhibition of NO synthase occurred. Enhanced NO release, and inhibition of PMN adherence, did not occur after platelets were exposed to light from a quartz lamp to photodissociate CO from heme proteins. The data suggest that the NO flux from platelets increased when CO became bound to heme-containing platelet proteins, which normally scavage intraplatelet NO and thus prevent diffusion beyond the platelet membrane.

Amino Acid Oxidoreductases↗
PubMed1994

On the pH-dependent yield of hydroxyl radical products from peroxynitrite.

Nitric oxide reacts rapidly with superoxide to give the strongly oxidizing peroxynitrite anion (ONOO-), which undergoes spontaneous first-order decomposition when protonated. The oxidative chemistry of peroxynitrite (ONOO-) is highly pH-dependent. At acidic pH, peroxynitrous acid (ONOOH) oxidizes dimethylsulfoxide to formaldehyde and 2,2'-azino-bis-(3-ethyl-1,2- dihydrobenzothiazoline 6-sulfonate) (ABTS) to the greenish-colored ABTS+ radical cation. The product yield from dimethylsulfoxide and ABTS decreased at more alkaline pH with apparent pK(a)s of 7.9 and 8.2, respectively. Decreasing yield with increasing pH could not be explained by the oxidation of either formaldehyde or ABTS+ by peroxynitrite. In the presence of 50 mM dimethylsulfoxide, nitrogen dioxide was formed in approximately equimolar amounts to the other reaction product, formaldehyde. The yield of nitrogen dioxide also decreased with an apparent pK(a) of 8.0. We propose that the complex oxidative chemistry of peroxynitrite is controlled by the pH-dependent isomerization of the relatively stable cis-configuration (predominant at high pH) to the trans-configuration. Trans-peroxynitrous acid can form a vibrationally excited intermediate capable of reacting like hydroxyl radical. The vibrationally excited intermediate can also directly rearrange to nitric acid, reducing the apparent hydroxyl radical yield to less than 30%. The loss of hydroxyl radical-like reactivity can be explained on the basis of ionization of trans-peroxynitrous acid to the trans-anion, which in turn undergoes internal rearrangement to nitrate without forming a strong oxidant.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzothiazoles↗
PubMed1994

Peroxynitrite-mediated oxidation of dihydrorhodamine 123.

Nitric oxide reacts with superoxide to form peroxynitrite, which may be an important mediator of free radical-induced cellular injury. Oxidation of dihydrorhodamine to fluorescent rhodamine is a marker of cellular oxidant production. We investigated the mechanisms of peroxynitrite-mediated formation of rhodamine from dihydrorhodamine. Peroxynitrite at low levels (0-1000 nM) induced a linear, concentration-dependent, oxidation of dihydrorhodamine. Hydroxyl radical scavengers mannitol and dimethylsulfoxide had minimal effect (< 10%) on rhodamine production. Peroxynitrite-mediated formation of rhodamine was not dependent on metal ion catalyzed reactions because studies were performed in metal ion-free buffer and rhodamine formation was not enhanced in the presence of Fe3+ ethylenediaminetetraacetic acid (EDTA). Thus, rhodamine formation appears to be mediated directly by peroxynitrite. Superoxide dismutase slightly enhanced rhodamine production. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dihydrorhodamine oxidation through competetive oxidation of free sulfhydryls. Urate was also an efficient inhibitor (KI approximately 2.5 microM), possibly by reduction of an intermediate dihydrorhodamine radical and recycling of dihydrorhodamine. Under anaerobic conditions, nitric oxide did not oxidize dihydrorhodamine and inhibited spontaneous oxidation of dihydrorhodamine. In the presence of oxygen, nitric oxide induces a relatively slow oxidation of dihydrorhodamine due to the formation of nitrogen dioxide. We conclude that dihydrorhodamine is a sensitive and efficient trap for peroxynitrite and may serve as a probe for peroxynitrite production.

Animals↗
PubMed1994

Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells.

Active sodium (Na+) transport by alveolar type II (ATII) cells plays an important role in limiting the volume of alveolar fluid. Reactive oxygen and nitrogen species, released in the epithelial lining fluid by activated inflammatory cells or present in inspired gases, may damage Na+ transporters and decrease fluid reabsorption. To test this hypothesis we exposed ATII cells to xanthine and xanthine oxidase (1 or 10 mU/ml), or to boluses of peroxynitrite (0.1-1 mM final concentration) for 15 min and measured 1) cellular oxygen consumption (VO2); 2) amiloride-inhibitable 22Na+ uptake, as an index of Na+ movement through apically located Na+ channels; and 3) ouabain-sensitive 86Rb+ uptake, as an index of the activity of the basolaterally located Na(+)-K(+)-ATPase. After exposure of ATII cells to 0.5 or 1 mM peroxynitrite, amiloride-inhibitable 22Na+ uptake decreased to 68 +/- 7 and 56 +/- 11 of their control values, respectively (mean +/- SE; n > or = 6). Exposure to 0.5 mM peroxynitrite decreased ATII cell VO2 from 76 +/- 6 to 25 +/- 5 microM.h-1 x 10(6) cells-1 (mean +/- SE; n = 5). Cell viability and ouabain-sensitive 86Rb+ uptake remained at control levels for either peroxynitrite concentration. Exposure of ATII cells to 10 mU/ml xanthine oxidase decreased their VO2 from 94 +/- 8 to 63 +/- 6 (mean +/- SE; n = 5), but did not alter amiloride-inhibitable 22Na+ uptake. These findings indicate that physiological concentrations of peroxynitrite, but not of reactive oxygen species, decrease ATII cell Na+ transport by damaging apically located amiloride-sensitive Na+ channels.

Amiloride↗
PubMed1994

Role of neutrophils and nitric oxide in lung alveolar injury from smoke inhalation.

We examined potential mechanisms responsible for the parenchymal lung injury seen in an animal model of smoke inhalation with concurrent inflammation. Rats injected with sterile glycogen and exposed to smoke generated by the nonflaming pyrolysis of combined Douglas fir wood and polyvinylchloride showed a 74% increase in 125I-albumin lung permeability and a fivefold increase in lung myeloperoxidase (MPO) compared with control rats. There was also a significant increase in plasma indices of oxidative injury in these animals. Compared with control animals, plasma concentrations of thiobarbituric acid reactive substances (TBARS) were elevated by 62%, the concentrations of reduced sulfhydryl groups declined by 37%, and the levels of dinitrophenylhydrazine-reactive proteins (DNPH-RP) were doubled. In addition, the plasma concentrations of nitrate (NO3-) in rats exposed to glycogen plus smoke were increased three times that of control animals. Injection of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), immediately after smoke exposure or induction of neutropenia using either nitrogen mustard or antineutrophil antiserum, abolished the increase in concentrations of circulating NO3-, and prevented changes in plasma concentrations of TBARS, DNPH-RP, lung MPO activity, and tissue permeability index. These data suggest that neutrophil activation and the production of nitric oxide-derived oxidants contribute to the lung and plasma indices of oxidative injury in this smoke inhalation model.

2,4-Dinitrophenol↗
PubMed1993

Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells.

2',7'-Dichlorofluorescein and dihydrorhodamine 123 were evaluated as probes for detecting changes in intracellular H2O2 in cultured endothelial cells. Stable intracellular levels of these probes were established within 15 min of exposure to the probe in culture medium. With continued presence of the probe in the medium, intracellular levels were unchanged for 1 h. However, if medium without the probes was used after intracellular loading had occurred, there was a greater than 90% loss of intracellular dichlorofluorescin, dichlorofluorescein, and dihydrorhodamine 123 while intracellular rhodamine 123 decreased by only 15%. Exposure of endothelial cells to exogenous 100 microM H2O2 for 1 h increased intracellular rhodamine 123 by 83%, but there was a reproducible decrease of 53% in intracellular dichlorofluorescein. Exposure to 0.05 mM BCNU plus 10 mM aminotriazole for 2 h increased intracellular rhodamine 123 by 111%. In vitro studies of dihydrorhodamine 123 oxidation were similar to previous reports of dichlorofluorescin oxidation. Oxidation of dihydrorhodamine 123 does not occur with H2O2 alone, but is mediated by a variety of secondary H2O2-dependent intracellular reactions including H2O2-cytochrome c and H2O2-Fe2+. Our results suggest that detection of increased oxidation of these probes in endothelial cells is most useful as a marker of a change in general cellular oxidant production.

Aerobiosis↗
PubMed1993

Mechanisms of peroxynitrite-induced injury to pulmonary surfactants.

Activated alveolar macrophages secrete both nitric oxide and superoxide in the alveolar lining fluid which combine rapidly to form peroxynitrite, a potent oxidizing agent capable of damaging lipids and proteins in biological membranes. Peroxynitrite (1 mM) plus 100 microM Fe3+EDTA inhibited calf lung surfactant extract (CLSE) from reaching a minimum surface tension below 10 mN/m on dynamic compression. Peroxynitrite and its by-products reacted with the unsaturated lipid components of CLSE, as evidenced by the appearance of conjugated dienes and thiobarbituric acid products, and damaged all surfactant proteins. A mixture of the hydrophobic proteins [surfactant protein B (SP-B) and surfactant protein C (SP-C)] exposed to peroxynitrite became incapable of lowering phospholipid minimum surface tension on dynamic compression. Exposure of SP-A to peroxynitrite decreased its ability to cause lipid aggregation and to act synergistically with SP-B and SP-C in lowering surface tension of surfactant lipids. Western blot analysis of SP-A exposed to peroxynitrite was consistent with fragmentation and polymerization of the 28- to 36-kDa triplet band, and amino acid analysis revealed the presence of significant levels of 3-nitro-L-tyrosine. We conclude that peroxynitrite and its reactive intermediates inhibit pulmonary surfactant function by lipid peroxidation and damaging surfactant proteins.

Animals↗
PubMed1992

Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.

Animals↗
PubMed1992

Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite.

Superoxide dismutase and Fe3+EDTA catalyzed the nitration by peroxynitrite (ONOO-) of a wide range of phenolics including tyrosine in proteins. Nitration was not mediated by a free radical mechanism because hydroxyl radical scavengers did not reduce either superoxide dismutase or Fe3+EDTA-catalyzed nitration and nitrogen dioxide was not a significant product from either catalyst. Rather, metal ions appear to catalyze the heterolytic cleavage of peroxynitrite to form a nitronium-like species (NO2+). The calculated energy for separating peroxynitrous acid into hydroxide ion and nitronium ion is 13 kcal.mol-1 at pH 7.0. Fe3+EDTA catalyzed nitration with an activation energy of 12 kcal.mol-1 at a rate of 5700 M-1.s-1 at 37 degrees C and pH 7.5. The reaction rate of peroxynitrite with bovine Cu,Zn superoxide dismutase was 10(5) M-1.s-1 at low superoxide dismutase concentrations, but the rate of nitration became independent of superoxide dismutase concentration above 10 microM with only 9% of added peroxynitrite yielding nitrophenol. We propose that peroxynitrite anion is more stable in the cis conformation, whereas only a higher energy species in the trans conformation can fit in the active site of Cu,Zn superoxide dismutase. At high superoxide dismutase concentrations, phenolic nitration may be limited by the rate of isomerization from the cis to trans conformations of peroxynitrite as well as by competing pathways for peroxynitrite decomposition. In contrast, Fe3+EDTA appears to react directly with the cis anion, resulting in greater nitration yields.

Animals↗
PubMed1992

Peroxynitrite formation from macrophage-derived nitric oxide.

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.

Animals↗
PubMed1992

Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.

Animals↗