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R Radi

Publications and source records attributed to R Radi.

At least 55 records · Page 3Linked to original sources
PubMed1997

Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation.

Peroxynitrite anion, the reaction product of superoxide and nitric oxide, is a potent biological oxidant, which inactivates mammalian heart mitochondrial NADH-coenzyme Q reductase (complex I), succinate dehydrogenase (complex II), and ATPase, without affecting cytochrome c oxidase (complex IV). In this paper, we evaluated the effect of peroxynitrite on mitochondrial membrane integrity and permeability under low calcium concentration. Phosphate buffer was used in most of our experiments since Hepes, Tris, mannitol, and sucrose were found to inhibit the oxidative chemistry of peroxynitrite. Peroxynitrite (0.1-1.0 mM) caused a dose-dependent decrease in the ability of mitochondria to build up a membrane potential when N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate were used as substrate. Elimination of the membrane potential was accompanied by penetration of the osmotic support (KCl/NaCl) into the matrix as judged by the parallel occurrence of mitochondrial swelling. This swelling was partially inhibited by dithiothreitol (DTT) or butylated hydroxytoluene (BHT) and was insensitive to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ADP, and cyclosporin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicated that alterations in membrane permeability were associated with the production of protein aggregates due to membrane protein thiol cross-linking. The protective effect of DTT on both mitochondrial swelling and protein polymerization suggests the involvement of disulfide bonds in the membrane permeabilization process. In addition, the increase in thiobarbituric acid-reactive substances and the partial inhibitory effect of BHT indicate the occurrence of lipid peroxidation. These results support the idea that under our experimental conditions peroxynitrite causes mitochondrial structural and functional alterations by Ca2+-independent mechanisms through lipid peroxidation and protein sulfhydryl oxidation.

Animals↗
PubMed1997

Mercaptoethylguanidine and guanidine inhibitors of nitric-oxide synthase react with peroxynitrite and protect against peroxynitrite-induced oxidative damage.

Nitric oxide (NO) produced by the inducible nitric-oxide synthase (iNOS) is responsible for some of the pathophysiological alterations during inflammation. Part of NO-related cytotoxicity is mediated by peroxynitrite, an oxidant species produced from NO and superoxide. Aminoguanidine and mercaptoethylguanidine (MEG) are inhibitors of iNOS and have anti-inflammatory properties. Here we demonstrate that MEG and related compounds are scavengers of peroxynitrite. MEG caused a dose-dependent inhibition of the peroxynitrite-induced oxidation of cytochrome c2+, hydroxylation of benzoate, and nitration of 4-hydroxyphenylacetic acid. MEG reacts with peroxynitrite with a second-order rate constant of 1900 +/- 64 M-1 s-1 at 37 degrees C. In cultured macrophages, MEG reduced the suppression of mitochondrial respiration and DNA single strand breakage in response to peroxynitrite. MEG also reduced the degree of vascular hyporeactivity in rat thoracic aortic rings exposed to peroxynitrite. The free thiol plays an important role in the scavenging effect of MEG. Aminoguanidine neither affected the oxidation of cytochrome c2+ nor reacted with ground state peroxynitrite, but inhibited the peroxynitrite-induced benzoate hydroxylation and 4-hydroxyphenylacetic acid nitration, indicating that it reacts with activated peroxynitrous acid or nitrogen dioxide. Compounds that act both as iNOS inhibitors and peroxynitrite scavengers may be useful anti-inflammatory agents.

Animals↗
PubMed1997

Xanthine oxidase binding to glycosaminoglycans: kinetics and superoxide dismutase interactions of immobilized xanthine oxidase-heparin complexes.

Xanthine oxidoreductase (XDH + XO, EC 1.2.3.2) is released into the circulation from organs rich in XO activity. Herein we report the specific high affinity binding of XO to glycosaminoglycans (GAGs) and the preferential association of XO with heparin, compared with heparan sulfate, chondroitin sulfate, and dematan sulfate. The binding of XO to Sepharose 6B-conjugated heparin (HS6B) occurs at physiological ionic strength and increased with pH, with Scatchard analysis revealing a nonlinear binding pattern at pH 7.4. The dissociation constant (Kd) for XO binding was 0.4 to 1.8 x 10(-7) M, similar to the heparin-reversible binding of lipoprotein lipase to vascular endothelium. The binding energy of 9-13 kcal/mol was concordant with noncovalent electrostatic interactions. Xanthine oxidase immobilization to HS6B rendered a catalytically active enzyme from that had kinetic characteristics distinct from XO in free solution. While the Km and Ki for xanthine in phosphate buffer at pH 7.4 were 3 microM and 1.6 mM, respectively, for free XO, they were 15 microM and 2.8 mM for immobilized XO. Inhibition constants for guanine and uric acid were also increased upon XO binding to HS6B. Changes in kinetic parameters were related to a real and not apparent decrease in binding affinity for substrate and inhibitors and were not due to diffusion-controlled processes within the gel matrix. Changes in Km and Ki for xanthine also had a significant influence on the relative quantities of O2.- and H2O2 generated by a given substrate concentration. Superoxide formed by HS6B-bound XO was partially consumed within the gel microenvironment which electrostatically excluded CuZn SOD. Immobilization of XO increased the half-life of enzyme activity in buffer and in the absence of substrate from 67 to 120 h at 4 degrees C. These data indicate that binding to cell surfaces will strongly influence the catalytic properties, oxidant producing capacity, and stability of XO.

Animals↗
PubMed1997

Pathways of peroxynitrite oxidation of thiol groups.

Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.

Catalase↗
PubMed1997

Peroxynitrite-mediated decarboxylation of pyruvate to both carbon dioxide and carbon dioxide radical anion.

There has been a recent renewal of interest in the antioxidant properties of pyruvate which are usually attributed to its capacity to undergo oxidative decarboxylation in the presence of hydrogen peroxide. The interaction of pyruvate with other oxidizing biological intermediates, however, has been scarcely considered in the literature. Here we report that peroxynitrite, the oxidant produced by the reaction between superoxide anion and nitric oxide, reacts with pyruvate with an apparent second-order rate constant of 88 +/- 7 M-1 s-1 at pH 7.4 and 37 degrees C. Kinetic studies indicated that pyruvate reacts with peroxynitrite anion (k = 100 +/- 7 M-1 s-1, peroxynitrous acid (k = 49 +/- 7 M-1 s-1, and a highly oxidizing species derived from peroxynitrous acid. Pyruvate decarboxylation was proved by anion exchange chromatography detection of acetate in incubations of peroxynitrite and pyruvate at pH 7.4 and 5.5. Formation of carbon dioxide radical anion was ascertained by EPR spin-trapping studies in the presence of GSH and the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The use of pyruvate labeled with 13C at the 1-position led to the detection of the labeled DMPO carbon dioxide radical anion adduct. In the absence of GSH, oxygen consumption studies confirmed that peroxynitrite mediates the decarboxylation of pyruvate to free radical intermediates. Comparing the yields of acetate and free radicals estimated from the oxygen uptake studies, it is concluded that pyruvate is oxidized by both one- and two-electron oxidation pathways, the latter being preponderant. Hydrogen peroxide-mediated pyruvate oxidation does not produce detectable levels of carbon dioxide radical anion except in the presence of iron(II)-ethylenediamine-N,N,N',N'-tetraacetate (EDTA). The apparent second-order rate constant of the reaction between pyruvate and hydrogen peroxide was determined to be 1 order of magnitude lower than that of the reaction between pyruvate and peroxynitrite. The latter process may contribute to the antioxidant properties of pyruvate.

Anions↗
PubMed1997

Ternary copper complexes and manganese (III) tetrakis(4-benzoic acid) porphyrin catalyze peroxynitrite-dependent nitration of aromatics.

Peroxynitrite is a powerful oxidant formed in biological systems from the reaction of nitrogen monoxide and superoxide and is capable of nitrating phenols at neutral pH and ambient temperature. This peroxynitrite-mediated nitration is catalyzed by a number of Lewis acids, including CO2 and transition-metal ion complexes. Here we studied the effect of ternary copper-(II) complexes constituted by a 1,10-phenanthroline and an amino acid as ligands. All the complexes studied accelerate both the decomposition of peroxynitrite and its nitration of 4-hydroxyphenylacetic acid at pH > 7. The rate of these reactions depends on the copper complex concentration in a hyperbolic plus linear manner. The yield of nitrated products increases up to 2.6-fold with respect to proton-catalyzed nitration and has a dependency on the concentration of copper complexes which follows the same function as observed for the rate constants. The manganese porphyrin complex, Mn(III)tetrakis(4-benzoic acid)porphyrin [Mn(tbap)], also promoted peroxynitrite-mediated nitration with an even higher yield (4-fold increase) than the ternary copper complexes. At pH = 7.5 +/- 0.2 the catalytic behavior of the copper complexes can be linearly correlated with the pKa of the phenanthroline present as a ligand, implying that a peroxynitrite anion is coordinated to the copper ion prior to the nitration reaction. These observations may prove valuable to understand the biological effects of these transition-metal complexes (i.e., copper and manganese) that can mimic superoxide dismutase activity and, in the case of the ternary copper complexes, show antineoplastic activity.

Chelating Agents↗
PubMed1996

Oxidative modification of nicotinamide nucleotide transhydrogenase in submitochondrial particles: effect of endogenous ubiquinol.

The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.

Adenosine Diphosphate↗
PubMed1996

Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations.

Peroxynitrite is a strong oxidant produced in vivo as the reaction product of superoxide anion and nitric oxide (k approximately 5 x 10(9) M-1 s-1) and can be formed and mediate reactions in the extracellular environment. It has recently been reported that peroxynitrite and carbon dioxide react in a second-order process (S. V. Lymar and K. Hurst (1995) J. Am. Chem. Soc. 117, 8867-8868). Since one of the most abundant constituents of the extracellular milieu is bicarbonate anion (25 mM in plasma) which is in equilibrium with carbon dioxide (1.3 mM in plasma) we have further studied the kinetics of the reaction between peroxynitrite and carbon dioxide/ bicarbonate and the effect of bicarbonate on different peroxynitrite-mediated oxidations. The apparent second-order rate constant for the reaction is (2.3 +/- 0.1) x 10(3) M-1 s-1 at 37 degrees C and pH 7.4 and a pH-independent second-order rate constant of (5.8 +/- 0.2) x 10(4) M-1 s-1 at 37 degrees C was obtained considering peroxynitrite anion and carbon dioxide as the reacting species. The enthalpy and entropy of activation are delta H* = +10.7 +/- 0.8 kcal mol-1 and delta S* = -6.5 +/- 0.5 cal mol-1 K-1, respectively. The presence of bicarbonate had variable influence on peroxynitrite-mediated oxidations. While bicarbonate significantly enhanced peroxynitrite-mediated nitration of aromatics, it partially inhibited the oxidation of thiols, dimethylsulfoxide, oxyhemoglobin, and cytochrome c+2 and totally inhibited the hydroxylation of benzoate. Spontaneous chemiluminescence studies suggest the formation of bicarbonate radicals during the interactions of peroxynitrite with carbon dioxide/ bicarbonate. Our results support that peroxynitrite anion rapidly reacts with carbon dioxide to yield an adduct (ONOOCO2-) which can participate in oxidation and nitration processes, thus redirecting the primary reactivity of peroxynitrite.

Bicarbonates↗
PubMed1996

Modulatory role of nitric oxide on superoxide-dependent luminol chemiluminescence.

Reactive oxygen species are involved in luminol chemiexcitation induced in biological systems, but the contribution of nitrogen-derived oxidants in the process still remains unclear. Herein, we report that luminol chemiluminescence (LCL) induced by a superoxide (O2.-)- and hydrogen peroxide (H2O2)-generating system (2-25 mU/ml xanthine oxidase plus acetaldehyde and oxygen) was markedly inhibited by nitric oxide (.NO) added either as bolus (0-10 microM) or a continuous flow (0-10 microM/min). However, the inhibition of LCL was followed by an overshoot in light emission after most .NO was consumed or the infusion stopped and was due to reactions of remaining peroxynitrite, the product of the reaction between O2.- and .NO, with luminol. Nitric oxide also inhibited peroxynitrite- and glucose oxidase-induced LCL, but no overshoot was observed. On the other hand, a continuous flux of pure peroxynitrite, at 2 to 10 microM/min, induced LCL with quantum yields close to those obtained by identical micromolar fluxes of O2.-, while peroxynitrite formed from the decomposition of the sydnonimine SIN-1 yielded 76% of the chemiluminescence obtained with authentic peroxynitrite. Peroxynitrite-induced LCL was 80 and 55% inhibitable by SOD and catalase, respectively, showing that there were O2.- and H2O2-dependent routes of chemiexcitation. The hydroxyl radical scavengers dimethyl sulfoxide, mannitol, and ethanol and the metal chelator diethylenetriaminepentaacetic acid did not inhibit peroxynitrite-induced LCL while desferrioxamine was an efficient inhibitor of light emission by reaction with an activated state of peroxynitrous acid which is responsible of performing the initial one-electron oxidation of luminol. Our results are consistent with a dual role of .NO in O2.(-)-induced LCL: (I) formation of peroxynitrite which in turn promotes the light-emitting route and (II) reaction with luminol radical intermediates directing the system toward a dark pathway. These considerations are of critical importance when analyzing cell- and tissue-derived LCL in .NO-, O2.(-)-, and peroxynitrite-producing systems.

Acridines↗
PubMed1996

Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport.

Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.

Adenosine Triphosphatases↗
PubMed1996

Nitric oxide diffusion in membranes determined by fluorescence quenching.

Quenching of pyrene derivative fluorescence by nitric oxide was used to evaluate the apparent diffusion coefficients of nitric oxide in artificial and biological membranes. The apparent second-order quenching constants of nitric oxide were obtained from Stern-Volmer plots using methyl- and undecylpyrene derivatives incorporated into liposomes and erythrocyte plasma membranes in order to assess the ability of nitric oxide to interact with molecules located at different positions in the membrane. Diffusion coefficients were estimated from the determined second-order quenching constants and compared to that of oxygen obtained under the same conditions. Oxygen and nitric oxide presented similar diffusional behavior in agreement with their similarity in structures, with the differences observed attributable to the higher lipophilicity of oxygen compared to nitric oxide. In solution, both showed the same quenching efficiency while in liposomes and erythrocyte ghosts oxygen diffusion was twice that of nitric oxide (k(O2)/k(NO) = 2). Nitric oxide diffusion coefficients determined at 20 degrees C ranged from 1.3 x 10(-5) cm2 s-1 in liposomes to 0.4 x 10(-5) cm2 s-1 in surface erythrocyte plasma membranes. Both nitric oxide and oxygen had larger quenching constants for the undecyl derivative compared to the methylpyrene compound incorporated into erythrocyte plasma membranes, indicating an increased solubility of both gases toward the center of the membrane.

Animals↗
PubMed1996

Peroxynitrite-dependent tryptophan nitration.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2.-) and nitric oxide (.NO), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. At high peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from peroxynitrite increased at acidic pH, with an apparent pKa of 7.8. High concentrations of Fe(3+)-EDTA were required to enhance peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 microM Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration. Plots of the observed rate constant of nitrotryptophan formation vs tryptophan concentration presented downward curvatures. Thus, the kinetics of metal-independent nitration reactions were interpreted in terms of two parallel mechanisms. In the first one, ground-state peroxynitrous acid nitrated tryptophan with a second-order rate constant of 184 +/- 11 M-1 s-1 at 37 degrees C. The activation enthalpy was 9.1 +/- 0.3 kcal mol-1, and the activation entropy was -19 +/- 1 cal mol-1 K-1. In the second mechanism, ONOOH*, an activated intermediate derived from trans-peroxynitrous acid formed in a steady state, was the nitrating agent.

Deferoxamine↗
PubMed1995

Kinetics of cytochrome c2+ oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems.

Cytochrome c3+ has been extensively used for the detection of superoxide produced in biological systems due to its fast superoxide-mediated reduction to cytochrome c2+. However, another biomolecule which is sometimes cogenerated with superoxide, nitric oxide, reacts with superoxide at almost diffusion-controlled rates (6.7 x 10(9) M-1 s-1), leading to the production of a highly oxidizing species, peroxynitrite anion (ONOO-). In this work we report that peroxynitrite readily oxidizes cytochrome c2+ to cytochrome c3+ in an ascorbate-reversible manner. The reaction between peroxynitrite and cytochrome c2+ occurs with a second-order rate constant of 2.3 x 10(5) M-1 s-1. The pH dependence of the apparent second-order rate constants as well as the effect of different scavengers indicated that peroxynitrous acid (ONOOH) in the ground state was the actual species responsible of cytochrome c2+ oxidation. The activation enthalpy, free energy, and entropy were +10.8 kcal mol-1, +11.8 kcal mol-1, and -3.15 cal mol-1 K-1, respectively, in agreement with the proposed reaction mechanism. Additionally, our results imply that when quantitating superoxide by the cytochrome c3+ reduction method, the existence of a simultaneous generation of nitric oxide and peroxynitrite may lead to an underestimation of the rates of superoxide production.

Cytochrome c Group↗
PubMed1995

Desferrioxamine inhibition of the hydroxyl radical-like reactivity of peroxynitrite: role of the hydroxamic groups.

Nitric oxide reacts with superoxide to form peroxynitrite, a strong oxidizing species. Peroxynitrite can either directly oxidize molecules such as thiols or protonate to peroxynitrous acid, which can yield an oxidant with a reactivity similar to that of hydroxyl radical in a transition metal-independent mechanism. This oxidative chemistry of peroxynitrite, however, is inhibited by the metal chelator desferrioxamine. Indeed, desferrioxamine, was a potent inhibitor of dimethylsulfoxide, hydrogen peroxide, 5,5-dimethyl-1-pyrroline-N-oxide, and luminol oxidation, whereas the metal chelator diethylenetriaminepentaacetic acid, and ferrioxamine, the iron complex of desferioxamine, were not. Two other hydroxamates, acetohydroxamate and salicylhydroxamate, were also effective inhibitors. Stopped-flow experiments showed that there is no direct reaction between peroxynitrite anion or cis-peroxynitrous acid with desferrioxamine. Electron paramagnetic resonance (EPR) studies showed the formation of the desferrioxamine nitroxide radical in incubations containing desferrioxamine, but not ferrioxamine, indicating that the hydroxamic group acts as a one-electron donor to peroxynitrite-derived oxidants. Taken together, our results led us to propose that desferrioxamine can inhibit the oxidative chemistry of peroxynitrite by reaction of the hydroxamic acid moieties with trans-peroxynitrous acid.

Cyclic N-Oxides↗
PubMed1995

Reaction between peroxynitrite and hydrogen peroxide: formation of oxygen and slowing of peroxynitrite decomposition.

Peroxynitrite, the reaction product of nitric oxide and superoxide, is a potent and versatile oxidant that can attack a wide range of targets. In this work, we studied the oxidation of hydrogen peroxide by peroxynitrite, which led to oxygen evolution. Oxygen yields increased at alkaline pH with an apparent pKa of 7.05 +/- 0.04. The maximum yields were 16% and 32% of added peroxynitrite at pH 5.9 and 7.4, respectively, assuming that two molecules of peroxynitrite are needed to produce one of oxygen. Hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol, formate, and acetate) inhibited oxygen evolution to a similar extent to that predicted from their rate constants with hydroxyl radical. The apparent rate constant of peroxynitrite decomposition was zero-order in hydrogen peroxide at acidic pH. At neutral and alkaline pH, the rate of peroxynitrite disappearance decreased in the presence of millimolar concentrations of hydrogen peroxide by up to 50%. The apparent activation enthalpy and entropy for peroxynitrite decomposition increased by 1.7 kcal mol-1 and 4.7 cal mol-1 K-1, respectively, in the presence of hydrogen peroxide. We propose that an activated intermediate of peroxynitrous acid is responsible for hydrogen peroxide oxidation at acidic pH, while at more alkaline pH the formation of a stabilizing complex between hydrogen peroxide and transperoxynitrite anion is involved.

Hydrogen Peroxide↗
PubMed1995

Peroxynitrite-induced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors.

Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (> 2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 microM) intially (3-4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.

Animals↗