Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions. Evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite

Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2).     

Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.     

Formation of peroxynitrite from reaction of nitroxyl anion with molecular oxygen.

Peroxynitrite (ONOO(-)/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O(2)) and nitric oxide ((*)NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO(-)), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate, (-)ON=NO(2)(-)), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO(-) in the presence of oxygen is mainly characterized by peroxynitrite and not by HO( small middle dot) radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of (*)NO and O(2), probably by an electron-transfer mechanism. This prediction is supported by the fact that H(2)O(2) is formed during the decay of NO(-) in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO(-) in addition to the "classical" ( small middle dot)NO + O(2)() pathway.     

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

Oxidative chemistry of fluorescent dyes: implications in the detection of reactive oxygen and nitrogen species

HE (hydroethidine), a widely used fluorescent dye for detecting intracellular superoxide, undergoes specific oxidation and hydroxylation reactions. The reaction between HE and O2?- (superoxide radical) yields a diagnostic marker product, 2-hydroxyethidium. This is contrary to the popular notion that O2?- oxidizes HE to form ethidium. HE, however, undergoes a non-specific oxidation to form ethidium in the presence of other oxidants (hydroxyl radical, peroxynitrite and perferryl iron) and other dimeric products. The mitochondria-targeted HE analogue Mito-SOX? undergoes the same type of oxidative chemistry to form products similar to those formed from HE. On the basis of the oxidative chemical mechanism of HE and Mito-SOX?, we conclude that flurorescence microscopy or related techniques are not sufficient to measure the superoxide-specific hydroxylated products. HPLC methodologies are required to separate and identify these products. Peroxynitrite reacts rapidly and stoichiometrically with boronates to form specific products. Assays using fluorescent-based boronate probes will be more reliable for peroxynitrite determination than those using either dichlorodihydrofluorescein or dihydrorhodamine.     

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.     

Redox regulation of vascular prostanoid synthesis by the nitric oxide-superoxide system

Oxygen is involved in cell signaling through oxygenases and oxidases and this applies especially for the vascular system. Nitric oxide (*NO) and epoxyarachidonic acids are P450-dependent monooxygenase products and prostacyclin is formed via cyclooxygenase and a heme-thiolate isomerase. The corresponding vasorelaxant mechanisms are counteracted by superoxide which not only traps *NO but through the resulting peroxynitrite blocks prostacyclin synthase by nitration of an active site tyrosine residue. In a model of septic shock, this leads to vessel constriction by activation of the thromboxane A2-prostaglandin endoperoxide H2 receptor. This sequence of events is part of endothelial dysfunction in which the activated vascular smooth muscle counteracts and regenerates vessel tone by cyclooxygenase-2-dependent prostacyclin synthesis. Peroxynitrite was found to activate cyclooxygenases by providing the peroxide tone at nanomolar concentrations. Such new insights into the control of vascular function have allowed us to postulate a concept of redox regulation in which a progressive increase of superoxide production by NADPH-oxidase, mitochondria, xanthine oxidase, and even uncoupled NO-synthase triggers a network of signals originating from an interaction of *NO with superoxide.     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.     

Reaction of human hemoglobin with peroxynitrite. Isomerization to nitrate and secondary formation of protein radicals

Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 x 10(4) m(-1) s(-1)) with oxyhemoglobin. The reaction mechanism of peroxynitrite with oxyhemoglobin that results in the formation of methemoglobin remains to be elucidated. In this work, we studied the reaction under biologically relevant conditions using millimolar oxyhemoglobin concentrations and a stoichiometric excess of oxyhemoglobin over peroxynitrite. The results support a reaction mechanism that involves the net one-electron oxidation of the ferrous heme, isomerization of peroxynitrite to nitrate, and production of superoxide radical and hydrogen peroxide. Homolytic cleavage of peroxynitrite within the heme iron allows the formation of ferrylhemoglobin in approximately 10% yields, which can decay to methemoglobin at the expense of reducing equivalents of the globin moiety. Indeed, spin-trapping studies using 2-methyl-2-nitroso propane and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated the formation of tyrosyl- and cysteinyl-derived radicals. DMPO also inhibited covalently linked dimerization products and led to the formation of DMPO-hemoglobin adducts. Hemoglobin nitration was not observed unless an excess of peroxynitrite over oxyhemoglobin was used, in agreement with a marginal formation of nitrogen dioxide. The results obtained support a role of oxyhemoglobin as a relevant intravascular sink of peroxynitrite.     

Reactivity of peroxynitrite and nitric oxide with LDL

Low density lipoprotein (LDL) oxidation by peroxynitrite is a complex process, finely modulated by control of peroxynitrite formation, LDL availability and free-radical scavenging by nitric oxide (*NO), ascorbate and alpha-tocopherol (alpha -TOH). In the presence of CO2, lipid targets are spared at the expense of surface constituents. Since surface damage may lead to oxidation-induced LDL aggregation and particle recognition by scavenger receptors, CO2 cannot be considered an inhibitor of peroxynitrite-dependent LDL modifications. Chromanols, urate and ascorbate cannot scavenge peroxynitrite in the vasculature, although intermediates of urate oxidation and high ascorbate concentrations may do soin vitro. Most if not all of the protection against peroxynitrite-induced LDL oxidation afforded by urate, ascorbate, chromanols and also*NO should be considered to depend on their free radical scavenging abilities, including inactivation of lipid peroxyl radicals (LOO),*NO2, and CO3*-; as well as their capacity to reduce high oxidation states of metal centers. Peroxynitrite direct interception by reduced manganese (II) porphyrins is possibly the most powerful although unspecific strategy to inhibit peroxynitrite reactions. In light of the recent demonstration of nitrated bioactive lipids in vivo, renewed interest in the mechanisms of peroxynitrite- and nitric oxide-mediated lipid nitration and nitrosation is guaranteed.     

Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.     

Formation of lipid-protein adducts in low-density lipoprotein by fluxes of peroxynitrite and its inhibition by nitric oxide.

Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.     

Nitric oxide enhances catechol estrogen-induced oxidative stress in LNCaP cells

Catechol estrogens (CEs), such as 4-hydroxyestradiol (4-OHE2), undergo redox cycling during which reactive oxygen species (ROS) such as superoxide (O2*-) and the chemically reactive estrogen semiquinone (CE-SQ) and quinone (CE-Q) intermediates are produced. The quinone's putative mutagenicity may be enhanced by ROS and/or reactive nitrogen species. High concentrations of nitric oxide (NO) present during inflammatory conditions may react with (O2*-) to form peroxynitrite (ONOO-), a potent oxidant implicated in many pathological conditions. In this study, the possible generation of peroxynitrite from the interaction of CEs and NO and its effect on plasmid DNA and intact cells were investigated. A combination of 4-OHE2 and NO increased the level of single strand breaks (SSB) in plasmid DNA by more than 60% compared to vehicle controls in a metal-free buffer system. 4-OHE2 alone or NO alone had no effect. Results obtained from use of different antioxidants and ROS scavengers suggested a role of peroxynitrite in oxidative stress. In cells, 4-OHE2 or NO alone induced dose-dependent DNA damage as assessed by single cell gel electrophoresis. Co-treatment with 4-OHE2 and NO had an additive effect at lower doses. Generation of intracellular ROS was measured by the oxidation of carboxy-2',7'-dichlorofluorescein diacetate to the fluorescent compound carboxy-2',7'-dichlorofluorescein. NO alone, in oxygenated media, generated little ROS whereas 4-OHE2 produced approximately 70% increase in fluorescence. When added together 4-OHE2 and NO, produced a 2-fold increase in ROS. The generation and involvement ofperoxynitrite to this increase was implied since uric acid inhibited it. Generation ofperoxynitrite was also observed by use of dihydrorhodamine 123. Therefore, we conclude that combined treatments with 4-OHE2 and NO generated peroxynitrite seen from increased fluorescence and its inhibition by uric acid or combined SOD and catalase treatments. Results reported here suggest a role of peroxynitrite in causing damage to biomolecules when CEs and NO are present simultaneously. This may have biological relevance as high concentrations of NO formed during inflammatory conditions may exacerbate cancers due to estrogens.     

CO2 impairs peroxynitrite-mediated inhibition of human caspase-3

Peroxynitrite (ONOO-) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide (.NO) with superoxide (O2.-). The peroxynitrite reactivity is modulated by carbon dioxide (CO2) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO2 on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the Sgamma atom of the Cys catalytic residue. In the absence of CO2, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO2 (=1.3x10(-3) M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the kcat value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering Km. Both in the absence and presence of CO2, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent the first evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO2, supporting the role of CO2 in fine tuning of cell processes (e.g., apoptosis).     

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-).     

Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.

Endothelial cells, macrophages, neutrophils, and neuronal cells generate superoxide (O2-) and nitric oxide (.NO) which can combine to form peroxynitrite anion (ONOO-). Peroxynitrite, known to oxidize sulfhydryls and to yield products indicative of hydroxyl radical (.OH) reaction with deoxyribose and dimethyl sulfoxide, is shown herein to induce membrane lipid peroxidation. Peroxynitrite addition to soybean phosphatidylcholine liposomes resulted in malondialdehyde and conjugated diene formation, as well as oxygen consumption. Lipid peroxidation was greater at acidic and neutral pH, with no significant lipid peroxidation occurring above pH 9.5. Addition of ferrous (Fe+2) or ferric (Fe+3) iron did not enhance lipid peroxide formation over that attributable to peroxynitrite alone. Diethylenetetraminepentacetic acid (DTPA) or iron removal from solutions by ion-exchange chromatography decreased conjugated diene formation by 25-50%. Iron did not play an essential role in initiating lipid peroxidation, since DTPA and iron depletion of reaction systems were only partially inhibitory. In contrast, desferrioxamine had an even greater concentration-dependent inhibitory effect, completely abolishing lipid peroxidation at 200 microM. The strong inhibitory effect of desferrioxamine on lipid peroxidation was due to direct reaction with peroxynitrous acid in addition to iron chelation. We conclude that the conjugate acid of peroxynitrite, peroxynitrous acid (ONOOH), and/or its decomposition products, i.e., .OH and nitrogen dioxide (.NO2), initiate lipid peroxidation without the requirement of iron. These observations demonstrate a potential mechanism contributing to O2-(-)and .NO-mediated cytotoxicity.     

Interactions of nitric oxide and peroxynitrite with low-density lipoprotein

Nitric oxide (*NO) is a free radical species that diffuses and concentrates in the hydrophobic core of low-density lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusion-limited reaction between *NO and superoxide (O2*-) represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between *NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and *NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.     

Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate

Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.