Physiological Effects of Peroxynitrite

Abstract

P eroxynitrite is a potent oxidant formed from the reaction between superoxide radicals and NO in a one-to-one stoichiometry. This reaction occurs at 6.710 9 mol/L 1 s 1 , 1 and it is essentially irreversible because of its highly exothermic nature. Therefore, the reaction is diffusion-limited, and it out-competes the reaction of superoxide dismutase for superoxide radicals, which proceeds at a rate of 210 9 mol/L 1 s 1. A key regulator of peroxynitrite production is the concentration of NO, and the reaction proceeds when the concentration of NO increases and can overcome dismutation by superoxide dismutase. This situation occurs when NO achieves micromolar concentrations as when there is a burst of NO production during ischemia/reperfusion and when NO is produced by cytokine-stimulated inducible NO synthase (iNOS) activity. The toxic effect of ONOO and its protonated form, peroxynitrous acid, may stem from its oxidation of zinc fingers, protein thiols, membrane lipids, and iron and sulfur clusters of biological molecules. In addition, hydroxyl-like and nitrogen dioxide radicals are produced by homolytic cleavage, increasing the potential for oxidant-mediated tissue injury. Intermediates are also formed from the heterolytic cleavage of ONOO to hydroxyl anion and nitronium ion (NO 2), catalyzed by the transition metal centers of superoxide dismutase and myeloperoxidase. The nitration of protein tyrosine residues gives rise to 3-nitrotyrosine, which is frequently used as an assay for ONOO in tissues, blood, and perfusates. Like NO, peroxynitrite has been associated with both deleterious and beneficial effects. An advantage of the oxidant-mediated deleterious effects of peroxynitrite is that it has been suggested to contribute to the host-defense response to bacterial invasion. Both neutrophils and macrophages produce peroxynitrite by the rapid biradical reaction between NO and superoxide anions generated simultaneously from those cells. 2 However, oxidant injury is also a primary mechanism of myocardial dysfunction and infarction, and several studies have implicated peroxynitrite as a major cause of injury in the heart subjected to ischemia/reperfusion or cytokines. Liu et al 3 demonstrated that NO and superoxide anions and the production of peroxynitrite were elevated in the ischemic-reperfused heart in vivo, which at least places peroxynitrite at the scene of the injury and is a prerequisite for suggesting that it participates in deleterious actions on tissue. The study by Ferdinandy et al 4 in this issue of Circulation Research demonstrates that the production of peroxynitrite (and the substrates NO and superoxide anion) is contributory to the cardiodepressant effects of the proinflammatory cytokines interleukin-1, interferon-, and tumor necrosis factor-. This study correlated the temporal appearance of peroxynitrite with cytokine-induced contractile dysfunction, the latter of which was reversed by attenuating the production of either of peroxyni-trite's precursors, NO (by L-nitroarginine) or superoxide anion (by titron). In addition, the degradation of peroxynitrite using 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato-iron(III), a catalyst of peroxynitrite decomposition to nitrate, attenuated cytokine-induced cardiodepression and decreased perfusate ni-trotyrosine levels, used as a surrogate measure of peroxynitrate. The depressed cardiac function induced by peroxynitrite may be related to a decrease in cardiac efficiency. 5 The authors concluded that peroxynitrite promotes myocardial contractile dys-function ostensibly secondary to its oxidant effects or alternatively to nitration of important contractile proteins. The central thesis of the study by Ferdinandy et al 4 relies on the endogenous production of peroxynitrite from superox-ide anion and NO stimulated by the mixture of cytokines. The authors verified that the substrates for peroxynitrite were, indeed, generated by measuring the presence of superoxide anions and NO in ventricular tissue. They also removed the substrates of peroxynitrite using an appropriate inhibitor of NOS and scavenger of superoxide anion. They additionally showed that xanthine oxidoreductase and NAD(P)H oxi-doreductase were sources of superoxide anions, and increased iNOS activity was associated with the increased generation of NO. Measuring the end product, peroxynitrite, is more problematic in that it cannot be measured directly, and the surrogate measures of nitrotyrosine footprints and dityrosine used by Ferdinandy et al 4 are not uniquely specific for peroxynitrite. 6 Nitrotyrosine and dityrosine were measured in perfusate rather than in tissue. Alternative methods (ie, oxidation of dihydrorhodamine-123) also have limitations in that there may be significant interference with biological molecules. Tyrosine residues can be nitrated by other nitrogen-centered oxidants, but this requires relatively high concentrations. Myeloperoxidase activity can form nitroty-rosine possibly by oxidation of nitrite to NO 2. However, myeloperoxidase activity likely is not important in the isolated buffer-perfused heart devoid of neutrophils. Hence, the most likely source of nitrotyrosine residues is peroxynitrite.