Nitric Oxide Reacting with Superoxide

CHEMISTRY OF NITRIC OXIDE REACTING WITH SUPEROXIDE 

Among the primary lines of evidence for demonstrating the action of EDRF is enhanced biological activity in the presence of superoxide dismutase (Moncada et al., 1991). Furthermore, superoxide-generating compounds are well known to inactivate EDRF. When nitric oxide was proposed to be the principal form of EDRF, the reason for its inactivation by superoxide was obvious. Both superoxide 

Many pathological conditions, including ischemia/reperfusion, inflammation, and sepsis may induce tissues to simultaneously produce both superoxide and nitric oxide. For example, ischemia allows intracellular calcium to accumulate in endothelium (Fig. 20). If the tissue is reperfused, the readmission of oxygen will allow nitric oxide as well as superoxide to be produced (Beckman, 1990). For each 10-fold increase in the concentration of nitric oxide and superoxide, the rate of peroxynitrite formation will increase by 100-fold. Sepsis causes the induction of a second nitric oxide synthase in many tissues, which can produce a thousand times more nitric oxide than the normal levels of the constitutive enzyme (Moncada et al., 1991). Nitric oxide and indirectly peroxynitrite have been implicated in several important disease states. Blockade of nitric oxide synthesis with N-methyl or N-nitroarginine reduces glutamate-induced neuronal degeneration in primary cortical cultures (Dawson et al., 1991). Nitroarginine also decreases cortical infarct volume by 70% in mice subjected to middle cerebral artery occlusion (Nowicki et al., 1991). Myocardial injury from a combined hy- 

Generation of peroxynitrite in the vascular compartment as the result of ischemia/reperfusion. The introduction of oxygen following ischemia will initiate the simultaneous production of superoxide and nitric oxide. Neutrophils and macrophages may also generate nitric oxide and peroxynitrite directly. The rate of forming peroxynitrite will increase as the pnxluct of nitric oxide and superoxide concentration, and thus will increase rapidly under conditions when both are produced simultaneously. 

How Does Superoxide Dismutase Reduce Tissue Injury  

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V. CHEMISTRY OF NITRIC OXIDE REACTING WITH SUPEROXIDE... 

Nitric oxide reacts with superoxide at a rate of 6.7 X 10 M" sec (Huie and Padmaja, 1993) and is typically produced around 1 X 10 M for cell signaling (Shibuki, 1990 Shibuki and Okada, 1991). Thus, the nitric oxide target area is normally 6.7 X 10 sec, or 30-fold lower than that for SOD. However, under pathological conditions such as ischemia/reperfusion. 

In the process, the iron is reduced to the ferrous form. Ferric cytochrome c is reduced by nitric oxide through a nitrosyl intermediate to produce ferrous cytochrome c and nitrite (Orii and Shimada, 1978). The nitrosyl cytochrome c absorbs at 560 nm, which is slightly higher than the 550-nm peak observed for reduced cytochrome c. Nitric oxide may be an interference in the assay of superoxide from cultured cells by the cytochrome c method. When nitric oxide reacts with cytochrome c, there is an initial decrease in absorbance at 550 nm as the nitrosyl complex is formed followed by a rise in absorbance as the complex decomposes to nitrite and reduced cytochrome c. This is a potential artifact in studies measuring the release of superoxide from cultured endothelial cells or other cells that make nitric oxide. 

The direct reaction of superoxide with nitric oxide is only one of at least four possible pathways that can form peroxynitrite (Fig. 40). For example, superoxide should also efficiently reduce nitrosyldioxyl radical to peroxynitrite. Alternatively, nitric oxide may be reduced to nitroxyl anion, which reacts with oxygen to form peroxynitrite. Superoxide dismutase could even catalyze the formation of peroxynitrite, since reduced (Cu or cuprous) superoxide dismutase can reduce nitric oxide to nitroxyl anion (Murphy and Sies, 1991). Thus, superoxide might first reduce superoxide dismutase to the cuprous form, with nitric oxide reacting with reduced superoxide dismutase to produce nitroxyl anion. A fourth pathway to form peroxynitrite is by the rapid reaction of nitrosonium ion (NO" ) with hydrogen peroxide. This is a convenient synthetic route for experimental studies (Reed et al., 1974), but not likely to be physiologically relevant due to the low concentrations of hydrogen peroxide and the difficulty of oxidizing nitric oxide to nitrosonium ion. 

Nitric oxide also reacts with superoxide to form the stable peroxynitrite anion, which, however, decomposes on protonation [23]. 

Synthesis and reactions of nitric oxide (NO). l-NMMA inhibits nitric oxide synthase. NO complexes with the iron in hemoproteins (eg, guanylyl cyclase), resulting in the activation of cGMP synthesis and cGMP target proteins such as protein kinase G. Under conditions of oxidative stress, NO can react with superoxide to nitrate tyrosine. 

Nitrite is also an important source of nitric oxide, molecule that could rapidly react with superoxide to form peroxynitrite (ONOO ), a potent cytokine which is very reactive (Kohn et al., 2002). Reactive species of oxygen and nitrogen could initiate a toxic oxidative chain, including lipid peroxidation, protein oxidation, directly inhibiting some enzymes from the mitochondrial respiratory chain, and causing dysfunctions of the antioxidant defense systems. 

Fig 24.23. A model for the role of ROS and RNOS in neuronal degradation in Parkinson s disease. 1. Dopamine levels are reduced by monoamine oxidase, which generates H2O2. 2. Superoxide also can be produced by mitochondria, which SOD will convert to H2O2. Iron levels increase, which allows the Fenton reaction to proceed, generating hydroxyl radicals. 3. NO, produced by inducible nitric oxide synthase, reacts with superoxide to form RNOS. 4. The RNOS and hydroxyl radical lead to radical chain reactions that result in Upid peroxidation, protein oxidation, the formation of lipofuscin, and neuronal degeneration. The end result is a reduced production and release of dopamine, which leads to the clinical symptoms observed. 

Production of nitric oxide (NO ), by hydroxylation of arginine, is a part of normal cell signalling. In addition to being a radical, and hence potentially damaging in its own right, nitric oxide can react with superoxide to form peroxynitrite, which in turn decays to yield the more damaging hydroxyl radical. Nitric oxide was first discovered as the endothelium-derived relaxation factor, and this loss of nitric oxide by reaction with superoxide may be an important factor in the development of hypertension. 

A related oxidative hypothesis implicates the molecules nitric oxide, superoxide, and peroxynitrite in the modification of proteins in cells 24, 25). Nitric oxide (NO), produced by intemeurons surrounding motor nuclei, reacts with superoxide (Oi ) approximately three times faster than superoxide does with native SODl, to form the strong oxidant peroxynitrite (ONOO ). It has been proposed that peroxynitrite can react with SODl to form a nitronium-like intermediate wldch can in turn nitrate tyrosine residues (reactions 3 and 4). 

At temperatures near 100° Horiguchi and associates (117) have shown that CO reacts with the superoxide ion on ZnO, but the Or spectrum remained at a fixed level following the addition of a mixture of CO and 02. Hydrogen did not react with 02- at that temperature. Upon addition of nitric oxide at room temperature the Oi signal instantaneously disappeared. 

At present, new developments challenge previous ideas concerning the role of nitric oxide in oxidative processes. The capacity of nitric oxide to oxidize substrates by a one-electron transfer mechanism was supported by the suggestion that its reduction potential is positive and relatively high. However, recent determinations based on the combination of quantum mechanical calculations, cyclic voltammetry, and chemical experiments suggest that °(NO/ NO-) = —0.8 0.2 V [56]. This new value of the NO reduction potential apparently denies the possibility for NO to react as a one-electron oxidant with biomolecules. However, it should be noted that such reactions are described in several studies. Thus, Sharpe and Cooper [57] showed that nitric oxide oxidized ferrocytochrome c to ferricytochrome c to form nitroxyl anion. These authors also proposed that the nitroxyl anion formed subsequently reacted with dioxygen, yielding peroxynitrite. If it is true, then Reactions (24) and (25) may represent a new pathway of peroxynitrite formation in mitochondria without the participation of superoxide. 

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