Reaction of superoxide and nitric oxide with peroxynitrite - Implications for peroxynitrite-mediated oxidation reactions in vivo

Peroxynitrite (ONOO-/ONOOH), the product of the diffusion-limited reaction of nitric oxide ((NO)-N-.) with superoxide (O-2(radical anion)), has been i mplicated as an important mediator of tissue injury during conditions assoc iated with enhanced (NO)-N-. and O-2(radical anion) production. Although se veral groups of investigators have demonstrated substantial oxidizing and c ytotoxic activities of chemically synthesized peroxynitrite, others have pr oposed that the relative rates of (NO)-N-. and production may be critical i n determining the reactivity of peroxynitrite formed in situ (Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., and Grisham, At B. (1996) J. Biol. Chem. 271, 40-47). In the present study, we examined the mechanisms by which excess O-2(radical anion) or (NO)-N-. production 2 inhibits peroxynitrite-mediated oxidation reactions. Peroxynitrite was gene rated in situ by the co-addition of a chemical source of (NO)-N-., spermine NONOate, and an enzymatic source of O-2(radical anion), xanthine oxidase, w ith either hypoxanthine or lumazine as a substrate. We found that the oxida tion of the model compound dihydrorhodamine by peroxynitrite occurred via t he free radical intermediates OH and NO2, formed during the spontaneous dec omposition of peroxynitrite and not via direct reaction with peroxynitrite. The inhibitory effect of excess O-2(radical anion) on the oxidation of dih ydrorhodamine could not be ascribed to the accumulation of the peroxynitrit e scavenger urate produced from the oxidation of hypoxanthine by xanthine o xidase. A biphasic oxidation profile was also observed upon oxidation of NA DH by the simultaneous generation of (NO)-N-. and O-2(radical anion). Conve rsely, the oxidation of glutathione, which 2 occurs via direct reaction wit h peroxynitrite, was not affected by excess production of (NO)-N-.. We conc lude that the oxidative processes initiated by the free radical intermediat es formed from the decomposition of peroxynitrite, are inhibited by excess production of (NO)-N-. or O-2(radical anion), whereas oxidative pathways in volving a direct reaction with peroxynitrite are not altered. The physiolog ical implications of these findings are discussed.