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) with superoxide (O(-*)(2)), has been implicated as an important mediator of tissue injury during conditions associated with enhanced *NO and O(-*)(2) production. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized peroxynitrite, others have proposed that the relative rates of *NO and production may be critical in determining the reactivity of peroxynitrite formed in situ (Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., and Grisham, M. B. (1996) J. Biol. Chem. 271, 40-47). In the present study, we examined the mechanisms by which excess O(-*)(2) or *NO production inhibits peroxynitrite-mediated oxidation reactions. Peroxynitrite was generated in situ by the co-addition of a chemical source of *NO, spermineNONOate, and an enzymatic source of O(-*)(2), xanthine oxidase, with either hypoxanthine or lumazine as a substrate. We found that the oxidation of the model compound dihydrorhodamine by peroxynitrite occurred via the free radical intermediates OH and NO(2), formed during the spontaneous decomposition of peroxynitrite and not via direct reaction with peroxynitrite. The inhibitory effect of excess O(-*)(2) on the oxidation of dihydrorhodamine could not be ascribed to the accumulation of the peroxynitrite scavenger urate produced from the oxidation of hypoxanthine by xanthine oxidase. A biphasic oxidation profile was also observed upon oxidation of NADH by the simultaneous generation of *NO and O(-*)(2). Conversely, the oxidation of glutathione, which occurs via direct reaction with peroxynitrite, was not affected by excess production of *NO. We conclude that the oxidative processes initiated by the free radical intermediates formed from the decomposition of peroxynitrite are inhibited by excess production of *NO or O(-*)(2), whereas oxidative pathways involving a direct reaction with peroxynitrite are not altered. The physiological implications of these findings are discussed.
Jourd'heuil
D
D
Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA. jourdhd@mail.amc.edu
Jourd'heuil
F L
FL
Kutchukian
P S
PS
Musah
R A
RA
Wink
D A
DA
Grisham
M B
MB
D002374:Catalase / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000737:chemistry; D009566:Nitrates / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry*; D009589:Nitrogen Oxides; D010084:Oxidation-Reduction; D011621:Pteridines / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D013096:Spermine / Q000031:analogs & derivatives / Q000737:chemistry; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry*; D014969:Xanthine Oxidase / Q000378:metabolism
D009566:Nitrates; D009589:Nitrogen Oxides; D011621:Pteridines; D011994:Recombinant Proteins; D012235:Rhodamines; D009243:NAD; C058319:dihydrorhodamine 123; D013481:Superoxides; C091861:spermine nitric oxide complex; C008297:lumazine; C039642:peroxynitric acid; D013096:Spermine; D019271:Hypoxanthine; D009569:Nitric Oxide; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D005978:Glutathione