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 CO2, thereby yielding NO2. and CO3<(.)over bar> 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 mu M)-mediated oxidation of NADH (200 mu M) was half-maximally inhibited by low ascorbate concentrations (61-75 mu M), both in the absence and presence of CO2. Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO2 in situ-generated peroxynitrite exclusively oxidized NADH via the CO3<(.)over bar> radical. This fact is attributed to the formation of peroxynitrate (O2NOO-/O2NOOH) from reaction of NO2. with O-2(<(.)over bar>) which is formed from reaction of CO3<(.)over bar> with NADH. SIN-1 (25 mu M)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 mu M), irrespective of the presence of CO2. 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 CO2 by both terminating CO3<(.)over bar>/HO . 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 NO2. by O-2(<(.)over bar>) to yield O2NOO-, which further decomposes into NO2- and O-2.