Inhibition of acetylcholine synthesis and tyrosine nitration induced by peroxynitrite are differentially prevented by antioxidants

Evidence of an overload of reactive oxygen species and peroxynitrite, a der ivative of nitric oxide, in sporadic amyotrophic lateral sclerosis suggests that peroxynitrite could impair cholinergic functions. Because of the impo ssibility of obtaining synaptosomes from vertebrate neuromuscular junctions , we used cholinergic synaptosomes purified from Torpedo marmorata electron eurons to characterize the defects triggered by peroxynitrite in more detai l. Addition of peroxynitrite or its donor 3-morpholinosydnonimine abolished high-affinity choline uptake and synthesis of acetylcholine from acetate. T. marmorata choline acetyltransferase (ChAT) was impaired to the same exte nt as bovine brain ChAT. A hallmark of peroxynitrite action is the nitratio n of tyrosine residues in proteins. Peroxynitrite induced a concentration-d ependent appearance of nitrotyrosines in several neuronal proteins from syn aptosomes and, more readily, from synaptic vesicles. Peroxynitrite also tri ggered tyrosine nitrations in purified ChAT. Peroxynitrite-dependent nitrat ions were impaired when synaptosomes were pretreated with thioreductants (g lutathione, N-acetyl cysteine, dithiothreitol) or antioxidants (uric acid, melatonin, bovine serum albumin, desferrioxamine). Deleterious effects of p eroxynitrite on choline transport and ChAT activity were prevented by the t hioreductants but only partially by the antioxidants, suggesting a mechanis m other than tyrosine nitration, which may involve cysteine oxidation. Furt her development of protective agents acting on choline transport and on ChA T activity may offer interesting therapeutic possibilities with respect to cholinergic dysfunction occurring in neurodegenerative diseases.