PEROXYNITRITE FORMATION FROM MACROPHAGE-DERIVED NITRIC-OXIDE

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-l-arginine prevented the Cu,Zn Superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 ± 2 pmol · 106 cells-1 · min-1 (mean ± STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol · 106 cells-1 · min-1. This upper estimate was consistent with N-methyl-l-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol · 106 cells-1 · min-1. The rate of nitrite and nitrate accumulation was 0.10 ± 0.001 nmol · 106 cells-1 · min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury. © 1992.