Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca(2+)-Independent Mechanism

Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca(2+) accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca(2+) from the ryanodine receptor and enforcing its mitochondrial accumulation. l-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca(2+) and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca(2+), or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca(2+)-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite.