Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria.

Abstract

1 In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O(2)(-)) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P)H, cytochrome c release, and caspase activity. 2 Addition of the reactive oxygen species (ROS) generator KO(2) produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca(2+) independent. 3 Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O(2)(-) production detected with hydroethidine in mitochondrial preparations exposed to Ca(2+) and the fact that ROS were required for Ca(2+)-induced mitochondrial swelling. 4 Superoxide anions, but not Ca(2+), decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD(P)H levels. 5 Calcium, but not O(2)(-), triggered a rapid loss of mitochondrial potential. Calcium-induced Deltapsi(m) dissipation was inhibited by Ruthenium Red, but not by CSA. 6 Calcium- and superoxide-induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A-sensitive manner. 7 In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization.