Uncoupling is without an effect on the production of reactive oxygen species by in situ synaptic mitochondria

Earlier reports that generation of reactive oxygen species (ROS) by isolated mitochondria supported by succinate was sensitive to small changes in the mitochondrial membrane potential (ΔΨm) served as a basis for the concept of 'mild uncoupling' suggesting that a few millivolts decrease in ΔΨm would be beneficial in neuroprotection because of reducing the production of ROS by mitochondria. In this study, we tested whether ROS generation by in situ mitochondria, which function in a normal cytosolic environment and oxidize glucose-derived physiological substrates, is also dependent on changes in ΔΨm. The release of H₂O₂ was measured by the Amplex red fluorescence assay in freshly prepared isolated nerve terminals, synaptosomes incubated in a glucose-containing medium. ΔΨm was decreased by the uncoupler carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) (10-200 nmol/L), which accelerated the oxygen consumption, decreased the NADH level and induced depolarization, as shown by the fluorescence indicator JC-1, in in situ mitochondria. These changes were detected at already the smallest FCCP concentration. H₂O₂ generation, however, was found to be unaltered by FCCP at any of the applied concentration. Depolarization of mitochondria was also induced by veratridine (40 μmol/L), which enhances the cytosolic Na⁺ concentration and imposes an ATP demand in synaptosomes. The accelerated oxygen consumption and the small depolarization of in situ mitochondria by veratridine were not paralleled by any significant alteration in the ROS generation. These findings indicate that a basal ROS generation by in situ mitochondria is not sensitive to changes in ΔΨm challenging the rational of the 'mild uncoupling' theory for neuroprotection and suggest that the ΔΨm-dependent characteristics of ROS generation is limited mainly to well-coupled succinate-supported isolated mitochondria.