Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

Abstract

The effect of Ca2+ applied in high concentrations (50 and 300µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H2O2 release from mitochondria was induced by Ca2+ applied in 50µM, but not in 300µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150sec after exposure to Ca2+. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca2+ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca2+ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca2+ given in 50µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H2O2 was significantly higher than that observed in a medium containing ADP and Ca2+ no longer increased but rather decreased the rate of H2O2 release. With 300µM Ca2+ only a loss but no tendency of a recovery of ΔΨm was detected and H2O2 release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca2+ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H2O2 release is induced by calcium, but only in ≤ 100µM concentration, when after a transient Ca2+-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca2+-induced depolarization, Ca2+ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300µM) are unrelated to mitochondrial permeability transition.