Ca2+ ion accumulation precedes formation of O·−2. in isolated brain mitochondria

The relationship between mitochondrial Ca accumulation and reactive oxygen species formation is studied by means of fast kinetics with fluorescence detection. Succinate or elevated [Na] and [Ca] provided alternative conditions for induction of reactive oxygen species in isolated rat cerebrocortical mitochondria. Depending on Na/Ca exchanger and superoxide dismutase activities, formation of reactive oxygen species in mitochondria exposed to elevated [Na] and [Ca] levels was characterized by t1/2=12.3±2.9 ms. Production of reactive oxygen species by xanthine oxidase followed a similar time course. Both the processes were blocked by para-benzoquinone, indicating the primary formation of O2·. Substantial increase in mitochondrial O2· was observed at [Ca]≥2.5 μM. Mitochondrial Ca accumulation obeyed similar [Ca] dependence but shorter t1/2 (2.5±0.3 ms), providing kinetic evidence for Ca accumulation-dependent primary formation of O2· in brain mitochondria.