Ca2+ ion accumulation precedes formation of O2. in isolated brain mitochondria

The relationship between mitochondrial Ca2+ accumulation and reactive oxygen species formation is studied by means of fast kinetics with fluorescence detection. Succinate or elevated [Na+] and [Ca2+] provided alternative conditions for induction of reactive oxygen species in isolated rat cerebrocortical mitochondria. Depending on Na+/Ca2+ exchanger and superoxide dismutase activities, formation of reactive oxygen species in mitochondria exposed to elevated [Na+] and [Ca2+] levels was characterized by t(1/2) = 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 [Ca2+] > or = 2.5 microM. Mitochondrial Ca2+ accumulation obeyed similar [Ca2+] dependence but shorter t(1/2) (2.5+/-0.3 ms), providing kinetic evidence for Ca2+ accumulation-dependent primary formation of O2-* in brain mitochondria.