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±2.9ms. 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]2.5μM. Mitochondrial Ca accumulation obeyed similar [Ca] dependence but shorter t1/2 (2.5±0.3ms), providing kinetic evidence for Ca accumulation-dependent primary formation of O2• in brain mitochondria.
- Isolated brain mitochondria
- Mitochondrial calcium ion accumulation
- Stopped lowmeasurements
- Subsecond kinetics
- Superoxide production in mitochondria
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