Isolated mitochondria were energized with pyruvate (Panel A), succinate (Panel B), or succinate in the presence of rotenone (Panel C). Rotenone was added to the mitochondrial suspension at time 0. In a modified protocol, isolated mitochondria were energized with succinate, and then rotenone was added followed by pyruvate (Panel D). A similar protocol was used but with the addition of antimycin A after all other additions and in the presence of amplex red to measure H2O2 (Panel E). Mitochondria were pre-incubated with either 0 μM CaCl2 (Trace 1) or with 100 μM CaCl2 (Trace 2). The number of animals ranged between 6–8 per group. All NADH figures have the same scale. Abbreviations: MIT, mitochondria (0.5 mg/ml); CON, control (H2O); PYR, pyruvate (10 mM); SUC, succinate (10 mM); ROT, rotenone (10 μM); AA, antimycin A (5 μM).