Effect of Peroxide, Sodium, and Calcium on Brain Mitochondrial Respiration In Vitro: Potential Role in Cerebral Ischemia and Reperfusion

Mitochondrial pyruvate‐supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca²⁺, Na⁺, and peroxide. Ca²⁺ alone (7–10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert‐butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na⁺ or tBOOH in combination with Ca²⁺ dramatically altered respiration. Respiratory inhibition induced by Ca²⁺ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R= 0.96). Calcium potentiated tBOOH‐induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na⁺ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium‐ as well as tBOOH‐induced state 3 inhibition required mitochondrial Ca²⁺ uptake because ruthenium red addition before Ca²⁺ addition negated the effect. The increase in state 4 respiration involved Ca²⁺ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca²⁺‐, Na⁺‐, and tBOOH‐induced alterations in pyruvate‐supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.