Potential role of mitochondrial calcium metabolism during reperfusion injury

Ischemia-reperfusion injury has been associated with intracellular H₂O₂ and superoxide radical production from accumulated hypoxanthine (HX) and xanthine oxidase (XO). The effect of H₂O₂ and superoxide radical on mitochondrial Ca²⁺ efflux was characterized in isolated renal mitochondria using a HX-XO system. Mitochondria were suspended in buffered medium containing 200 μM HX. Extramitochondrial Ca²⁺ was monitored kinetically at 660-685 nm using the Ca²⁺ indicator arsenazo III. After preloading mitochondria with 18-25 nmol Ca²⁺/mg protein, addition of XO to the medium caused a rapid oxidation of mitochondrial NAD(P)H followed by Ca²⁺ release. Ca²⁺ efflux was attributed to mitochondrial metabolism of H₂O₂ because efflux could be prevented with catalase but not superoxide dismutase. The Ca²⁺ efflux rate (r = 0.995) and lag time to Ca²⁺ efflux (r = 0.987) both correlate well with the NAD(P)H oxidation rate. Exogenous ATP prevents Ca²⁺ efflux in a dose-dependent fashion (K(m) = 35 μM ATP) without affecting NAD(P)H oxidation; ATP plus oligomycin, however, had no effect. The protective effect of ATP on Ca²⁺ efflux was diminished by ruthenium red (RR). XO-induced Ca²⁺ efflux increased state 4 respiration 148% via a futile Ca²⁺ cycle involving the Ca²⁺ uniport. The increase in state 4 respiration could be reversed with RR (α < 0.001) or ATP (α < 0.01); ATP plus oligomycin, however, had no efect. The results are discussed in relation to the oxygen free radical theory of reperfusion injury.