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 microM 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 (Km = 35 microM 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 (alpha less than 0.001) or ATP (alpha less than 0.01); ATP plus oligomycin, however, had no effect. The results are discussed in relation to the oxygen free radical theory of reperfusion injury.
Vlessis
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Department of Surgery, Oregon Health Sciences University, Portland 97201.
Mela-Riker
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002374:Catalase / Q000494:pharmacology; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D051381:Rats; D011919:Rats, Inbred Strains; D015427:Reperfusion Injury / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism
D007042:Hypoxanthines; D014970:Xanthines; D009243:NAD; D019820:Xanthine; D019271:Hypoxanthine; D009249:NADP; D002374:Catalase; D013482:Superoxide Dismutase; D002118:Calcium