Uptake, retention, and efflux of Ca 2+ by mitochondrial preparations from skeletal muscle

Abstract

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca 2+-handling properties examined. When judged by their capacity to retain large Ca 2+ loads and the magnitude of basal and Na +-stimulated Ca 2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca 2+-dependent respiratory control and tolerance of the mitochondria to Ca 2+ loads, probably by removing Mg 2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca 2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca 2+/mg while maintaining a low basal Ca 2+ efflux. Addition of BSA to the assay medium slightly improved Ca 2+ retention but was not essential either during isolation or assay. Ca 2+-dependent state 3 respiration was maximal at pH 6.5–7.0 while respiratory control and Ca 2+ O were optimal at pH 7.0–7.5. Neither P i nor oxaloacetate induced Ca 2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na +-stimulated Ca 2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.