CALCIUM CONTENT AND RESPIRATORY CONTROL INDEX OF ISOLATED SKELETAL-MUSCLE MITOCHONDRIA - EFFECTS OF DIFFERENT ISOLATION MEDIA

Calcium (Ca) content measured with graphite furnace atomic absorption spectrometry and polarographical measurement of respiratory control in dex (RCI) were performed on isolated skeletal muscle mitochondria. Fou r different isolation media were used in order to develop a method for isolating physiologically intact mitochondria in small samples with a n endogenous Ca content as close as possible to in vivo conditions. Wi th ruthenium red in the isolation medium, the mean Ca content decrease d fourfold to 19.3 +/- 6.8 nmol/mg protein and RCI increased about 25% to 6.9 +/- 1.1 compared with a standard medium. With EGTA or EG;TA pl us ruthenium red, the mean Ca content decreased further to 5.8 +/- 1.0 and 5.1 +/- 0.9 nmol/mg protein and the mean RCI values increased to 8.4 +/- 1.2 and 8.8 +/- 1.3, respectively. Isolated mitochondria from human muscle samples using EGTA and ruthenium red in the isolation med ium had mean Ca values of 11.0 +/- 0.9 nmol/mg protein and mean RCI va lues of 12.2 +/- 1.6. The data indicate that the Ca concentration in t he skeletal muscle mitochondria is inversely proportional to RCI, and the mitochondrial functional property is significantly improved upon l owering the intramitochondrial Ca accumulation by the incorporation of a chelating agent (EG;TA) and a potent Ca antagonist, ruthenium red, in the isolation media. (C) 1996 Academic Press, Inc.