The effect of azastene, cyanoketone and trilostane upon respiration and cleavage of the cholesterol side chain in mitochondria from bovine adrenal cortex.

Abstract

Mitochondria prepared from bovine adrenal cortex and incubated with ADP and phosphate respired at about 45% of the rat observed in the presence of an uncoupler of oxidative phosphorylation; there was, however, little inefficiency in the reactions involved in the phosphorylation of ADP. Three inhibitors of the 3 beta-hydroxysteroid dehydrogenase (azastene, cyanoketone and trilostane) were employed with a view to preventing pregnenolone metabolism and thus aiding the assay of cholesterol side-chain cleavage. Freshly made solutions of these inhibitors did not modify mitochondrial respiratory rates, at concentrations of 10 microM. In contrast, solutions maintained at 0-4 degrees C for one week subsequently inhibited the respiratory rate of uncoupled mitochondria. When fresh solutions of the inhibitors were used in the assays of cholesterol side-chain cleavage, 10 microM azastene did not significantly inhibit pregnenolone metabolism. Cyanoketone and trilostane were both significant inhibitors of pregnenolone metabolism, but 10 microM cyanoketone reduced the initial rate of cholesterol side-chain cleavage by 50% in the presence of 10 mM malate, although this inhibition did not occur in the presence of 10 mM DL-isocitrate. Thus trilostane may be the preferred inhibitor of the 3 beta-hydroxysteroid dehydrogenase during studies of cholesterol side-chain cleavage in vitro.