The mechanism of the prolonged stimulatory effect of corticotrophin on pregnenolone production by guinea-pig adrenocortical mitochondria

Abstract

ABSTRACT The postulated prolonged stimulatory influence of ACTH on the adrenocortical mitochondrial synthesis of pregnenolone in response to ACTH was studied in adrenal mitochondria isolated from control guinea-pigs and from animals treated s.c. with 100 μg ACTH(1–24) twice daily on the day before the animals were killed. The animals from both groups were injected with 100 μg ACTH s.c. 30 min before killing. The mitochondrial production of pregnenolone (expressed in nmol per mg mitochondrial protein after 10-min incubation) increased from 1·52 ± 0·46 (s.e.m.) in the control group to 4·50 ± 0·59 for mitochondria from ACTH-treated animals, despite a similar free cholesterol content in the mitochondria, even when determined after a previous in-vivo treatment with aminoglutethimide to block further metabolism of cholesterol into pregnenolone. In addition, in the presence of an excess of exogenous cholesterol (100 μmol/l), the production of pregnenolone remained higher for mitochondria from ACTH-treated animals. In contrast, when the calcium concentration in the incubation medium was raised to 1 mmol/l, with subsequent enhancement in pregnenolone synthesis, the mitochondrial pregnenolone production became similar for both groups (8·28 ± 1·11 nmol in the ACTH-treated group and 9·55 ± 1·90 nmol in the control group), even in the presence of 100 μmol cholesterol/l (13·5 ±1·80 nmol in ACTH-treated animals and 14·8 ± 1·93 nmol in controls). Cycloheximide treatment administered on the day before the animals were killed was without any effect on pregnenolone production in control animals (3·51 ± 0·43 nmol before and 3·65 ± 0·63 nmol after cycloheximide treatment). In contrast, the pregnenolone production by mitochondria from ACTH-treated guinea-pigs injected with cycloheximide was reduced to a level similar to that observed in the control group (5·82± 0·23 nmol in the absence and 3·09± 0·32nmol in the presence of cycloheximide treatment). The results of the present study suggest that the prolonged stimulatory effect of ACTH on pregnenolone production involves an increase in the synthesis of a protein factor which promotes the availability of cholesterol to the side-chain cleavage system. Other mechanisms such as a stimulated synthesis of the side-chain cleavage complex itself or an increased total mitochondrial cholesterol content are not supported by our data. J. Endocr. (1986) 108, 377–384