Intracellular movement of cholesterol in rat adrenal cells. Kinetics and effects of inhibitors.

Adrenocorticotropin (ACTH)-stimulated cholesterol transport to mitochondria has been quantitated in rat adrenals in vivo and in adrenal cell suspensions. The initial rate of transport (1.25 pg/mg of mitochondria/ min) is similar in vivo and in cell suspensions, but the mitochondrial cholesterol level reaches higher levels in uivo. ACTH stimulation of cholesterol transport into mitochondria when cholesterol side chain cleavage is inhibited by aminoglutethimide (AMG) (3.1 nmol/107 cells/min) greatly exceeds both ACTH-stimulated steroidogenesis (0.5 nmol/107 cells/min) in absence of AMG and, by implication, the rate of cholesterol transport during steroidogenesis in absence of AMG. Both cholesterol transport within adrenal cells and steroidogenesis are inhibited by cytochalasin B (antimicrofilament agent) and vinblastine (antimicrotubule agent) while only steroidogenesis is inhibited by cycloheximide (protein synthesis inhibitor). Cytochalasin inhibits cholesterol transport to mitochondria more effectively ( I D S O = 6 to 7 SM) than total steroidogenesis in rat adrenals. These inhibitory effects occur both with in vitro cell suspensions and in vivo. Both inhibitors acted rapidly in uitm: cytochalasin, tl/2 = 4% min; vinblastine, t l I f = 2% min. Release from AMG inhibition in superfused cells generated an “overshoot” in steroid efflux from the cells in which steroidogenesis reached 160% of the normal maximum ACTH-stimulated rate. A direct correspondence has been established between excess steroidogenesis in this overshoot period and the accumulation of mitochondrial cholesterol. Both cholesterol accumulation and overshoot reach peak values after 7 to 8 min of exposure to AMG, followed by a decline. This late decline in mitochondrial cholesterol levels was prevented by both cytochalasin B and vinblastine. It is concluded that microfilaments and microtubules are involved in the transport of cholesterol both to and from the mitochondria while coupling of protein synthesis and steroidogenesis is expressed ultimately entirely on the rate-limiting transference of cholesterol to cytochrome P-450 within the mitochondria. R a t adrenal cell suspensions did not show the marked stimulation of steroidogenesis by cytochalasin B in the presence of serum lipoproteins which has been reported for Y-1 adrenal tumor cells. The in vivo effects of cytochalasin on rat adrenals suggest that intracellular transfer of cholesterol rather than transfer from plasma lipoproteins is critical to the rate of corticosterone synthesis during short term stimulation of rat adrenals.