Highly purified anti-actin was prepared from sera of patients suffering from hepatitis. The antibody was entrapped within negatively charged vesicles made from phosphatidylcholine and phosphatidylserine (9:1 molar ratio). Leydig cells prepared from testes of adult rats were incubated for one hour with liposomes containing buffer (control) or buffer with anti-actin. Cells were washed and incubated with saline (control), LH (50 μg/ml) or db cyclic AMP (2 mM) for 30 min. Entrapped anti-actin inhibited three steroidogenic responses of the cells to LH: (i) increased production of testosterone by Leydig cells, (ii) increased side-chain cleavage (cholesterol → pregnenolone) by isolated mitochondria prepared from cells previously incubated with either of these stimulating agents, and (iii) increase in the cholesterol content of inner mitochondrial membranes isolated from cells incubated with aminoglutethimide (an inhibitor of side-chain cleavage) and LH. Free anti-actin (no liposomes) produced no more than slight inhibition, while boiled anti-actin, human IgG, anti-actin with excess actin and bovine serum albumin captured within liposomes were all without effect on the response to LH. In addition, anti-actin was without effect on side-chain cleavage when added to isolated mitochondria and on the conversion of [3H]-pregnenolone to [3H]-testosterone. These findings, together with previous evidence, strongly suggest that LH and cyclic AMP stimulate side-chain cleavage of cholesterol (and hence steroidogenesis) by a mechanism involving actin-presumably in microfilaments; the responding microfilaments promote transport of cholesterol to mitochondria.
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