The ability of cytochalasin B to inhibit the steroidogenic response of mouse adrenal tumor cells (Y-1) to adrenocorticotropin (ACTH) was examined with two aims: to consider the specificity of the inhibitor and to determine at what point(s) in the steroidogenic pathway it acts. Cytochalasin B did not inhibit protein synthesis or transport of [3H]-cholesterol into the cells nor did it alter total cell concentration of ATP. Together with previous evidence, this suggests that the effects of cytochalasin observed are relatively specific in these cells. Cytochalasin inhibits the increase in conversion of [3H]cholesterol to 20alpha-[3H]dihydroprogesterone (20alpha-hydroxypregn-4-en-3-one: a major product of the steroid pathway in Y-1 cells) produced by ACTH but does not inhibit conversion of cholesterol to pregnenolone by mitochondrial and purified enzyme preparations from Y-1 cells and bovine adrenal, respectively. Cytochalasin does not inhibit the conversion of pregnenolone to 20alpha-dihydroprogesterone but was shown to inhibit increased transport of [3H]cholesterol to mitochondria resulting from the action of ACTH. These findings indicate that cytochalasin acts after cholesterol has entered the cells and before it is subjected to side-chain cleavage in mitochondria. In view of the known action of cytochalasin on microfilaments, it is proposed that these organelles are necessary for the transport of cholesterol to the mitochondrial cleavage enzyme and that at least one effect of ACTH (and cyclic AMP) is exerted upon this transport process. The specificity of the effects of cytochalasin is considered in relation to this conclusion.

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Two approaches were used to study the possible role of calmodulin in the regulation of synthesis of testosterone by Leydig cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. The inhibitor prevented the expected responses of Leydig cells to LH and to cAMP. First the increase in synthesis of testosterone produced when these agents are added to Leydig cells was inhibited by the drug. Second, increased transport of cholesterol to mitochondria produced by LH and cAMP was inhibited by trifluoperazine. Third, increased side-chain cleavage of cholesterol (cholesterol leads to pregnenolone) produced by these agents in isolated mitochondria was also inhibited by the drug. When Leydig cells were incubated with liposomes containing calmodulin, production of testosterone, transport of cholesterol to mitochondria, and side-chain cleavage of cholesterol were all stimulated. The effect of calmodulin is greater if Ca2+ is added before incorporation into liposomes than if calmodulin and Ca2+ are introduced into the Leydig cells from separate liposomes. Stimulation of testosterone synthesis does not occur if calmodulin is dialyzed against EGTA, if calmodulin with excess anticalmodulin is present in the liposomes, if either calmodulin or Ca2+ is added to the medium (no liposomes), or if Ca2+ alone is present in liposomes. These observations suggest that calmodulin is involved in regulating the transport of cholesterol to mitochondria, a process that is stimulated by LH and cAMP and one that may account for the increased steroid synthesis produced by these agents.

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