The Influence of Calmodulin on Steroid Synthesis in Ley dig Cells from Rat Testis*

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Abstract
Two approaches were used to study the possible role of calmodulin in the regulation of synthesis of testosterone by Leydig culls: 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 → 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.