Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells： involvement of Ca^2＋ entry via the T-type Ca^2＋ channel

Intracellular cAMP and Ca^2＋ are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone （LH） and human ehorionic gonadotropin （hCG）. However, the identification of Ca^2＋ entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca^2＋ channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil （a putative T-type Ca^2＋ channel blocker） on steroidogenesis were assessed using reverse transcription （RT）-polymerase chain reaction analysis for the steroidogenic acute regulatory protein （STAR） mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L-1 extracellular Ca^2＋, hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion （P 〈 0.05）, and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner （P 〈 0.05）. Moreover; the hCG-induced increase in testosterone production was completely removed when external Ca^2＋ was omitted, implying that Ca entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca^24- currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, Our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca^2＋ entry carried out by the T-type Ca^2＋ channel in the Leydig cells of mice.