Volume 12, Issue 6 (November 2010) 12, 807–813; 10.1038/aja.2010.51
Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells: involvement of Ca2+ entry via the T-type Ca2+ channel
Jae-Ho Lee1, Jong-Uk Kim2, Changhoon Kim3, Churl K. Min4
1 Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 330714, South Korea
2 Department of Molecular Sciences and Technology, Ajou University, Suwon 443749, South Korea
3 Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA 30322, USA
4 Department of Biological Sciences, Ajou University, Suwon 443749, South Korea
Correspondence: Dr Churl K. Min,email@example.com
Received 10 March 2010; Revised 8 April 2010; Accepted 15 April 2010; Published online 9 August 2010.
Intracellular cAMP and Ca2+ are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone (LH) and human chorionic gonadotropin (hCG). However, the identification of Ca2+ entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca2+ 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 Ca2+ 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 Ca2+, 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 Ca2+ was omitted, implying that Ca2+ entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca2+ 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 Ca2+ entry carried out by the T-type Ca2+ channel in the Leydig cells of mice.
Leydig cells; mibefradil; StAR; steroidogenesis; T-type Ca2+ channel