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 chorionic 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(2+) entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca(2+) 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.
Lee
Jae-Ho
JH
Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 330714, South Korea.
Kim
Jong-Uk
JU
Kim
Changhoon
C
Min
Churl K
CK
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000187:drug effects / Q000502:physiology*; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D020748:Mibefradil / Q000494:pharmacology*; D051379:Mice; D008813:Mice, Inbred ICR; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism*; D013256:Steroids / Q000096:biosynthesis*; D000098682:Steroidogenic Acute Regulatory Protein
D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D006063:Chorionic Gonadotropin; D010750:Phosphoproteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D020748:Mibefradil; D002118:Calcium