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

Abstract

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.

Figure 1

Microphotographs of Leydig cells after acute isolation from interstitial tissues of mouse testis. (A): Mouse Leydig cells were mechanically isolated and viewed microscopically as indicated by the arrows. (B): Acutely dissociated mouse Leydig cells were subjected to immunostaining by 3-β-hydroxysteroid dehydrogenase (3-β-HSD), a cellular marker for Leydig cells, and viewed microscopically as indicated by the arrows. Bars = 100 μm, bars = 25 μm in inset panels.

Figure 2

Inhibitory effects of mibefradil on hCG-stimulated testosterone production in mouse Leydig cells. Purified mouse Leydig cells were incubated in DMEM/F12 medium in the presence (A, C) or absence of 1.0 mmol L⁻¹ Ca²⁺ (B). (A): The onset of steroidogenesis was induced by adding hCG at a concentration ranging from 1 to 100 IU for 30 min or for 1 h. (B): the control experiment is done with Ca²⁺ in the presence of 100 IU hCG whereas the other two are done without Ca²⁺. (C): The cells were all treated with 100 IU hCG to induce testosterone production in the presence of mibefradil, ranging from at 0.01 to 1.00 mmol L⁻¹. All data are means ± SD (n = 5) compared with controls respectively ([0 hCG, A]; [100 IU hCG with Ca²⁺, B]; [0 mibefradil, C]). ^*P < 0.05, compared with corresponding control.

Figure 3

Expression of StAR mRNA in mouse Leydig cells is inhibited by mibefradil. Acutely purified mouse Leydig cells were incubated in 1.0 mmol L⁻¹ Ca²⁺-containing DMEM/F12 medium and stimulated by 100 IU hCG treatment in the presence of mibefradil at 0.01, 0.1 or 1.0 mmol L⁻¹ for 1 h before StAR mRNAs were semi-quantitatively amplified in RT-PCR. (A) A representative RT-PCR profile of StAR mRNA expression. (B): The band intensities of StAR mRNA products were quantified densitometrically and expressed relative to control (that is, no mibefradil). Data are means ± SD of results from five independent experiments. ^*P < 0.05 compared with control (100%).

Figure 4

The blockade of T-type Ca²⁺ current (I_CaT) by mibefradil in mouse Leydig cells. Acutely dissociated mouse Leydig cells were bathed in a bath solution containing 2 mmol L⁻¹ CaCl₂, 5 mmol L⁻¹ 4-aminopyridine, 136 mmol L⁻¹ TEA-Cl, 1.1 mmol L⁻¹ MgCl₂, 25 mmol L⁻¹ HEPES and 22 mmol L⁻¹ glucose, pH 7.4, and held at −100 mV. A command pulse was applied to −30 mV for 40 msec. Representative I_CaT traces were generated in the presence or absence of 1.0 μmol L⁻¹ mibefradil. The capacitive transients were suppressed from the traces for the clarity.