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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.

SUBMITTER: Lee JH

PROVIDER: S-EPMC3739071 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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

Lee Jae-Ho JH Kim Jong-Uk JU Kim Changhoon C Min Churl K CK

Asian journal of andrology 20100809 6

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 indu ...[more]

PMID: 20694017

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Project description:At chemical synapses, voltage-activated calcium channels (VACCs) mediate Ca2+ influx to trigger action potential-evoked neurotransmitter release. However, the mechanisms by which Ca2+ regulates spontaneous transmission have not been fully determined. We have shown that VACCs are a major trigger of spontaneous release at neocortical inhibitory synapses but not at excitatory synapses, suggesting fundamental differences in spontaneous neurotransmission at GABAergic and glutamatergic synapses. Recently, VACC blockers were reported to reduce spontaneous release of glutamate and it was proposed that there was conservation of underlying mechanisms of neurotransmission at excitatory and inhibitory synapses. Furthermore, it was hypothesized that the different effects on excitatory and inhibitory synapses may have resulted from off-target actions of Cd2+, a nonselective VACC blocker, or other variations in experimental conditions. Here we report that in mouse neocortical neurons, selective and nonselective VACC blockers inhibit spontaneous release at inhibitory but not at excitatory terminals, and that this pattern is observed in culture and slice preparations as well as in synapses from acute slices of the auditory brainstem. The voltage dependence of Cd2+ block of VACCs accounts for the apparent lower potency of Cd2+ on spontaneous release of GABA than on VACC current amplitudes. Our findings indicate fundamental differences in the regulation of spontaneous release at inhibitory and excitatory synapses by stochastic VACC activity that extend beyond the cortex to the brainstem.SIGNIFICANCE STATEMENT Presynaptic Ca2+ entry via voltage-activated calcium channels (VACCs) is the major trigger of action potential-evoked synaptic release. However, the role of VACCs in the regulation of spontaneous neurotransmitter release (in the absence of a synchronizing action potential) remains controversial. We show that spontaneous release is affected differently by VACCs at excitatory and inhibitory synapses. At inhibitory synapses, stochastic openings of VACCs trigger the majority of spontaneous release, whereas they do not affect spontaneous release at excitatory synapses. We find this pattern to be wide ranging, holding for large and small synapses in the neocortex and brainstem. These findings indicate fundamental differences of the Ca2+ dependence of spontaneous release at excitatory and inhibitory synapses and heterogeneity of the mechanisms of release across the CNS.

Project description:BackgroundEtomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.MethodologyImmature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.Results and conclusionsIn intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.

Dataset Information

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

Publications

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

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