Modulation of Steroidogenesis by Chloride Ions in MA-10 Mouse Tumor Leydig Cells: Roles of Calcium, Protein Synthesis, and the Steroidogenic Acute Regulatory Protein*

Abstract

It has previously been shown that omission of extracellular chloride ions during culture of rat Leydig cells markedly enhances LH-stimulated steroidogenesis. In the present study, the mechanisms of the effect of chloride omission on (Bu)2cAMP-stimulated steroidogenesis in MA-10 mouse Leydig tumor cells have been investigated. It was found that chloride omission enhanced progesterone production 2- and 4-fold in the absence and presence, respectively, of submaximally stimulating levels of (Bu)2cAMP (0.1 mM) during incubation for 2 h. This enhancement of stimulation increased continuously with time, because after 6 h, (Bu)2cAMP-stimulated progesterone production was 15-fold higher in the absence of chloride. These effects were not found in the presence of maximum stimulating levels of (Bu)2cAMP (1 mM). Omission of calcium from the incubation medium decreased (Bu)2cAMP-stimulated progesterone production by over 70% in the presence and absence of chloride. Progesterone production was still enhanced by the omission of chloride in the absence of calcium, but the effects were less marked than those in the presence of calcium. Addition of the protein synthesis inhibitor, cycloheximide, completely inhibited (Bu)2cAMP-stimulated, but not basal, steroidogenesis in the absence and presence of chloride ions during 2- and 6-h incubation. Total protein synthesis (measured by the incorporation of [3H]methionine) was 4-fold higher in cells incubated in chloride-free medium compared with that in cells incubated in chloride-replete medium in the presence of 0.1 mM (Bu)2cAMP. No effects were found on basal levels. Several proteins specific to the steroidogenic machinery were quantified in mitochondria isolated from cells incubated with and without chloride by Western blot analysis after separation by PAGE. Omission of chloride increased (4-fold) the level of the steroidogenic acute regulatory (StAR) protein in the cells incubated with (Bu)2cAMP (0.1 mM). There was no increase in either the levels or activities of cytochrome P450 cholesterol side-chain cleavage enzyme (cytP450scc) or 3beta-hydroxysteroid dehydrogenase. No effects were found on the basal level of any of the proteins measured. These results are consistent with a cAMP-dependent regulatory role of chloride ion efflux in the control of steroidogenesis, which requires protein synthesis. It is proposed that this occurs by increases in StAR protein synthesis via a general increase in cAMP-dependent protein synthesis and/or by enhancement of the steroidogenic effects of StAR.

Regulation of the Steroidogenic Acute Regulatory Protein Expression: Functional and Physiological Consequences

“…In this study, the modulation of steroidogenesis was shown to be affected by the omission of Ca 2+ from the incubation medium in the presence or absence of chloride ions. Using the same experimental paradigms in mouse Leydig cells, a potential link has been demonstrated between steroid synthesis and StAR protein expression, but not with P450scc and 3β-HSD expression, further indicating the direct involvement of StAR in the regulation of steroidogenesis [93].…”

“…In addition, a chloride channel blocker 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulphonic acid inhibited LH induced steroidogenesis with the former condition, suggesting the independent involvement of chloride channels and cAMP pathways [241]. The omission of extracellular Cl -has been demonstrated to enhance steroid synthesis in response to submaximal cAMP analog treatment in MA-10 cells [93]. In this study, the modulation of steroidogenesis was shown to be affected by the omission of Ca 2+ from the incubation medium in the presence or absence of chloride ions.…”

“…Likewise, several additional signaling pathways have also been demonstrated to play important roles in regulating StAR expression. These include calcium regulated transduction systems [59,[86][87][88][89][90], arachidonic acid metabolites [91,92], chloride ion channels [93,94], and mitogen activated protein Kinase (MAPK)/extracellular signal-related kinases (ERKs) [95,96].…”

“…While the action of hCG has been shown to be independent of extracellular Ca 2+ , addition of Ca 2+ to mouse Leydig cells has been demonstrated to enhance the stimulatory action of hCG on progesterone production, StAR mRNA and StAR protein levels [88]. Regulation of StAR expression and aldosterone synthesis has been demonstrated to increase in response to the Ca 2+ -mobilizing agonists, angiotensin II (Ang II) and potassium (K + ), in adrenal cells [86,93,222]. In H295R adrenocortical cells, inhibition of Ang II and K + actions markedly inhibited aldosterone synthesis without altering the induction of StAR expression [86,222].…”

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

“…Hormonal signal transduction and steroidogenesis can be affected by changes in intracellular and extracellular concentrations of inorganic ions, cellular resting membrane potential, and cell size [29,30,35,54-59]. Blockade of 4-AP sensitive K + channels in non-nerve, non-muscle cells can lead to changes in transmembrane ion gradients, membrane potential and cell volume [42,60].…”