Modulation of steroidogenesis by chloride ions in MA-10 mouse tumor Leydig cells: roles of calcium, protein synthesis, and the steroidogenic acute regulatory protein.

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.