Cortisol acts directly in human granulosa-lutein cells to inhibit the support of steroidogenesis by LH and this interaction is modulated by ovarian 11 beta HSD in the majority of patients.

Progesterone production by dispersed luteal cells obtained from the marmoset monkey on day 14 after ovulation can be stimulated by both prostaglandin F2 alpha (PGF2 alpha) and its structural analogue, cloprostenol. To establish whether these responses can be attributed to cross-reaction with the prostaglandin E2 (PGE2) receptor, this study compared the involvement of cyclic adenosine-3',5'-monophosphate (cAMP) and protein kinase C (PKC) in the luteotrophic responses to PGE2, PGF2 alpha and cloprostenol. While all three prostaglandins stimulated similar increases in progesterone production (239.5 +/- 7.9% of control; P < 0.01), only PGE2 stimulated a significant increase in cAMP accumulation (373.2 +/- 28.4% of control; P < 0.01). This study is the first to demonstrate PKC activity in the marmoset ovary. Following down-regulation of PKC with a tumour-promoting phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA), basal progesterone production was significantly increased (150.9 +/- 8.2% of control; P < 0.05) and the luteotrophic effects of PGF2 alpha and cloprostenol were no longer evident, whereas the response to PGE2 was unaffected. These observations are consistent with the differential involvement of cAMP and PKC in the luteotrophic responses to PGE2 and PGF2 alpha/cloprostenol respectively. Hence, we conclude that the luteotrophic actions of prostaglandins E2 and F2 alpha on dispersed marmoset luteal cells are mediated via different receptors and signal transduction pathways.

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.

1. The type 2 isoform of 11beta-hydroxysteroid dehydrogenase, an enzyme which converts cortisol or corticosterone to inactive 11-ketosteroid metabolites, is thought to be responsible for preventing access of endogenous glucocorticoids to mineralocorticoid receptors in the distal nephron; although direct in vivo evidence for this is still lacking. We have examined whether graded inhibition of renal 11beta-hydroxysteroid dehydrogenase activities in vivo results in corresponding changes in urinary electrolyte excretion due to exposure of mineralocorticoid receptors to circulating endogenous glucocorticoids.2. Anaesthetized rats were infused intravenously with vehicle alone or with one of three doses of carbenoxolone: 0.06, 0.6 or 6 mg/h. After measurement of renal electrolyte excretion, the kidneys were snap-frozen in liquid nitrogen and 11beta-hydroxysteroid dehydrogenase activities were measured directly by enzyme assay in the presence of NAD+ or NADP+.3. A dose-dependent inhibition of renal 11beta-hydroxysteroid dehydrogenase activities was observed: the low, intermediate and high doses of carbenoxolone causing approximately 50%, 80% and >90% inhibition respectively. Only with the high dose was an effect on renal function observed (decreased fractional Na+ excretion and urinary Na+/K+ ratio).4. The poor correlation between the extent of inhibition of renal 11beta-hydroxysteroid dehydrogenase and altered urinary Na+ excretion, apparent at the lower doses of carbenoxolone, suggests either that 11beta-hydroxysteroid dehydrogenase has considerable functional reserve, or that it may not be the only mechanism determining mineralocorticoid receptor specificity in the distal nephron.