Inhibition of hormonal-induced cAMP and steroid production by inhibitors of pregnenolone metabolism in adrenal and leydig cells

doi:10.1016/0303-7207(88)90119-0

Molecular and Cellular Endocrinology

Volume 60, Issue 1, November 1988, Pages 55-60

https://doi.org/10.1016/0303-7207(88)90119-0 Get rights and content

Abstract

The effects of inhibitors of pregnenolone metabolism, WIN-24540 and spironolactone, on adrenocorticotropic hormone (ACTH)- and human chorionic gonadotropin (hCG)-induced cAMP and steroid production by bovine (BAC) and ovine (OAC) adrenal cells and pig Leydig cells (PLC) were investigated. The inhibitors reduced cAMP production by adrenal and Leydig cells by about 75% and 60%, respectively (P < 0.001). Further, the inhibitors also reduced the cholera toxin- and forskolin-induced cAMP production by pig Leydig cells. In the presence of the inhibitors, corticosterone and testosterone production by BAC and PLC, respectively, following hormonal stimulation was reduced by more than 90%. However, pregnenolone production by BAC and PLC under these conditions represented only 12% and 42% of the corticosterone and testosterone production, respectively, in the absence of inhibitors. Moreover, the inhibitors also reduced the steroidogenic response of PLC to 8-Br-cAMP and the conversion of 22(R)-hydroxycholesterol to pregnenolone by BAC and PLC. The reduced production of pregnenolone in the presence of inhibitors was in part due to the weak inhibition of 17α-hydroxylase by spironolactone. However, when OAC cells were incubated in the presence of WIN-24540 and SU-10603, a potent 17α-hydroxylase inhibitor, the amount of pregnenolone produced in response to ACTH or 22(R)-hydroxycholesterol was only 10% and 19%, respectively, of the steroids (corticosterone plus cortisol) secreted in the absence of inhibitors. The results show that the inhibitors of pregnenolone metabolism reduced, in both adrenal and Leydig cells, the response of adenylate cyclase to several effectors and the activity of the cholesterol side-chain cleavage. Therefore, caution should be taken in the interpretation of the results when these inhibitors are used.

References (25)

S.B. Cigorraga et al.
J. Biol. Chem.
(1978)
R.V. Farese et al.
Biochim. Biophys. Acta
(1978)
R.H. Menard et al.
Arch. Biochem. Biophys.
(1976)
R.H. Menard et al.
Steroids
(1978)
R.H. Menard et al.
J. Biol. Chem.
(1979)
M. Shikita et al.
Biochim. Biophys. Acta
(1965)
M. Bernier et al.
Eur. J. Biochem.
(1986)
M. Bernier et al.
Endocrinology
(1986)
P. Biffignandi et al.
Endocr. Rev.
(1984)
D.F. Child et al.
Acta Endocrinol
(1976)

A. Crozat et al.

Endocrinology

(1986)

E. De Peretti et al.

J. Clin. Endocrinol. Metab.

(1983)

Cited by (6)

Mineralocorticoid antagonists in the treatment of central serous chorioetinopathy: Review of the pre-clinical and clinical evidence
2019, Experimental Eye Research
Citation Excerpt :
Spironolactone and eplerenone have differential effects on steroidogenesis. Spironolactone at micromolar concentrations inhibits aldosterone biosynthesis (Netchitailo et al., 1985) and blocks enzymes involved in steroidogenesis (Penhoat et al., 1988). In contrast, eplerenone (1–30 mM) did not impair basal and angiotensin II-induced cortisol and aldosterone production in human adrenocortical H295R cells (Ye et al., 2009).
Central serous chorioretinopathy (CSCR) is part of the pachychoroid spectrum disorders, characterized by serous retinal detachments, retinal pigment epithelium alterations and dilation of choroidal vessels. No consensus exists regarding the clinical classification and the physiopathogenic mechanisms of the disease, delaying the comprehension of the most optimal treatment options. An overactivation of the mineralocorticoid receptor (MR) pathway in the choroid/retina has been suggested in CSCR. Since, MR antagonists could target the affected RPE/choroid in CSCR and have shown to act as disease modifier drugs inducing tissue remodeling in other organs (heart, kidney, vessels), we summarize here the pre-clinical and clinical evidence for using oral mineralocorticoid receptor antagonist in the treatment of CSCR.
Ontogenesis of cholesterol side-chain cleavage activity in the ovine adrenal during late gestation
1992, Journal of Steroid Biochemistry and Molecular Biology
The present study examined the activity of the cholesterol side-chain cleavage system, and the amount of cytochrome P450scc in adrenal glands of sheep fetuses and newborn lambs as well as the in vitro regulation of these parameters. Freshly isolated fetal adrenal cells incubated in the presence of 1 mM 8Br-cAMP or 25 μM 22R-OH cholesterol, produced 4- to 5-fold less pregnenolone than neonatal cells under similar conditions. Likewise, pregnenolone production by isolated fetal adrenal mitochondria was lower than that of neonatal mitochondria when endogenous cholesterol was used as a substrate or when 22R-OH cholesterol was added to the incubation medium. Also, the amount of P450scc, determined by immunoblot, was lower in fetal mitochondria than in neonatal mitochondria. In culture, ACTH, despite enhancing both the production of pregnenolone and the incorporation of [¹⁴C]acetate in cholesterol and its end-products by fetal adrenal cells, neither increased the amount of pregnenolone formed from 22R-OH cholesterol nor the amount of immunoreactive P450scc. By contrast, during the first 48 h of culture under standard conditions, there was a “spontaneous” increase in the activity of P450scc which reached values observed in neonatal adrenal cells. Such a development was inhibited when 5% ovine fetal serum was added to the culture medium. These results reinforce the view that in the ovine fetal adrenal gland, the development of P450scc is not ACTH-dependent but involves most probably a decrease in inhibitory factors present in fetal blood.
Pig Leydig cell culture: A useful in vitro test for evaluating the testicular toxicity of compounds
1991, Toxicology and Applied Pharmacology
In vivo studies have shown that the testis is a target organ for drugs and chemicals. In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model. Testosterone and progesterone secretion were measured to evaluate testicular function. Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs. Ketoconazole and aminoglutethimide inhibited both testosterone and progesterone secretion, but without modifying cell viability. Spironolactone specifically blocked testosterone secretion and increased progesterone concentration without inducing cell mortality. Cycloheximide altered testicular steroid secretion by another mechanism of action. Chlorpromazine, which interferes with the secretion of gonadotropins in vivo, produced a significant inhibition of progesterone and testosterone secretion as a result of the cytotoxic effects of the drug. In conclusion, this in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.
Measurement of steroidogenesis in rodent Leydig cells: a comparison between pregnenolone and testosterone production
1989, Molecular and Cellular Endocrinology
The efficiency and specificity of inhibition of pregnenolone metabolism in mature, immature rat Leydig cells, mouse and tumour Leydig cells by SU-10603, a 17α-hydroxylase inhibitor and epostane (WIN-32729), a 3β-hydroxysteroid dehydrogenase inhibitor, were studied.
Metabolism of [¹⁴C]pregnenolone by mature rat Leydig cells was inhibited for more than 95% in the presence of 20 μM SU-10603 and 5 μM epostane. The sum of the different steroids produced by Leydig cells from immature rats incubated in the presence of a 5α-reductase inhibitor was only 80% of pregnenolone production in the presence of SU-10603 and epostane. Pregnenolone metabolism could also be inhibited in tumour Leydig cells but not in mouse Leydig cells. Pregnenolone and testosterone production by Leydig cells from mature rats were similar when steroidogenesis is maximally stimulated by luteinizing hormone (LH). However, in the presence of LH and bovine serum albumin (bSA), or 22R-hydroxycholesterol and bSA, pregnenolone production was 1.7- and 6-fold higher, respectively, than testosterone production.
The data show that for measuring the steroidogenic activity of Leydig cells estimation of pregnenolone production is more reliable than measuring testosterone production. At high activities of the cholesterol side-chain cleavage (CSCC) the conversion of pregnenolone into testosterone may become the rate-limiting step for testosterone production. Under all conditions the conversion of cholesterol into pregnenolone is the (hormonal regulated) rate-determining step for steroidogenesis.
Finerenone: A new mineralocorticoid receptor antagonist without hyperkalemia: An opportunity in patients with CKD?
2016, Current Hypertension Reports
Emerging roles of the mineralocorticoid receptor in pathology: Toward new paradigms in clinical pharmacology
2016, Pharmacological Reviews

View full text

Research paperInhibition of hormonal-induced cAMP and steroid production by inhibitors of pregnenolone metabolism in adrenal and leydig cells

Abstract

J. Biol. Chem.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Steroids

J. Biol. Chem.

Biochim. Biophys. Acta

Eur. J. Biochem.

Endocrinology

Endocr. Rev.

Acta Endocrinol

Endocrinology

J. Clin. Endocrinol. Metab.

Research paper
Inhibition of hormonal-induced cAMP and steroid production by inhibitors of pregnenolone metabolism in adrenal and leydig cells