Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression.
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Transforming growth factor beta1 (TGFbeta1) has been shown to exert strong inhibitory effects on adrenocortical cell steroidogenesis. However, the molecular targets of TGFbeta1 in adrenocortical cells appear to differ between species. Here, we report the first characterization of the regulatory effects of TGFbeta1 on the steroidogenic functions of the human adrenocortical tumor cell line NCI-H295R. After treatment with 2 ng/ml TGFbeta1 for 24 h, basal production of corticosterone, cortisol and androstenedione was dramatically decreased. When TGFbeta1 was added simultaneously with forskolin, the production of cortisol and 11-hydroxyandrostenedione was decreased by 85% whereas that of deoxycortisol was increased. When TGFbeta1 was added simultaneously with angiotensin II, aldosterone production was reduced by 80%. We observed that TGFbeta1 strongly inhibits forskolin-induced steroid 11beta-hydroxylase activity and CYP11B1 mRNA levels, as well as angiotensin II-induced aldosterone synthase activity and CYP11B2 mRNA levels. CYP11B1 and CYP11B2 gene products thus appear as the major steroidogenic enzymes down-regulated by TGFbeta1 in the human adrenocortical tumor cell line NCI-H295R. (+info)
Steroid metabolism in ocular tissues of the rabbit.
(10/79)
The metabolism of cortisol and other steroids was studied in normal untreated rabbit iris-ciliary body and cornea as part of an investigation into the mechanism of glucocorticoid-induced glaucoma. Cortisol is readily converted to the inactive metabolite cortisone by these eye tissues indicating the presence of an 11beta-oxidoreductase system. This reaction is reversible with cortisone being converted to cortisol in the presence of appropriate cofactor. However, due to the absence of a (or as yet undetectable) cortisol-A-ring-reductase system (rate-limiting reaction) the steroid is not irreversibly metabolized to biologically inactive compounds. The 11beta-oxidoreductase system readily converts other C21-11beta-hydroxysteroids, such as corticosterone, to its appropriate C21-11-ketosteroid (11-dehydrocorticosterone). Some C21-steroids lacking the 11-hydroxyl group (11-deoxycortisol, 11-deoxycorticosterone) remain virtually unmetabolized (exception to this was found with progesterone). Evidence of a C21-steroid A-ring reductase system was found only when cortisone and progesterone were used as substrates. However, testosterone a C19 steroid was converted to clearly identifiable A-ring reduced and 17beta-and 3alpha(beta)-oxidoreduced metabolites, thus indicating the presence of testosterone A-ring reductase, 17beta-and 3alpha(beta)-oxidoreductase systems in the eye tissues studied. The presence of a steroid 5alpha(beta)-reductase for some steroids but not for cortisol indicates a distinct substrate specificity for this enzyme system in the eye tissues. (+info)
Low 11-deoxycortisol to cortisol conversion reflects extra-adrenal factors in the majority of women with normo-gonadotrophic normo-estrogenic infertility.
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BACKGROUND: Women with normogonadotrophic normo-estrogenic oligomenorrhoea often disclose a variety of clinical symptoms. Many of these individuals are obese with features of pseudo-hypercortisolism. In the current study, 11-deoxycortisol and cortisol concentrations were determined in this group and compared with ovulatory controls. METHODS AND RESULTS: Twenty-six women with clomiphene citrate-resistant infertility, 12 lean and 11 obese ovulatory controls were studied. Women with infertility had the highest 11-deoxycortisol concentrations (mean +/- SD: 4.1 +/- 1.5 ng/ml) compared with obese and lean controls (3.1 +/- 1.4 and 2.4 +/- 0.9 ng/ml) (P < 0.01), but similar morning cortisol concentrations (0.47 +/- 0.15, 0.45 +/- 0.16 and 0.47 +/- 0.18 nmol/l). Baseline 11-deoxycortisol/cortisol ratios (>90th percentile of ovulatory controls) were elevated in 23/26 infertile women (88%), and in 3/26 women (12%) after adrenocorticotrophic hormone (ACTH) stimulation. Three out of six lean infertile women had elevated baseline 11-deoxycortisol/cortisol ratios, but none of these women had elevated ratios after ACTH stimulation. Stepwise regression analysis, after exclusion of testosterone, revealed significant correlations between the groups (lean controls, obese controls, infertility) and ACTH-stimulated 11-deoxycortisol/cortisol ratio (P < 0.05), but not with fasting glucose, insulin, cortisol, 11-deoxycortisol and baseline 11-deoxycortisol/cortisol ratios. CONCLUSIONS: Congenital adrenal hyperplasia was not observed in the majority of infertile women. The data indicate that extra-adrenal factors were involved in most of the infertility syndromes that were studied. (+info)
Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity.
(12/79)
The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR alpha-isoform (hGRalpha) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRalpha ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRalpha antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRalpha mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRalpha deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRalpha differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132. (+info)
Delta sleep response to metyrapone in post-traumatic stress disorder.
(13/79)
Metyrapone blocks cortisol synthesis, which results in the stimulation of hypothalamic cortiocotropin-releasing factor (CRF) and a reduction in delta sleep. We examined the effect of metyrapone administration on endocrine and sleep measures in male subjects with and without chronic PTSD. We hypothesized that metyrapone would result in a decrease in delta sleep and that the magnitude of this decrease would be correlated with the endocrine response. Finally, we utilized the delta sleep response to metyrapone as an indirect measure of hypothalamic CRF activity and hypothesized that PTSD subjects would have decreased delta sleep at baseline and a greater decrease in delta sleep induced by metyrapone. Three nights of polysomnography were obtained in 24 male subjects with combat-related PTSD and 18 male combat-exposed normal controls. On day 3, metyrapone was administered during normal waking hours until habitual sleep onset preceding night 3. Endocrine responses to metyrapone were measured in plasma obtained the morning following sleep recordings, the day before and after administration. Repeated measures ANOVAs were conducted to compare the endocrine and sleep response to metyrapone in PTSD and controls. PTSD subjects had significantly less delta sleep as indexed by stages 3 and 4, and total delta integrated amplitude prior to metyrapone administration. There were no differences in premetyrapone cortisol or ACTH levels in PTSD vs controls. PTSD subjects had a significantly decreased ACTH response to metyrapone compared to controls. Metyrapone caused an increase in awakenings and a marked decrease in quantitative measures of delta sleep that was significantly greater in controls compared to PTSD. The decline in delta sleep was significantly associated with the magnitude of increase in both 11-deoxycortisol and ACTH. The results suggest that the delta sleep response to metyrapone is a measure of the brain response to increases in hypothalamic CRF. These data also suggest that the ACTH and sleep EEG response to hypothalamic CRF is decreased in PTSD. (+info)
Reversed-phase high-performance liquid chromatography separation of adrenal steroids prior to radioimmunoassay: application in congenital adrenal hyperplasia.
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21-Hydroxylase deficiency (21-OHD) is the most common form of congenital adrenal hyperplasia (CAH), followed by 11beta-hydroxylase deficiency (11beta-OHD). Diagnostic serum markers for these conditions are 17-hydroxyprogesterone (17-OHP) and 11-desoxycortisol (S), respectively. In 21-OHD, the large amounts of 17-OHP are further 11beta-hydroxylated to form 21-deoxycortisol (21-DF), making it also an excellent marker of this disease. These steroids can be measured in blood by radioimmunoassay (RIA). In this paper, we report the use of high-performance liquid chromatography (HPLC) for steroid purification, prior to RIA determinations of 21-DF, S, 17-OHP, and testosterone (T) in ether-extracted serum. The chromatographic separation is developed in a BDS-Hypersil column using water-methanol (53:47, v/v) as the mobile phase. The method is applied to 35 patients with the classic form of 21-OHD (18 females, 17 males, 5.1-14.2 years old) and 2 with 11beta-OHD (1 female, 1 male, 9.5 and 12.6 years old). Thirteen control children (5 females and 8 males, 5.2-15.2 years) are also studied. The results obtained for all measured steroids are compatible with those reported in the literature. The method is precise, and recovery is adequate. The HPLC technique proved to be of value for the purification of several steroids from single serum samples prior to RIA in patients with CAH. (+info)
Dexamethasone does not exert direct intracellular feedback on steroidogenesis in human adrenal NCI-H295A cells.
(15/79)
Experimental therapy of fetuses affected with congenital adrenal hyperplasia (CAH) has been reported by administering dexamethasone (Dex) to pregnant women at risk for carrying a CAH fetus. Such prenatal therapy can almost wholly eliminate virilization of the external genitalia of affected female fetuses, but only when treatment is started before 9 weeks of gestation. As it is not known whether the hypothalamic-pituitary-adrenal axis is functional at this time, and as the minimal effective doses of Dex are substantially supraphysiologic for the fetus, the mechanism of action of prenatal Dex treatment has been unclear. To assess the possibility that Dex might act directly on the adrenal, we used human adrenocortical NCI-H295A cells, an established model of the human fetal adrenal. Short term (6 h) incubation of these cells with Dex decreased synthesis of 11-deoxycortisol and 17alpha-hydroxyprogesterone and increased synthesis of deoxycorticosterone (DOC), but only at very high concentrations of Dex (> or =10 microM) that affect P450c17 (17alpha-hydroxylase/17,20 lyase) as a competitive inhibitor; no effects were seen at lower concentrations corresponding to those used in prenatal treatment. When NCI-H295A cells were treated with up to 100 microM Dex for 72 h, dot-blot analysis showed no changes in the abundance of the mRNAs for P450scc (cholesterol side-chain cleavage enzyme), P450c17, or 3beta-hydroxysteroid dehydrogenase II (3betaHSDII). When NCI-H295A cells were transfected with promoter/reporter constructs for the human genes for P450scc and P450c17, 24-h treatment with Dex at doses up to 100 microM had no effect on the transcription of these constructs. Because the regulation of steroidogenic processes in NCI-H295A cells closely resembles such regulation in the adrenal, we suggest that Dex may not act by an intra-adrenal mechanism in the experimental prenatal treatment of CAH. (+info)
The effect of intrafetal infusion of metyrapone on arterial blood pressure and on the arterial blood pressure response to angiotensin II in the sheep fetus during late gestation.
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While the impact of exogenous glucocorticoids on the fetal cardiovascular system has been well defined, relatively few studies have characterised the role of endogenous fetal glucocorticoids in the regulation of arterial blood pressure (BP) during late gestation. We have therefore infused metyrapone, an inhibitor of cortisol biosynthesis, into fetal sheep from 125 days gestation (when fetal cortisol concentrations are low) and from 137 days gestation (when fetal cortisol concentrations are increasing) and measured fetal plasma cortisol, 11-desoxycortisol and ACTH, fetal systolic, diastolic and mean arterial BP, heart rate, and the fetal BP responses to increasing doses of angiotensin II (AII). At 125 days gestation, there was a significant increase in fetal plasma ACTH and 11-desoxycortisol by 24 h after (+24 h) the start of the metyrapone infusion, and plasma cortisol concentrations were not different at +24 h when compared with pre-infusion values. Whilst the initial fall in circulating cortisol concentrations may have been transient, systolic, diastolic and mean arterial BP were ~5-6 mmHg lower (P < 0.05) in metyrapone- than in vehicle-infused fetuses at 24-48 h after the start of the infusion. When metyrapone was infused from 137/138 days gestation, there was a significant decrease in plasma cortisol concentrations by +6 h, which was followed by an increase back to pre-infusion values. While cortisol concentrations decreased, there was no change in fetal mean arterial BP during the first 24 h after the start of metyrapone infusion. Mean fetal arterial BP values at 137-139 days gestation were not different in fetuses that had been infused with either vehicle or metyrapone from 125 days gestation or with metyrapone from 137/138 days gestation. At 137-139 days gestation, however, arterial BP responses to increasing doses of AII were significantly blunted in fetuses that had been infused with metyrapone from 125 days gestation, when compared with fetuses that had been infused with metyrapone from 137/138 days gestation or with vehicle from 125 days gestation. The dissociation of the gestational age increase in arterial BP and the effects of intrafetal AII on fetal arterial BP indicates that increase in fetal BP with gestational age is not entirely a result of an increased vascular responsiveness to endogenous AII. Furthermore there may be a critical window during late gestation when the actions of cortisol contribute to the development of vascular responsiveness to AII. (+info)