Active Mineralocorticoids Research Articles

Ocular steroidome in human eyes and in eyes with complex central serous chorioretinopathy (CSCR)

The exact link between systemic and ocular endogenous corticoids (steroidome) is unclear and whether the ocular steroidome is altered in CSCR eyes is unknown. The aims of this study were to analyze the human steroidome in the aqueous humor as a function of age, sex and time of the day, to correlate systemic and ocular steroidome and to analyze the ocular steroidome in long lasting complex inactive CSCR. Based on our results, we present two CSCR cases treated by the combination of oral mineralocorticoid antagonist and glucocorticoids drops. In a cross-sectional study, aqueous humor (AH) was collected between 8am and 6 pm from 50 unaffected individuals (25 men and 25 women) and from 14 patients with chronic CSCR, during cataract surgery. In addition, simultaneous serum and AH were collected from 27 individuals undergoing cataract surgery and, simultaneous AH and vitreous were collected from 9 patients undergoing cataract and vitrectomy to estimate corticoids levels in the different compartments. The steroidome was determined using a LC–MS/MS method that quantifies 13 endogenous corticoids from the gluco, mineralocorticoid and androgen pathways. In AH and vitreous, the highest corticoid level is reached by cortisol (F), that represents less than 10% of F serum level. The cortisol levels in the serum did not correlate with ocular cortisol levels. Serum and ocular cortisone (E) levels correlate, although less than 5% of circulating E reaches the eye. The only mineralocorticoids measured in the AH were corticosterone (B) and its inactive form, the 11-desoxycorticosterone (A). There was no influence of circadian rhythm on cortisol ocular levels and there was no correlation between the age or the sex and the level of F, E, A, and B. In eyes with chronic inactive CSCR, the levels of the active glucocorticoid form F was lower than in control eyes and the F/E ratio was reduced by 50% but the B/A ratio was higher indicating imbalance towards active mineralocorticoids. Base on this observation, we propose to combine an antagonist of the mineralocorticoid receptor together with topical glucocorticoids in two CSCR patients, resistant to all other treatments, with favorable outcome. Our results indicate that the ocular psteroidome is highly regulated suggesting a local metabolism of ocular corticoids. In eyes with long-lasting complex inactive CSCR, the steroidome analysis shows lower active glucocorticoids and higher active mineralocorticoids.

High aldosterone-to-renin variants of CYP11B2 and pregnancy outcome

Increased aldosterone concentrations and volume expansion of normal pregnancies are hallmarks of normal pregnancies and blunted in pre-eclampsia. Accordingly, we hypothesized an active mineralocorticoid system to protect from pre-eclampsia. In pregnant women (normotensive n = 44; pre-eclamptic n = 48), blood pressure, urinary tetrahydro-aldosterone excretion and activating polymorphisms (SF-1 site and intron 2) of the aldosterone synthase gene (CYP11B2) were determined; 185 non-pregnant normotensive individuals served as control. Amino acid-changing polymorphisms of the DNA- and agonist-binding regions of the mineralocorticoid receptor were evaluated by RT-PCR, SSCP and sequencing. Urinary tetrahydro-aldosterone excretion was reduced in pre-eclampsia as compared to normal pregnancy (P < 0.05). It inversely correlated with blood pressure (r = 0.99, P < 0.04). Homozygosity for activating CYP11B2 polymorphisms was preferably present in normotensive as compared to pre-eclamptic pregnancies, identified (intron 2, P = 0.005; SF-1 site, P = 0.016). Two mutant haplotypes decreased the risk of developing pre-eclampsia (RR 0.16; CI 0.05-0.54; P < 0.001). In contrast, intron 2 wild type predisposed to pre-eclampsia (P < 0.0015). No functional mineralocorticoid receptor mutant has been observed. High aldosterone availability is associated with lower maternal blood pressure. In line with this observation, gain-of-function variants of the CYP11B2 reduce the risk of developing pre-eclampsia. Mutants of the mineralocorticoid receptor cannot explain the frequent syndrome of pre-eclampsia.

CYP11B2 polymorphisms and cardiovascular risk factors

CYP11B2 polymorphisms and cardiovascular risk factors

Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists.

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green fluorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclear-to-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were retained in nuclear matrix preparations after in vivo crosslinking. These data strongly suggest that hormone-activated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids.

Aldosterone responsiveness of A6 cells is restored by cloned rat mineralocorticoid receptor.

A6 cells, derived from Xenopus laevis renal tubule, form a high-resistance ion-transporting monolayer when grown on permeable supports and can generate a short-circuit current (SCC) that is stimulated by high levels of the mineralocorticoid aldosterone. Surprisingly, A6 SCC is more responsive to glucocorticoids than to mineralocorticoids, suggesting the possibility that these cells do not contain transcriptionally active mineralocorticoid receptor (MR) and that glucocorticoid receptor (GR) mediates MR-like responses in these collecting duct-like cells. We have examined the response of both SCC and a transfected reporter gene to mineralocorticoids and glucocorticoids in the presence and absence of transfected rat MR (rMR). We found that, in the absence of transfected MR, a reporter gene that can be activated by MR or GR was more responsive to glucocorticoids such as dexamethasone and RU-28362 than to mineralocorticoids such as aldosterone. Transfected rMR underwent mineralocorticoid-dependent nuclear localization and restored both transcriptional sensitivity of a reporter gene and SCC response to mineralocorticoids. These data demonstrate that A6 cells contain transcriptionally active GR but not MR and thus suggest a molecular basis for the defect in A6 cell SCC response to aldosterone. Our results also demonstrate that GR is capable of mediating hormone stimulation of SCC, a classic mineralocorticoid response. Finally, the observation that heterologous expression of rMR can localize normally to the A6 nucleus in a hormone-dependent fashion and restore both the transcriptional and SCC response to mineralocorticoids suggests that MR function is conserved in species as distantly related as toads and mammals.

11 beta-hydroxysteroid dehydrogenase and corticosteroid hormone receptors in the rat colon.

In the rat kidney 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) maintains normal in vivo specificity for mineralocorticoid receptor (MR) by converting the active steroid corticosterone to inactive 11-dehydrocorticosterone, leaving aldosterone to occupy the MR. Clinical observations support the hypothesis that 11 beta-HSD also protects the distal colonic MR from glucocorticoid excess. We have measured 11 beta-HSD mRNA and activity along the rat colon and have analyzed the distribution of 11 beta-HSD, MR, and glucocorticoid receptor (GR) mRNA within rat distal colon using in situ hybridization. Levels of 11 beta-HSD mRNA (1.7 and 3.4 kb) and activity were higher in distal vs. proximal colon, paralleling reported MR mRNA levels. Within the distal colon mucosa both 11 beta-HSD immunoreactivity and mRNA was observed in cells in the lamina propria but not in epithelial cells. MR mRNA was present in surface epithelial cells, but was also colocalized with the same 11 beta-HSD-expressing cells in the lamina propria. In contrast GR mRNA was more uniformly distributed. The localization of MR mRNA to nonepithelial cells in the lamina propria, possibly neuroendocrine cells, suggests that mineralocorticoid-regulated sodium transport across colonic epithelial cells may also involve a paracrine mechanism. As with the kidney, exposure of active mineralocorticoid to the MR in these cells in the lamina propria is dictated by 11 beta-HSD in an autocrine fashion.

Synthesis of 19-hydroxyaldosterone and the 3β-hydroxy-5-ENE analog of aldosterone, active mineralocorticoids

19-Hydroxyaldosterone ( 20) and the 3β-hydroxy-5-ene analog of aldosterone (HAA) ( 8) were synthesized from 21-acetoxy-4-pregnene-3,20-dion-20-ethylene ketal-18,11β-lactone ( 2) as follows: the double bond was transposed from the 4,5 to the 5,6-position by enol acetylation to 3, followed by sodium borohydride reduction. Further reduction of the resulting lactone 4a with diisobutylaluminum hydride (DIBAH) furnished the 20-ketal of HAA 6, from which free HAA ( 8) and the 18,21-anhydro compound 7 were obtained by acid treatment. The [ 1H]NMR spectrum of 8 in CDCl 3 showed it to be a mixture of two isomeric forms. Correlation with the known aldosterone-γ-etiolactone ( 10) was established by periodate oxidation of HAA to the corresponding etiolactone 9 followed by chromic acid oxidation. The preparation of 20 was next effected in the following manner: the diacetate 4b was converted into the 6β,19-oxido compound 13b by addition of hypobromous acid followed by the hypoiodite reaction of the bromohydrin 11. Mild saponification of 13b lead to the corresponcling diol 13a, and was followed by selective oxidation to the 3-one 14, readily dehydrobrominated to 15a. Reductive ring opening furnished a mixture of the 19,21-diol 16a and its 5-ene isomer 16b, which was directly converted to the diketal 17. Reduction with DIBAH gave the hemiacetal 18, and hydrolysis of the latter 19-hydroxyaldosterone ( 20) as a water-soluble solid, accompanied by the 18,21-anhydro compound 19. 19-Hydroxyaldosterone exists in CHCl 3 and water as a mixture of mainly two isomers. Periodate oxidation furnished the etiolactone 21. Preliminary results indicate that HAA and 19-hydroxyaldosterone are active mineralocorticoids in the Kagawa bioassay and short-circuit current measurements.

Orally active mineralocorticoid agonists and antagonists: delta 1-derivatives of aldosterone and 18-deoxyaldosterone.

The effect of delta 1 unsaturation on the oral effectiveness of a representative mineralocorticoid agonist and antagonist was investigated in an adrenalectomized rat bioassay. Dehydrogenation at the 1.2 position did not alter the qualitative nature of the mineralocorticoid activity of the parent compound. Thus delta 1-aldosterone (21,18-dihydroxy-11 beta, 18-oxido-1,4-pregnadiene-3,20-dione) retained pure mineralocorticoid agonism, and delta 1-18-deoxyaldosterone (21-hydroxy-11 beta, 18-oxido-1,4-pregnadiene-3,20-dione)demonstrated the same relative degree of predominant antagonism as 18-deoxyaldosterone (21-hydroxy-11 beta, 18-oxido-4-=pregnene-3,20-dione) itself. In each instance, receptor affinity was diminished by 1,2 unsaturation, but this effect was offset by the greater bioavailability of the delta 1 derivatives on oral administration. (Endocrinology 108: 517, 1981)

Licorice Raises Urinary Cortisol in Man*

The finding that urine cortisol excretion was increased in patients with hypokalaemic hypertension induced by licorice addiction led to this study on the effect of licorice in normal subjects. Thirteen normal volunteers ate either 100 or 200 g licorice for 1-4 weeks and assessment of pituitary-adrenal function was made before, during, and 1 week after cessation of licorice ingestion. Urine cortisol excretion more than doubled in 10 of 13 subjects (mean, 33.8 +/- 15.6 SD before and 83.3 +/- 56 SD micrograms/24 h at 1 week after commencing licorice) and excretion rates similar to those observed in Cushing's syndrome were seen in 7 subjects (range, 91-226, compared to normal range of 11-82 micrograms/24 h). Urine cortisol excretion remained significantly elevated (P less than 0.01) above control levels for at least 1 week after licorice was withdrawn. Despite these increases, urinary steroid metabolite (tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, 17-ketogenicsteroids, and 17-ketosteroids) excretion was not affected, plasma cortisol and ACTH values were unchanged, and normal 0800-1600-h diurnal variation of plasma cortisol was maintained. The direct intraadrenal infusion of the active mineralocorticoid component of licorice, glycyrrhetinic acid, in two sheep with autotransplanted adrenal glands failed to stimulate cortisol secretion acutely. It is concluded from these studies that the licorice-induced changes in cortisol excretion are not a result of adrenocoritcal stimulation but more likely represent a change in the renal handling of cortisol.

Antimineralocorticoid Action of Progesterone in the Rat: Correlation of the Effect on Electrolyte Excretion and Interaction with Renal Mineralocorticoid Receptors*

The ability of progesterone to increase sodium excretion only in the presence of active mineralocorticoids has led to the suggestion that progesterone antagonizes mineralocorticoid action at the renal tubular level. We have examined the effects of progesterone and aldosterone on sodium and potassium excretion in adrenalectomized rats and have compared these results with the ability of progesterone to inhibit the binding of aldosterone to cytoplasmic and nuclear mineralocorticoid receptors. The administration of aldosterone to saline-loaded, adrenalectomized male rats resulted in a dose-dependent fall in the urinary sodium to potassium ratio with a near maximum effect produced by 1 μg aldosterone. Progesterone alone in doses of 10, 100, and 1000 μg had no significant effect on the sodium to potassium ratio, but when administered simultaneously with 1 μg aldosterone, the same concentrations of progesterone inhibited the effect of aldosterone in a dose-dependent manner; 1 mg progesterone completely blocked t...

18-Hydroxy-Deoxycorticosterone in Human Hypertension

18-Hydroxy-ll-deoxycorticosterone (18-OH-DOC) was isolated from human adrenal vein blood in concentrations of the same magnitude as is aldosterone, and its structure was proved by a variety of methods including mass spectral analysis. Angiotensin infusions failed to evoke a rise in 18-OH-DOC secretion, whereas ACTH increased 18-OH-DOC secretion up to 20-fold. The secretion rate of 18-OH-DOC determined by isotope dilution in healthy subjects was similar to the range of aldosterone secretion. Excretion of the ring A reduced glucuronide metabolite, 18-hydroxy-tetrahydro-deoxycorticosterone (18-OH-TH DOC), in urine proved to be a valid index of secretion of 18-OH-DOC. 18-OH-DOC secretion was estimated in a variety of hypertensive disorders and found to be significantly elevated in a few (3 of 12) patients with essential hypertension, suppressed plasma renin activity, low or normal aldosterone secretion, and who had significant reduction in blood pressure with spironolactone. A single patient became normotensive on prolonged dexamethasone suppression of ACTH and 18-OH-DOC secretion. The possibility that 18-OH-DOC is a precursor of some more oxygenated and, perhaps, more active mineralocorticoid is suggested.

Macromolecular binding of aldosterone in the toad bladder

Protein-bound aldosterone has been extracted by sonication of nuclei from the mucosal cells of toad bladder. After incubation of hemibladders in 10 and 100 nM (+)-[ 3H]aldosterone, 1.5·10 −14 and 7.7·10 −14 moles bound aldosterone per 100 μg DNA can be extracted, respectively. Most of the bound aldosterone dissociates rapidly, and 4 h after isolation only a stable component of 20% remains. Dissociation is accelerated at room temperature. The stability of the complex is unaffected by dithiothreitol but enhanced by 25% glycerol. An excess of deoxycorticosterone, hydrocortisone, spirolactone (SC 14266) and unlabeled aldosterone, when added to the incubation medium, displace bound aldosterone while testosterone has no effect on binding by purified nuclei. This suggests mineralocorticoid specificity for the binding protein. [ 3H]Hydrocortisone (100 nM), a less active mineralocorticoid than aldosterone, binds primarily to the nonspecific, nondissociable binding sites at this concentration. One high-molecular-weight component and one component of molecular weight of approx. 100 000 were demonstrated by agarose column chromatography. Both components are stable and unaffected by deoxycorticosterone. The mineralocorticoid-specific complex can be equated with the rapidly dissociable fraction. When nuclei are sonicated and (+)-[ 3H]aldosterone is then added a nonsaturable nonspecific binding occurs which is increased with time and unaffected by temperature. Thus three aldosterone binding complexes have been isolated from toad-bladder mucosal-cell nuclei one of which is rapidly dissociable and mineralocorticoid specific.

Stereospecific nuclear and cytosol aldosterone-binding proteins of various tissues.

3H-Aldosterone-binding proteins (ABP) were isolated from nuclear fractions of rat kidney, duodenal mucosa, spleen, liver and brain by extraction in Tris-CaCI2 and precipitation in 50% (NH4)2SO4. Cytosol (105,000 × g supernatants) ABP were isolated from the same tissues by G-50 Sephadex chromatography. The ABP content of the nuclear fractions was in the order: kidney ≈duodenal mucosa > spleen ≈liver≈ brain (table I). The ABP content of the cytosol fractions was in the order: kidney≈duodenal mucosa > spleen ≈ brain≈liver (table III). The relative affinities of the competing steroids, cold d-aldosterone, 9α-fluorocortisol, 17-isoaldosterone and 17/S-estradiol for the 3H-aldosterone binding sites were similar in both the nuclear and cytosol fractions of kidney, duodenal mucosa, spleen and liver. The active mineralocorticoids competed successfully for the 3H-aldosterone binding sites but the inactive steroids were either without effect or tended to enhance the binding of 3H-aldosterone. The physiological significance of relatively low levels of stereospecific ABP in spleen, liver and brain remains to be elucidated.

On the nephrosclerotic action of 2-methyl-9-fluorocortisol, a highly active artificial mineralocorticoid.

2-Methyl-9-fluorocortisol (2Me-9F-COL) was shown to possess extraordinarily intense mineralocorticoid but comparatively slight glucocorticoid activity, as judged by various tests (1). This compound, like other active mineralocorticoids (e.g., desoxycorticosterone and aldosterone), also exhibits a mutual antagonism with glucocorticoids (e.g., cortisone, cortisol) in the regulation of inflammation, anabolism and lymphatic-tissue development (2). On the other hand, the production by typical mineralocorticoids of renal damage and hypertension is not counteracted but rather enhanced by glucocorticoids (3, 4). It was to be expected, therefore, that a compound such as 2Me-9F-COL would produce marked renal lesions and hypertension by virtue of its strong mineralocorticoid potency, despite the fact that it also possesses some glucocorticoid activity. The experiments to be described here demonstrate that this is actually the case; indeed, 2Me-9F-COL proved to be the most potent nephrotoxic steroid among all those h...

Further studies on the properties of a highly active mineralocorticoid

Further studies on the properties of a highly active mineralocorticoid

Isolation of a highly active mineralocorticoid from beef adrenal extract.

Grundy, Simpson and Tait1 have shown that most of the activity of beef adrenal extract, as measured by its effect on the mineral metabolism of adrenal-ectomized rats2, was coincident with 11-dehydro-17-hydroxy-corticosterone (cortisone), when the extract was fractionated by Zaffaroni's method of paper chromatography3. From quantitative measurements of the biological activity of the fraction, it was concluded that the activity was due to the presence of a compound other than cortisone.