Carlos P. Lantos

In vivo stimulation of aldosterone biosynthesis by endothelin: loci of action and effects of doses and infusion rate.

Infusion of endothelin-1 (ET-1) into rats increased adrenal mitochondrial synthesis of aldosterone from deoxycorticosterone and the adrenal cytosolic content of aldosterone. The dose-response relationships for these last two effects of ET-1 were found to be biphasic with a maximum (corresponding to 80 to 200% increase) at 50 to 80 ng ET-1/kg/min, and were also dependent on the infusion rate. Plasma aldosterone levels were also increased in a similar ratio. Previous infusion of the converting enzyme inhibitor enalapril did not affect the ET-1-induced increase in steroidogenesis. Finally, pregnenolene production was also increased in incubations of mitochondria from treated rats. These results indicate that ET-1 augments aldosteronogenesis by increasing the early as well as the late pathway. These effects were independent of the formation of angiotensin II. Isolated glomerulosa cells responded to ET-1 increasing aldosterone production in a dose-related fashion. These results confirm a direct effect of ET-1 on the adrenal gland in vivo.

New lead compounds in the search for pure antiglucocorticoids and the dissociation of antiglucocorticoid effects.

Antiglucocorticoids that act as antagonists at the glucocorticoid receptor (GR) level may be used to block or modulate the undesirable effects of glucocorticoid excess (from endogenous or exogenous origin). RU486 developed in the early 80s, is an antiglucocorticoid but also a potent antiprogestin and abortifacient, nevertheless it still remains as the only GR antagonist drug in the market. Further on, in view of the variety of physiological processes in which glucocorticoids are involved, selective antiglucocorticoids that can block only some of these processes (eventually with tissue specificity) would be highly desirable. The bridged pregnane 21-hydroxy-6,19-epoxyprogesterone, was developed as an alternative lead being an antagonist of the GR with no affinity for mineralocorticoid and progesterone receptors. Antagonistic activity was evidenced by partial blocking of dexamethasone induction of tyrosine aminotransferase (TAT) and thymocyte apoptosis. Replacement of the oxygen bridge by a sulfur bridge gave a less bent, more flexible molecule. 21-Hydroxy-6,19-epithioprogesterone exhibited improved antiapoptotic activity on thymocytes but was not effective blocking TAT induction. This selectivity was improved further by oxidation to the sulfone. The sulfone but not the reduced compound also reverted the dexamethasone-mediated inhibition of NFkappaB activity in HeLa cells. Blocking of the apoptotic effect of TNFalpha by dexamethasone in the L929 cell line (mouse fibroblasts), was only reverted partially by the sulfone which exhibited a mild agonistic/antagonistic activity in this assay. None of these compounds showed antiprogestin activity. Similar overall molecular shapes but more lipophylic and with higher metabolic stability were obtained by introduction of a methylene bridge (6,19-methanoprogesterone) or by a direct bond between C-6 and C-19 (6,19-cycloprogesterone and its 21-hydroxy derivative). The latter highly bent steroids showed affinity for the GR. Recently we performed molecular dynamics simulations of GR-ligand complexes to investigate the molecular basis of the passive antagonism exhibited by 21-hydroxy-6,19-epoxyprogesterone. On the basis of our findings, we proposed that the passive antagonist mode of action of this antiglucocorticoid analog resides, at least in part, in the incapacity of GR-21-hydroxy-6,19-epoxyprogesterone complex to dimerize, making the complex unable to activate gene transcription.

Molecular mechanism of activation and nuclear translocation of the mineralocorticoid receptor upon binding of pregnanesteroids.

The mineralocorticoid receptor (MR) is primarily localized in the cytoplasm of the cell in the absence of ligand. The first step in the genomic-dependent mechanism of action of mineralocorticoids is the binding of steroid to the MR, which in turn triggers MR nuclear translocation. The regulation of hormone-binding to MR is complex and involves a multifactorial mechanism, making it difficult to determine the optimal structure of a steroid for activating the MR and promoting its nuclear translocation. Here we review the structure-activity relationship for several pregnanesteroids that possess various functional groups, and suggest that a flat conformation of the ligand rather than the presence of particular chemical groups is a critical parameter for the final biological effect in vivo. We also discuss how the MR undergoes differential conformational changes according to the nature of the bound ligand, which in turn affects the dynein-dependent retrograde rate of movement for the steroid/receptor complex.

Less polar forms and derivatives of 18 hydroxy-corticosterone

Abstract The formation of various compounds upon reaction of 18 hydroxycorticosterone with neutral and acid-added alcohols as well as with acidic aqueous solutions is described. The time course of their formation in such media has been studied. Some of these compounds have been isolated and tentatively identified by paper chromatography employing double isotope techniques and by mass spectrometry. The more polar form has a C-20 hemiketalic structure; the single less polar form obtained upon storage in neutral methanol would be the methylketal at C-20 but the less polar fraction having the same mobility in the Chromatographie system employed, obtained upon reaction with acidic aqueous solutions, seems to be formed by two different dehydration products.

Mechanisms of action of endothelin-1 in rat adrenal

Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.

Eighteen-deoxyaldosterone and other less polar forms of 18-hydroxycorticosterone as aldosterone precursors in rat adrenals

Samples containing as precursors either 18-hydroxycorticosterone (18-OH-B) in its M form, or this converted to less polar forms at pH 2 (ACM), or M or ACM enclosed in liposomes from adrenal lipids were incubated at pH 7.4, 4.8 or 3.3 in the presence or absence of quartered rat adrenals for 1 and 2 h. Optimal (10%) yields of aldosterone were obtained when (a) ACM was incubated at pH 4.8 and (b) M enclosed in liposomes was suspended in buffer and shaken without enzyme at pH 3.3. When conditions (a) were supplemented with malate and NADP, 16% of ACM was converted to aldosterone. ACM contained 80% of a fraction which, according to 13C NMR spectroscopy, was identical to 18-deoxyaldosterone (18-DAL). Experiments in which radioactivity from corticosterone (B) or M was trapped by radioinert M or 18-DAL disclosed a pathway comprising sequentially B, 18-OH-B, 18-DAL and aldosterone, and the combined evidence of this work, an enzymatic hydroxylation of 18-DAL to aldosterone.

Regulatory effects of 5β-reduced steroids

The first section of this publication summarizes early work according to which 5β-pregnanedione is an important metabolite of progesterone in the early stages of the chick embryos adrenal steroidogenesis, then decreasing gradually as corticosteroidogenesis increases. In the second section a model is described in which adrenal 3β-ol hydroxylase-isomerase of the 17-day-old chicken is suppressed pharmacologically, this suppression being correlated with that of the synthesis of aminoevulinic acid (ALA), the first and rate-limiting step of the heme pathway. 5β-Pregnanedione (10−7–10−6M) restored ALA synthesis in this inhibited model to normal values. The effect of 5β-pregnanedione was specific since other steroids tested: progesterone; 5α-pregnanedione; corticosterone or estradiol, did not stimulate ALA. Since heme formation by steroidogenic glands contributes to the synthesis of cytochrome P450 rather than hemoglobin, 5β-pregnanedione was also assayed as a stimulator of this enzyme system and was found to increase cytochrome P450 in adrenals and testes but not in the liver. In view of these results a hypothesis is advanced according to which 5β-reduced progestagens and androgens stimulate cytochrome P450 formation, i.e. the synthesis of progesterone and higher hydroxylated steroids, by steroidogenic glands in the event of an excessive precursor reduction.

The glucocorticoid properties of the synthetic steroid pregna-1,4-diene-11β-ol-3,20-dione (ΔHOP) are not entirely correlated with the steroid binding to the glucocorticoid receptor

The natural steroid 11beta-hydroxyprogesterone is not only a modulator of 11beta-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor. 11beta-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, deltaHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that deltaHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, deltaHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of deltaHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. deltaHOP is similarly inefficient in inducing chloramphenicol-acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, deltaHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes deltaHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.

A highly lipophilic form of aldosterone. isolation and characterization of an aldosterone dimer

A highly lipophilic form of aldosterone obtained both from incubations of 18-hydroxycorticosterone with quartered rat adrenals and by treatment of aldosterone with acid, was identified as an aldosterone dimer based on its 1H, 13C NMR and mass spectra.

Inhibition of aldosterone formation by cortisol in rat adrenal mitochondria

In this work we confirm by a metabolic method the existence of at least two enzymes with 11 beta- and 18-hydroxylase activities in rat adrenal mitochondria. The method was based on the ability of cortisol (F), a foreign alternative substrate, to inhibit competitively metabolite productions from various precursors. F inhibited a) aldosterone (ALDO) production from 11-deoxycorticosterone (DOC) without affecting the yields of corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18-OHDOC); b) 18-hydroxycorticosterone and aldosterone productions from B (Ki = 2.5 +/- 0.5 microM); and c) ALDO production from 18-OHDOC. These results suggest the existence of two categories of enzymes with both 11 beta- and 18-hydroxylase activities, one comprising those that catalyze the conversions of DOC to B and 18-OHDOC (F-insensitive reactions [FIS]) and the other one comprising the enzymes involved in the conversions of B to 18-OHB and ALDO and that of 18-OHDOC to ALDO (F-sensitive reactions [FS]). The cloned enzymes CYP11B1 and CYP11B2 would pertain respectively to the FIS and FS categories.