M. Palem-Vliers

Molecular configuration and conformation of aldosterone, 18-hydroxy-11-deoxycorticosterone and a new urinary 18-hydroxy-steroid—an n.m.r. study

Abstract The 1 Hmr and 13 Cmr spectra of aldosterone in various solvents show only the tautomeric forms with hemi-acetal[11–18] and hemi-ketal [18–20] bridges. The predominant form of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) in CDCl 3 (compound M of Dominguez) is the hemi-ketal [18–20] tautomer. Two forms are detected in CD 3 OD: the hemiketal tautomer and very likely a dimer. Other derivatives are found in the presence of water and small amounts of acid. The 18-hydroxy-tautomer with a 2O-C O is not found. Two unknown steroids (compound x and compound y ) were isolated from the urine of patients with hypertension and/or adrenal disorders. The 1 Hmr spectroscopy indicates that the following structure can be proposed for compound x : a : a derivative of dihydro-18-hydroxy-deoxycorticosterone hydroxylated either at 4β and 9α or less probably at 5α and 9α. b : a derivative of dihydro-18-hydroxy-dehydrocorticosterone hydroxylated either at 4β or 5α or 9α. The behaviour of compound x in solution is very similar to that of 18-OH-DOC (predominance of the 18–20 hemi-ketal, formation of dimer and of other derivatives).

Structure-activity relationships for agonistic and antagonistic mineralocorticoids

Abstract The molecular structure of natural and synthetic steroids which bind to the cytosolic mineralocorticoid receptor and possess a biological effect in vivo , have been studied by means of nuclear magnetic resonance spectroscopy and X-ray examination. C 5 , C 7 , C 12 , C 14 (and probably C 15 ) and the corresponding α-axial hydrogens constitute a stereospecific site able to bind to the mineralocorticoid cytosolic receptor by means of Van Der Waals forces. Three diffferent polar groups in DOC, aldosterone and 9α-fluorocortisol although contributing most of the binding energy during the steroid receptor interaction are less specific. A slight conformational change in the hydrophobic site by dehydrogenation at C 6 C 7 leaves the affinity to the receptor practically unmodified but induces a dramatic decrease in mineralocorticoid activity in vivo and results in an antagonistic effect against DOCA, 9α-fluorocortisol and aldosterone.

Physico-chemical characterization of cytosol and nuclear mineralocorticoid receptors in rat kidney.

Abstract Cytosolic and nuclear mineralocorticoid receptors in rat kidney have been studied by means of ultracentrifugation and gel electrophoresis. In cytosol preparations, mineralocorticoid receptors are acidic proteins; sucrose gradient centrifugation and polyacrylamide gel electrophoresis studies show two forms of the mineralocorticoid receptor. One of the two cytosol forms is electrophoretically identical to the aldosterone-protein complex isolated from the soluble nuclear fraction.

An in vitro study of mineralocorticoid binding proteins from rat kidney

Various studies have shown that specific proteins of target cells bind added steroids: oestrogens [ 1,2] testosterone [3,4] and progesterone [5,7] . These proteins have been isolated from the cytosol of cell homogenates. When the active steroid has bound to the cytosol receptor protein, the complex migrates into the nucleus, as initially shown by Jensen [2] and Gorski [l] where it encounters another acceptor the nature of which is still unknown but may be a protein or DNA itself [7] . A similar succession of events appears to be involved in the mechanism of action of aldosterone at peripheral receptor sites. The studies of Edelman [g] and Swaneck [9, lo] led to the isolation of proteins obtained from the nuclear and cytoplasmic fractions of the rat kidney that bind tritiated aldosterone in vivo and these proteins have also been found in homogenates of other tissues of the rat. Alberti [ 1 l] extracted protein-bound aldosterone from nuclei of the mucosal cells of the toad bladder by sonication. Rousseau [12] showed, both in vivo and in vitro, that there were two classes of renal cytosol receptors for aldosterone. The type I receptors have a high affinity for aldosterone while type 2 receptors have a high affinity for glucocorticoids. The present study shows that tritiated aldosterone binds to proteins of the cytosol when incubated with rat-kidney slices. The binding of tritiated aldosterone to soluble proteins of the nucleus and to the chromatin has also been observed; this binding is reduced in the presence of active steroids and not in the presence of inactive steroids.

Role of hydrophobic effects and polar groups in steroid-mineralocorticoid receptor interactions

To test whether hydrophobic interactions are the main driving forces in the association of steroids with mineralocorticoid receptors, the binding free energy calculated (delta Gc) using the surface area of the steroids accessible to water was compared with the observed free energy (delta GM) obtained from the Kd at the equilibrium estimated in the rat cytosol. The results are consistent with a binding process involving principally hydrophobic effects implying association of both faces of the steroid with the receptor. The discrepancies between calculated and observed values probably depend upon the mechanism of hydrogen bonding of the polar groups of the steroids to those on the receptor. Hydrogen bonds in an aqueous environment are likely between polar groups of the receptor and the 21-OH, 18-OH and 11-18 hemiacetal oxygen of the steroids. Strong hydrogen bonding in a hydrophobic environment from the 3-CO and 20-CO groups is a possibility (the latter with a dihedral angle C16-C17-C20-O20 of approximately equal to 7 degrees). Such a strong hydrogen bond from the 9 alpha-fluorine atom could account for the observed high affinity receptor binding of 9 alpha-fluorocortisol and 9 alpha-fluoroprednisolone. The loss of affinity in 11-deoxycortisol and cortisol may be explained by modification of the C17 side chain and repulsion forces from the 11 beta-OH.

Activation of Cytosol Aldosterone Receptors in Rat Kidney

Cytosolic aldosterone-protein complexes are isolated from rat kidney slices after incubation with [3H]aldosterone and dexamethasone. Activated and unactivated forms of the complex are characterized by gel electrophoresis and hydroxyapatite chromatography after incubation at 4 degrees C and 25 degrees C respectively. It is found that the activated form reaches a maximum after 30 min at 25 degrees C and can be separated as an homogeneous peak by electrophoresis. Intermediate forms can also be identified. In the presence of 10 mM ATP, activation immediately occurs at 4 degrees C and is almost complete. In the presence of 10 mM molybdate, the activation is strongly enhanced and the increase in activated form may be about fifteen-fold whether molybdate is added during kidney homogenization or just before incubation at 25 degrees C. On the other hand molybdate reduces to one third the binding of the aldosterone-receptor complexes to nuclei. In the presence of the steroid RU 26988 which is a pure glucocorticoid, experiments done on aldosterone-receptors complexes and their binding to nuclei are confirmed. This proves that aldosterone is specific for mineralocorticoid sites. The general pattern of the mineralocorticoid receptor activation is discussed and its resemblance to the case of other steroid hormones is emphasized.

Binding of natural spirolactone derivatives to mineralo- and glucocorticoid receptors of the rat kidney

AbstractIn previous papers (Genard et al., 1985 & 1986) were reported the discovery and identification of three biologically active natural spirolactone derivatives in man and in animal i.e. 6α, 7α-, 6β, 7β- and 6β, 7α- dihydroxy-6,7-dihydrocanrenone (DHC).

MOLECULAR CONFIGURATION AND CONFORMATION OF ALDOSTERONE, 18-HYDROXY-11-DEOXYCORTICOSTERONE AND A NEW URINARY 18-HYDROXY-STEROID—AN N.M.R. STUDY

The 1Hmr and 13Cmr spectra of aldosterone in various solvents show only the tautomeric forms with hemi–acetal [11–18] and hemi-ketal [18–20] bridges. The predominant form of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) in CDCl3 (compound M of Dominguez) is the hemi-ketal [18–20] tautomer. Two forms are detected in CD3OD: the hemi-ketal tautomer and very likely a dimer. Other derivatives are found in the presence of water and small amounts of acid. The 18-hydroxy-tautomer with a 20−C = O is not found. Two unknown steroids (compound × and compound y) were isolated from the urine of patients with hypertension and/or adrenal disorders. The 1Hmr spectroscopy indicates that the following structure can be proposed for compound x: a: a derivative of dihydro-18-hydroxy-deoxycorticosterone hydroxylated either at 4s and 9α or less probably at 5α and 9α. b: a derivative of dihydro-18-hydroxy-dehydrocorticosterone hydroxylated either at 4s or 5α or 9α. The behaviour of compound × in solution is very similar to that of 18-OH-DOC (predominance of the 18–20 hemi-ketal, formation of dimer and of other derivatives).