Aldosterone Binding Sites Research Articles

Mineralocorticoid regulation of salt intake is preserved in hippocampectomized rats.

Considering the high concentration of binding sites for [3H]-aldosterone in the hippocampus, a relationship between these sites and the regulation of sodium appetite by aldosterone was investigated. After surgical hippocampectomy, rats showed an approximately 80% depletion of [3H]-aldosterone binding sites. In spite of this reduction, hippocampectomized rats developed a sodium appetite after adrenalectomy; this sodium appetite was suppressed by continuous administration of aldosterone, a response similar to the pattern found in normal rats. These results suggest that the hippocampus is not the main target of the effect of the hormone on salt intake.

Aldosterone receptors.

In 1972, in a collaborative study between the Edelman and Tomkins laboratories, aldosterone was shown to bind two classes of sites in rat kidney preparations.1 Those with higher affinity were proposed as physiological mineralocorticoid Type I receptors, and those with lower affinity for aldosterone Type II, glucocorticoid receptors. The Type I aldosterone binding sites were subsequently shown to have very high affinity for 9α-fluorocortisol and deoxycorticosterone, as well as for aldosterone; moderate affinity for the physiological glucocorticoids cortisol and corticosterone; and relatively low affinity for the synthetic glucocorticoids dexamethasone and triamcinolone acetonide.KeywordsMammary GlandGlucocorticoid ReceptorMineralocorticoid ReceptorTriamcinolone AcetonideAldosterone ReceptorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Further studies of brain aldosterone binding sites employing new mineralocorticoid and glucocorticoid receptor markers in vitro

We have used synthetic markers of the glucocorticoid (GC) receptor (RU 28362) and of the mineralocorticoid (MC) receptors (RU 26752 and RU 28318) to characterize the specificity of the sites binding aldosterone (ALDO), dexamethasone (DEX) and corticosterone (CORT) in cytosol of hippocampus. The results obtained suggest that ALDO was bound mostly to a MC receptor, as the relative binding affinity (RBA) of the GC receptor marker (and that of the previously studied RU 26988) was negligible for this site, in contrast to the high RBA displayed by RU 26752. DEX was bound for a large part to a GC receptor, as RU 28362 competed for this site, although the MC receptor marker still showed some affinity. An intermediate effect of both marker types was obtained with CORT. RU 28318 was a weak competitor for either the GC or the MC binding site. Thus, RU 28362 and RU 26752 allowed the discrimination of two to three receptors in the hippocampus, similarly to those described in the kidney. Finally, we have demonstrated the usefulness of these synthetic markers in identifying MC binding sites in several brain regions and also in the hippocampus during ontogenetic development.

Comparison of aldosterone binding in aortic cells from Dahl salt-susceptible and salt-resistant rats

The Dahl salt-resistant substrain of Sprague-Dawley rats represents a uniform population of animals that are resistant to salt and mineralocorticoid induced hypertension. Aldosterone binding in the aortae of these rats is contrasted to that of age- and sex-matched rats of the Dahl salt-susceptible strain in an effort to identify a mechanism for resistance to salt induced hypertension. Cultured smooth muscle cells of both substrains contain two classes of aldosterone binding sites: corticoid receptor I with high affinity and low capacity, and corticoid receptor II with low affinity and high capacity. No differences were found between the two substrains in the affinities or binding capacities of these receptors. Both groups of Sprague-Dawley rats had a significantly higher corticoid receptor I affinity than the salt resistant Fischer 344 rats, a strain with a two-fold lower affinity than salt sensitive strains. These results indicate that an intrinsic defect in mineralocorticoid binding in aortic smooth muscle cells could not account for the resistance to salt and mineralocorticoid induced hypertension seen in Sprague-Dawley rats and that the biochemical abnormality underlying salt resistance is likely to be different from that of Fischer 344 rats.

Characterization of aldosterone binding sites in circulating human mononuclear leukocytes.

Aldosterone binding sites in human mononuclear leukocytes were characterized after separation of cells from blood by a Percoll gradient. After washing and resuspension in RPMI-1640 medium, cells were incubated at 37 degrees C for 1 h with different concentrations of [3H]aldosterone plus a 100-fold concentration of RU-26988 (11 alpha, 17 alpha-dihydroxy-17 beta-propynylandrost-1,4,6-trien-3-one), with or without an excess of unlabeled aldosterone. Aldosterone binds to a single class of receptors with an affinity of 2.7 +/- 0.5 nM (means +/- SD, n = 14) and a capacity of 290 +/- 108 sites/cell (n = 14). The specificity data show a hierarchy of affinity of desoxycorticosterone = corticosterone = aldosterone greater than hydrocortisone greater than dexamethasone. The results indicate that mononuclear leukocytes could be useful for studying the physiological significance of these mineralocorticoid receptors and their regulation in humans.

Cytoplasmic and nuclear uptake of aldosterone in toad bladder: a mathematical modeling approach.

The mechanism of aldosterone uptake in the epithelial cells of toad bladder was studied using mathematical modeling. Two complementary approaches were used. The first involved analysis of cytosolic aldosterone binding at steady state according to models defined by the sum of independent noninteractive binding sites. The best model describing the experimental data corresponded to two specific binding sites with mean dissociation constant values of 0.20 and 60 nM for types 1 and 2, respectively. The second approach was based on the analysis of cytoplasmic and nuclear aldosterone uptake kinetics at 25 and 0 degrees C in intact bladder. Two models (A and B) were studied. They both implied the existence of two types of aldosterone binding sites as precursors of the corresponding chromatin bound complexes. In model A, nuclear translocation of the two types of receptors was assumed to obey first-order kinetics. In model B, the translocation process for type 1 sites involved a time lag leading to delayed binding to chromatin. Both models were found to fit the experimental data satisfactorily. The fit obtained for model B appeared to be better at low aldosterone concentrations.

Ontogeny of renal mineralocorticoid receptors and urinary electrolyte responses in the rat.

We examined the ontogeny of cytosol mineralocorticoid receptors in renal inner medulla-papilla (IM-PAP) and outer medulla-cortex (OM-CX) and of a biological response to administered corticosteroids (urinary K+/Na+ ratio). Rats at early (7-9 days), intermediate (13-15 days), and late (23-30 days) stages of maturation were used. Young rats were found to be essentially insensitive to aldosterone, in contrast to rats aged 13-15 days and older. Scatchard analysis of IM-PAP and OM-CX cytosol showed equivalent high-affinity aldosterone binding sites at all ages. The affinity of these sites was constant (Kd, 4 degrees C approximately 0.5 nM) and their concentration varied only slightly over maturation. In a series of in vivo nuclear transfer studies, the hyposensitivity to aldosterone in young rats did not seem to be due to a difference in the nuclear binding of aldosterone-receptor complexes. The resistance of the immature (7-9 days) rat kidney to aldosterone thus appears to be a postreceptor phenomenon. The maturational events allowing expression of mineralocorticoid-inducible genes in the kidney remain to be determined.

Autoradiographic study of nuclear localization of aldosterone binding sites in intact renal cells: Lack of temperature dependency

We examined by autoradiography on intact target cells the nucleocytoplasmic distribution of aldosterone-receptor complexes and the thermodependency of the nuclear translocation process. Autoradiographs (dry films) were performed on cortical collecting tubules isolated by microdissection, after incubation of rabbit kidney pyramids with [ 3H]aldosterone (2 × 10 −9M) in the presence or absence of an excess unlabelled aldosterone (× 100). Paired experiments were done at 30°C (1 h) or 4°C (2 h). In both cases, the specific labelling was exclusively nuclear. Values were higher at 30°C (14.5 ° 1.5 specific silver grains per 100 μ 2) than at 4°C (5.4 ± 0.6), with no concommitant cytoplasmic labelling (4°C:0.7 ± 0.3; 30°C: − 0.04 ± 0.6). At 30°C, addition of unlabelled spirolactone (× 200) prevented the observed autoradiographic nuclear accumulation of aldosterone-receptor complexes, suggesting that cytoplasmic binding preceded the nuclear entry. The results suggest that, in intact cells, almost all aldosterone receptor complexes accumulate in nuclei, and that this process does not depend on temperature. In parallel biochemical binding series we found the classical distribution of aldosterone receptor complexes both in cytoplasm and nuclei, and the classical thermodependency of nuclear translocation. The present autoradiographic results, together with similar observations reported for sex steroids (Martin P.M. and Sheridan P. J., J. steroid Biochem. 16 (1982) 215–229), question the classical model of thermodependent nuclear translocation, based on biochemical experiments.

Corticosteroid receptors in the kidney of chick embryo: III. Nature, properties, and ontogeny of aldosterone receptor

An aldosterone receptor in the cytosol from kidney of chick embryos which had a sedimentation coefficient of 8.2 S and a molecular weight higher than 100,000 was identified. Kinetic analysis at 4° revealed a rapid association of the hormone to the receptor that followed second-order reaction kinetics and a dissociation of pseudo-first-order reaction kinetics. The association ( k a) and dissociation ( k d) rate constants were, respectively, 4.94 × 10 5 M −1 sec −1 and 8.33 × 10 −6 sec −1. From their ratio a K A value of 5.9 × 10 10 M −1 was calculated. In a series of experiments performed with kidneys of 17-day-old embryos, the K A at equilibrium, obtained from the Scatchard plot, was 3.1 ± 1.2 × 10 8 M −1, whereas the N max was 172 ± 14 fmol/mg protein. Competition studies with various steroids demonstrated that corticosterone had an affinity for the receptor close to that of aldosterone, thus suggesting a degree of resemblance of the mineralo- and glucocorticoid receptors in the chick embryo. However, the profiles of the binding affinities and capacities during the embryogenesis showed that the aldosterone-binding sites had a pattern completely different from that of the glucocorticoid receptor, indicating that the two receptors are most likely separate entities.

Autoradiographic study of thermo-dependent nucleo-cytoplasmic distribution of aldosterone binding sites in intact target cells

Autoradiographic studies of [ 3H]aldosterone ([ 3H-A] and [ 3H]dexamethasone binding sites in intact target cells (isolated collecting tubules of rabbit and rat kidney) revealed an almost exclusive nuclear localization of the hormone-receptor complexes. In the present work we compared the nucleo-cytoplasmic repartition of [ 3H]A-receptor complexes studied in parallel by biochemical and autoradiographic methods. In addition, the thermo-dependency of the nuclear translocation was examined. Kidney pyramids were incubated in vitro with [ 3H]A (2 × 10 −9 M) in the presence or absence of a 100-fold excess unlabelled A, at 30°C for 1 h or 4°C for 2 h. Then tissue was processed for isolation of nuclear and cytoplasmic fractions, on the one hand, or for obtention of microdissected tubular segments on which autoradiographs on dry films were performed. Autoradiographs showed that the specific labelling was almost exclusively nuclear without significant cytoplasmic labelling, at both 30 or 4°C. This indicates that almost all binding sites migrated rapidly into nuclei, and that this translocation did not depend on temperature. In contrast, parallel biochemical experiments yielded classical results, that is, at 30°C, the presence of specific binding sites in both cytoplasm and nuclei with a predominance in cytoplasm. At 4°C, the cytoplasmic binding was unchanged, but nuclear binding was drastically reduced, indicating thermodependency of nuclear translocation, when studied by biochemical methods including cell disruption. Autoradiographic results thus questioned the classical notion of thermo-dependent nuclear translocation of aldosterone-receptor complexes, based on results obtained by biochemical methods.

Aldosterone-binding sites in pregnant and lactating rat mammary glands.

We have identified and characterized aldosterone-binding sites in the mammary gland of pregnant and lactating rats by using the highly specific glucocorticoid RU26988 (11 beta, 17 beta-dihydroxy-17 alpha-propynyl-androsta-1,4,6-triene-3-one) to eliminate aldosterone binding to classical glucocorticoid (type II)-binding sites. The hierarchy of affinity of steroids competing for aldosterone in the presence of RU26988 was similar in mammary glands of pregnant and lactating rats and kidneys of lactating rats: deoxycorticosterone greater than aldosterone greater than or equal to corticosterone greater than dexamethasone; this pattern is similar but not identical to that for the mineralocorticoid (type I) receptor of a classical mineralocorticoid target organ, the kidney of the virgin female rat, where aldosterone has a higher affinity than corticosterone. This pattern (aldosterone greater than corticosterone) was also found in renal cytosols of pregnant rats. To define these aldosterone-binding sites in terms of dissociation constant and number of sites, it was necessary to pretreat cytosols with charcoal. Without this treatment, curvilinear Scatchard plots were obtained. After pretreatment with charcoal, subsequent Scatchard analysis indicated a single class of site with low capacity (3-33 fmol/mg protein) and dissociation constants of about 1-3 nM in kidneys and mammary glands of pregnant and lactating rats. The occupancy of this site in vivo may depend on the relative tissue concentrations of aldosterone or corticosterone, which, in turn, are modulated by the levels of extravascular transcortin.

Mineralcorticoid receptor in rat kidney and hippocampus: characterization and quantitation by isoelectric focusing.

The mineralcorticoid receptor in cytosol from the kidneys and hippocampi of adrenalectomized rats was characterized and quantified by use of isoelectric focusing in slabs of polyacrylamide gel. The mineralcorticoid receptor was labeled with [3H]aldosterone after selective blockade of the glucocorticoid receptor sites with a synthetic pure glucocorticoid RU 26988 [11 beta, 17 beta-dihydroxy-17 alpha-(1-propionyl)androsta-1,4,6-trien-3-one]. The apparent dissociation constant for aldosterone was 5.1 X 10(-9) M, and the ligand specificity studies revealed a mineralcorticoid pattern. The glucocorticoid and mineralocorticoid receptors were compared by limited trypsin digestion, followed by isoelectric focusing. Completely different fragmentation patterns were obtained for the two receptors in the same cytosol from either kidney or hippocampus. However, comparison of the mineralcorticoid receptor from the kidney with that from the hippocampus gave rise to identical fragmentation patterns. An antiglucocorticoid receptor antibody showed no crossreactivity with the mineralcorticoid receptor. Cytosol from kidney and hippocampus contained 28 +/- 24 and 155 +/- 115 fmol mineralcorticoid receptors/mg cytosol protein, respectively. Significant amounts of RU 26988-resistant [3H]aldosterone-binding sites were also present in cerebrum, cerebellum, spleen, and colon.

Structure-activity relationship of new steroidal aldosterone antagonists: Comparison of the affinity for mineralocorticoid receptors in vitro and the antialdosterone activity in vivo

The ability of 18 steroids with structures similar to spironolactone, progesterone or aldosterone to compete with [ 3H]aldosterone for binding at rat renal cytosol receptors in vitro and the antialdosterone activity in vivo were tested in comparison with spironolactone. The affinity of these compounds for mineralocorticoid receptors was then compared with their pharmacological action in rats. Replacement of the 17-spirolactone ring by a 17α-hydroxypropyl group and a 17β-hydroxyl group resulted in a loss of affinity for the [ 3H]aldosterone binding sites but not in a reduction in antialdosterone activity in vivo. Compared to spironolactone, C 6/C 7 unsaturated compounds showed a reduced activity both in vitro and in vivo. Substitution of the 7α-thioacetyl group of spironolactone by a 6, 7-methylene group in the β position (prorenone) increased the affinity to the receptor as well as the biological activity by 52 and 41%, respectively. Introduction of a methyl-group at the D-ring of spironolactone resulted in similar significant drops in activity both in vitro and in vivo. The progesterone like steroids were weak competitors for aldosterone in vitro and in vivo. Two of three aldosterone like steroids (18-deoxyaldosterones) still exhibit mineralocorticoid activity and one analogue could be classified as a weak aldosterone antagonist. The studies show in general that the comparison of the affinity for mineralocorticoid receptor sites with the antimineralocorticoid activity in vivo is a valuable procedure in the search for new antimineralocorticoid substances.

Binding and antimineralocorticoid activities of spirolactones in toad bladder.

The role of the soluble pool (cytoplasmic or cytosolic) of [3H]-aldosterone binding sites in the toad bladder was assessed by the use of two spirolactones, prorenone and spironolactone as a reference drug. Prorenone fulfills all the criteria for a specific competitive antagonist of aldosterone for its effect on Na+ transport. Compared with spironolactone (Ki approximately equal to 1 microM), prorenone was about eightfold less potent (Ki approximately equal to 8 microM). Competition for [3H]aldosterone binding sites by spironolactone and prorenone revealed an order of potency (spironolactone greater than prorenone) that corresponded to their antagonist activities in the Na+ transport assay. There was a linear correlation between the effects of the two spirolactones on the aldosterone-stimulated Na+ transport and their ability to displace [3H]aldosterone from its binding sites in the soluble pool. Finally [3H]prorenone binding sites were detected in the soluble pool but an insignificant number of antagonist-receptor complexes were found associated with the nuclear pool. Our study indicates that the aldosterone binding sites of the soluble pool are indeed mineralocorticoid receptors, which are probably the first intracellular mediators leading to an increased Na+ reabsorption.

Identification of mineralocorticoid binding sites in rat brain by competition studies and density gradient centrifugation.

A search for mineralocorticoid binding sites in neural tissue was carried out on the basis of competition by two mineralocorticoid antagonists (spironolactone and glycyrrhetinic acid), a mineralocorticoid agonist (9 alpha-fluorocortisol) and a pure glucocorticoid (RU 26988). The hippocampus was selected for these studies, in view of its preferential concentration of binding sites for [3H]-aldosterone (ALDO) and two glucocorticoids: [3H]-corticosterone (CORT) and [3H]-dexamethasone (DEX). Inhibition studies performed with 5 X 10(-4)-5 X 10(-9) M spironolactone and with 5 X 10(-4)-5 X 10(-8) M glycyrrhetinic acid, showed a dose-response reduction of [3H]-ALDO, [3H]-CORT and [3H]-DEX binding, implying that in brain these compounds did not behave as exclusive ALDO antagonists. Two concentrations of 9 alpha-fluorocortisol (5 X 10(-9) and 2.5 X 10(-8) M) preferentially displaced [3H]-ALDO in comparison to [3H]-DEX or [3H]-CORT. Relative binding affinity (RBA) of 9 alpha-fluorocortisol was also higher for the mineralocorticoid than for the glucocorticoids. RU 26988 (5 X 10(-6)-2.5 X 10(-8)M) gave differential inhibition of the three ligands and its RBA for [3H]-DEX site was twice as high as for [3H]-CORT, and three orders of magnitude higher than for [3H]-ALDO binding sites, thus clearly separating sites for gluco- and mineralocorticoids. Further evidence for the presence of separate binding molecules for mineralo- and glucocorticoids in hippocampal cytosol was provided by ultracentrifugation on 16-41% glycerol gradients containing molybdate, which yielded sedimentation values of 9.88 +/- 0.27 for [3H]-DEX (n = 5), 10.48 +/- 0.27 for [3H]-CORT (n = 9) and 11.3 +/- 0.13 for [3H]-ALDO (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the glucocorticoid-receptor complex.

Activation of the glucocorticoid-receptor complex.

Aldosterone binding sites in aortic cell cultures from spontaneously hypertensive rats.

Spontaneously hypertensive rats and rats made hypertensive by deoxycorticosterone-salt treatment have in common increased Na+ and K+ permeability and transport in their aortic cells. These changes may be important factors in the development of the hypertensive state and may be mediated by mineralocorticoid binding to intracellular sites in the aorta. Therefore, we examined 3H-aldosterone binding in aortic cell cultures from spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. Vascular corticoid binding sites in the two strains were compared by Scatchard analysis of Kd and Bmax, pH and temperature stability, and subcellular binding. By all of these criteria normotensive rats. These results indicate that the underlying genetic defect in spontaneous hypertension is not an intrinsic cellular defect which alters mineralocorticoid binding in the aorta.

116. Autoradiographic study of nuclear translocation of aldosterone binding sites in intact target cells: Lack of temperature dependency

116. Autoradiographic study of nuclear translocation of aldosterone binding sites in intact target cells: Lack of temperature dependency

Aldosterone and corticosterone binding and effects on Na+ transport in cultured kidney cells.

A continuous line of cells (A6) derived from toad kidney has been shown to form epithelia in culture that manifest aldosterone-stimulatable transepithelial sodium transport. In this study an efficient filtration assay for nuclear binding of [3H]aldosterone was validated. Specific high-affinity aldosterone and corticosterone binding sites in the particulate (nuclear-enriched) fraction were characterized in intact epithelia. Despite metabolism of both steroids, two high-affinity binding sites for each were demonstrable: aldosterone, K'd1 = 0.85 (+/- 0.19) X 10(-10) and K'd2 = 1.6 (+/- 0.42) X 10(-8) M; corticosterone, K'd1 = 0.5 (+/- 0.31) X 10(-10) and K'd2 = 0.32 (+/- 0.19) X 10(-8) M. Analogue competition-binding studies indicated a qualitative difference in the two sites and co-occupancy of both sites by the two steroids. The sodium transport response to aldosterone and corticosterone approximated a linear function of occupancy of the lower affinity sites. Although the lower affinity sites resemble mammalian glucocorticoid receptors in terms of relative binding affinities for analogues, we conclude that they are the receptors which mediate the aldosterone and corticosterone stimulation of Na+ transport in these epithelia.

Aldosterone binding in isolated tubules I. Biochemical determination in proximal and distal parts of the rabbit nephron.

Microbiochemical methods were applied to proximal tubules (PCT) and a mixture of distal and cortical collecting tubules (D + C) of rabbit kidney in order to define aldosterone binding sites. For each experiment, after incubation of kidney pyramids with [3H]aldosterone ([3H]A), either alone or in the presence of an excess unlabeled A, 100-150 mm of both categories of tubules were microdissected using collagenase. Specific binding was determined on the nuclear fraction of each sample. Aldosterone concentrations ranged from 2 X 10(-9) to 4.5 X 10(-8) M. No specific binding was detectable in PCT. Specific binding in D + C increased rapidly as a function of [3H]A concentration up to 5 X 10(-9) M and then more slowly. No plateau was reached. Both the absence of saturation of the binding curve and the curvilinear aspect of the Scatchard plot suggested the presence of two binding sites, one of high affinity, presumably a mineralocorticoid site, and the other of lower affinity, possibly a glucocorticoid site. These experiments suggest that the distal structures of the nephron, located in the cortex, are the main sites of binding of aldosterone and contain a high number of specific binding sites for this hormone.