Mineralocorticoid Binding Sites Research Articles

Poster 73: Association of Mineralocorticoid Binding Sites (Type 1 Receptors) with Vestibular Blood Vessels

Poster 73: Association of Mineralocorticoid Binding Sites (Type 1 Receptors) with Vestibular Blood Vessels

Glucocorticoid and mineralocorticoid receptors in rat neocortical and hippocampal brain cells in culture: characterization and regulatory studies

Glucocorticoid and mineralocorticoid binding sites were characterized in cell cultures derived from neocortical and hippocampal brain tissue from fetal (E18) rats. Specific and saturable binding was detected in living cells with a sensitive whole cell binding method using [ 3H]dexamethasone ([ 3H]DEX) and [ 3H]aldosterone ([ 3H]ALDO) (in the presence of RU 28362, a selective glucocorticoid receptor (GR) agonist) as ligands for the measurement of glucocorticoid and mineralocorticoid receptors (MRs), respectively. Specific corticosteroid binding was demonstrated as early as day 4 in culture in neocortical cells, with a time-dependent increase in binding sitesduring further culturing time. At 7–9 days in vitro Scatchard analysis of [ 3H]DEX binding revealed a maximum binding capacity ( B max) of 83.4 ± 5.0 fmol/mg protein and a binding affinity ( K d) of 3.6 ± 0.4 nM in neocortical brain cells. Competition binding studies with [ 3H]DEX demonstrated a glucocorticoid specificity of receptor sites (relative binding affinity: RU 28362 = DEX>PROG>ALDO). Similar binding characteristics were demonstrated for GRs in cultures derived from fetal hippocampal tissue ( B max49.1 ± 5.8fmol/mg protein, K d of 3.5 ± 0.2 nM). Analysis of MRs with [ 3H]ALDO (+ RU 28362) revealed specific and saturable binding in hippocampal cultures, with a B max of 8.0 ± 0.5 fmol/mg protein and a K d of 0.2 ± 0.1nM. Competition studies with [ 3H]ALDO showed a mineralocorticoid-like pattern of receptor binding (relative binding affinity: CORT = ALDO>PROG>DEX). In addition, small numbers of MRs were detectable in cortex-derived cultures ( B max: 3.7 ± 0.8fmol/mg protein, K d: 0.3 ± 0.2nM). After incubation for 24 h, corticosterone dose-dependently down-regulated [ 3H]DEX binding sites in neocortical and hippocampal cultures, indicating the homologous regulation of these receptors under in vitro conditions. Continous incubation of cultures with serotonin (5-hydroxytryptamine) resulted in an increase in [ 3H]DEX binding sites in both neocortical and hippocampal cultures. The present study demonstrates that corticosteroid receptors are constitutively expressed in neocortical and hippocampal brain cells in culture. They are regulated by hormones and neurotransmitters, indicating that the in vitro system employed is well suited for studies on the mechanisms of GR and MR regulation and the characterization of steroid receptor-mediated effects in neuronal cells.

Role of central mineralocorticoid binding sites in development of hypertension

The possibility that central mineralocorticoid binding sites are involved in the development of mineralocorticoid hypertension was examined using chronic blockade of these sites with a specific mineralocorticoid receptor antagonist RU 28318 administered by intracerebroventricular (icv) infusion. The antagonist significantly attenuated the development of deoxycorticosterone acetate (DOCA)-salt hypertension, but the development of one-kidney, one-clip renal hypertension was not affected. This antihypertensive action was attributable to a central action, since intraperitoneal infusion of the same dose of mineralocorticoid antagonist did not alter the peak development of DOCA-salt hypertension. The icv infusion of RU 28318 did not change either the increase of fluid intake induced by DOCA-salt treatment or the pressor reactivity to centrally or peripherally injected arginine vasopressin and angiotensin II and peripherally administered phenylephrine. The antihypertensive action of icv infusion of the mineralocorticoid antagonist was associated with a reduction of neurogenic vasomotor tone and a restoration of impaired arterial baroreflexes. We conclude that functional integrity of central mineralocorticoid binding sites is required for the full development of DOCA-salt hypertension.

Steroid C-20 oxidoreductase activity of duck intestinal mucosa: The interrelations of the enzymatic activity with steroid binding

The binding of corticosterone and aldosterone to domestic duck ( Anas platyrhynchos)-dispersed colonic mucosal cells at 37° was investigated. It was found that in contrast to experiments using cell-free intestinal preparations, corticosterone was extensively metabolized and it was the metabolite, not the native steroid that became receptor bound and all the bound ligand was in the nuclear fraction. The metabolite turned out to be identical with 4-pregnene-11β,20β,21-triol-3-one (20β-dihydrocorticosterone, 20β-DHB). Binding experiments with [ 3H]corticosterone yielded the following kinetic parameters: K d = 87.6 n M, N max = 337,900 sites/cell. When synthetic [ 3H]20β-DHB was used as the ligand a curvilinear-binding isotherm was obtained. This could be resolved into a high affinity-low capacity (HA) and a low affinity-high capacity (LA) component with the following binding parameters: K d,HA = 91 n M, N max, HA = 130,800 sites/cell; K d,LA = 5.4 × 10 −6 M, N max,LA = 3.7 × 10 6 sites/cell. Binding of the metabolite to cell-free preparations, at 0°, gave the following results: for cytosol, linear-binding isotherm, K d = 14.0 n M, N max = 26.5 fmol/mg protein; and for crude nuclei, curvilinear-binding isotherm, K d,HA = 45.0 n M, N max,HA = 5.33 pmol/mg DNA; K d,LA = 2.2 × 10 −6 M, N max,LA = 286.6 pmol/mg DNA. [ 3H]Aldosterone was also bound by the dispersed whole cells and again, this binding was only nuclear ( K d = 9.3 n M, N max = 10,042 sites/cell). The bound ligand was unchanged aldosterone. Competition experiments have shown that aldosterone did not compete with 20β-DHB for binding sites and vice versa. The intracellular 20β-hydroxysteroid oxidoreductase responsible for the transformation of corticosterone was found mostly in the cytoplasm. Kinetic studies with the enzyme yielded classical Michaelis-Menten kinetics ( K m = 15.7 μ M, V max = 2.6 nmol/min/mg protein). The enzyme had an apparent M r of 35 kDa and a R s of 25.5 Å. It is believed that our results might explain the binding of aldosterone to mineralocorticoid-binding sites in the presence of overwhelming concentrations of corticosterone and that experiments with cell-free tissue preparations, performed at 0°, do not reflect the true cellular-binding events.

Central interactions between aldosterone and vasopressin on cardiovascular system.

Although the presence of mineralocorticoid binding sites have been demonstrated in brain, little is known about their physiological role. The purpose of this study was to evaluate possible interactions between a relatively short 2-day central infusion of aldosterone (5 ng/h) and a diverse group of centrally acting pressor agents. The intracerebroventricular infusion of aldosterone selectively attenuated the pressor response produced by the injection of arginine vasopressin (AVP, 10-400 ng) into the lateral ventricle without altering the responses to ventricular administration of 50-200 ng angiotensin II (ANG II), 150 ng carbachol, and 0.5 and 1 M hypertonic sodium chloride. No aldosterone-vasopressin interaction occurred in rats receiving a simultaneous central infusion of aldosterone and RU 28318 [7 beta-hydroxy-3-oxo-7 alpha-propyl(17 alpha)-pregn-4-ene-21-potassium carboxylate], a specific mineralocorticoid receptor antagonist. Baroreflex reactivity and the pressor response to intravenous AVP were not modified by the aldosterone treatment, indicating that overall cardiovascular reactivity was not depressed. Because the vascular reactivity of the mesenteric artery to AVP and norepinephrine remained unchanged after 2 days of central aldosterone infusion, and because plasma levels of aldosterone were not altered, the selective inhibition by this mineralocorticoid of the central AVP response appears to be purely central in origin. This specific central effect of aldosterone is mediated through interaction with mineralocorticoid receptors.

Aldosterone receptors in different types of primary hyperaldosteronism.

The number of mineralocorticoid-binding sites on mononuclear leukocytes and plasma aldosterone (aldo) concentrations were measured in patients with different types of primary hyperaldosteronism. Patients with unilateral adenoma and patients with bilateral adrenal hyperplasia had a significantly lower (P less than 0.001) mean number of binding sites for aldo [144 +/- 36 (+/- SD; n = 6) and 140 +/- 28 sites/cell (n = 4), respectively] compared with normal subjects (292 +/- 110 sites/cell; n = 25). In four patients with dexamethasone-suppressible hyperaldosteronism, mineralocorticoid-binding sites in mononuclear leukocytes were normal (291 +/- 108 sites/cell). In all patients undergoing surgery for unilateral adenoma, the receptors normalized 3 months after the operation. In two patients the reduction in receptors persisted for a short time after surgery even though the plasma aldo level had already normalized. We conclude that mineralocorticoid excess produces down-regulation of mineralocorticoid receptors, which, in turn, might contribute to the genesis of the aldo escape phenomenon.

ATYPICAL 11β-HYDROXYLASE DEFICIENCY: ABSENCE OF RENIN SUPPRESSION AND HYPERTENSION DESPITE HIGH DOC

Congenital adrenal hyperplasia due to 11β-hydroxylase (11β-OHase) deficiency usually produces hypertension and virilization. The hormonal profile is typified by elevated serum and urinary DOC and compound S, and suppressed renin (PRA) and aldosterone (aldo). We report 2 cases in which extreme elevation of serum and urinary precursors of 11β-OHase produced neither hypertension nor suppression of PRA and aldo. These findings correlated with low number of mineralocorticoid binding sites per cell (MRBS) in peripheral blood leukocytes. DOC suppressed following dexamethasone administration, and additional DCC infusion caused transient hypertension and expected renin suppression. We postulate that down-regulation of MRBS is an adaptive response to high DOC, inhibiting mineralocorticoid effects. Normal basal aldo levels indicate intact glomerulosa 11β-hydroxylation, suggesting a defect in tissue-specific expression of the enzyme, rather than a structural gene defect.

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)

Aldosterone binding along the rabbit nephron: an autoradiographic study on isolated tubules.

The sites of action of aldosterone (A) along the tubule of rabbit kidney were studied by autoradiographic localization of mineralocorticoid-binding sites on microdissected tubular segments. Kidney pyramids were incubated at 30 degrees C for 1 h in a collagenase solution with [3H]aldosterone at a concentration of 1.5 X 10(-9) M with and without an excess unlabeled A. Tubular segments were then microdissected and transferred onto dry film; fixation and staining were done only after exposure of the film 4 mo later in order to avoid diffusion. Specific nuclear labeling was 19.0 +/- 1.3 silver grains/100 micrometers2 in distal convoluted tubules (n = 28) and 21.0 +/- 1.8 in cortical collecting ducts (n = 18). No difference between these two structures was observed (P greater than 0.1, paired t test, n = 15). No specific binding was found in the proximal tubule (0.5 +/- 0.4, n = 17). In the thick ascending limb of Henle's loop, the labeling was low (3.9 +/- 0.9, n = 16). We conclude that, in the rabbit kidney, nuclear mineralocorticoid-binding sites, presumably receptors, are present in the distal and cortical collecting tubule.

The Mineralocorticoid Antagonist Activity of an 11β, 18-Oxidopregnane*

Removal of the 18-hydroxy group of the hemiacetal form of aldosterone transforms its activity from that of pure agonist to predominant antagonist. The 18-deoxy derivative possesses one third of the binding affinity of aldosterone for the cytoplasmic mineralocorticoid receptor of rat kidney and exhibits an approximate 2:1 antagonist to agonist ratio in both toad bladder and adrenalectomized rat bioassay systems. The promising properties of the 11 beta,18-oxidopregnane tested included very low androgen receptor affinity and approximately equal effectiveness in vitro and in vivo in displacing aldosterone from mineralocorticoid-binding sites in the rat.

Mechanism of Action of a New Antialdosterone Compound, Prorenone*

Two new aldosterone antagonists, K-prorenoate [potassium 3(17 beta-hydroxy-6 beta, 7 beta-methylen-3-oxo-4-androsten-17 alpha-yl)propionate] and prorenone [3(17 beta-hydroxy-6 beta, 7 beta-methylen-3-oxo-4-androsten-17 alpha-yl) propionic acid gamma-lactone], its lactonic form, were studied in rat kidney using in vitro systems. Study of [3H]prorenone binding by a recently developed computer method indicated a high affinity, low capacity class of sites which are, seemingly, mineralocorticoid receptors. In competition experiments performed on [3H]aldosterone- and [3H]dexamethasone-binding sites, prorenone appeared to be a good competitor for mineralocorticoid-binding sites and a poor competitor for glucocorticoid-binding sites. The specificity of this molecule was further confirmed by its poor ability to displace [3H]dihydrotestosterone from rat prostate androgenic receptors compared to spironolactone [3-(3-oxo-7 alpha-acetylthio-17 beta-hydroxy-4-androsten-17 alpha-yl) propionic acid gamma-lactone]. In the same experiments, K-prorenoate demonstrated a very low affinity for the two types of receptors. The behavior of [3H]prorenone cytosolic complex was also studied in kidney mince experiments, which showed that the [3H]prorenone complex was not able to translocate into the nucleus. Prorenone inhibited the binding of [3H]aldosterone to the receptor and, consequently, the nuclear binding of aldosterone was not observed.