Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.     

Heterogeneity of steroid hormone receptor in adult rat lung cytosol

Chromatography on cellulose DEAE-52 columns revealed that the glucocorticoid receptor from rat lung cytosol consisted of a component in the 0.001 M prewash, revealed with synthetic steroids and natural mineralocorticoids, a second component eluted with 0.04 M PO₄, labelled with triamcinolone, dexamethasone, and a third moiety in the 0.06 M PO₄ region, evident with natural glucocorticoids (corticosterone, cortisol) as well as mineralocorticoids (aldosterone, deoxycorticosterone). The thrid component coelutedf with rat blood serum transcortin in double labelled experiments. Rat lung was devoid of another component in the 0.02 M PO₄ found in rat liver supernate and of the mineralocorticoid receptor evident only in rat kidney. Chromatography on Sephadex G-200 columns revealed a shift of radioactivity from a higher to a lower molecular weight region in the presence of 0.4 M KCl. Collectively, these studies indicate the subunit nature of the lung receptor as evidenced in most tissues hitherto tested. Moreover, polymorphism within a given subunit component can not be revealed by competition alone, as attempted by others, but can be revealed under selected conditions of physical separation.     

Isolation and characterization of a subchloroplast particle enriched in iron-sulfur protein and P700.

Transcortin-bound gluco- and mineralocorticoids were fractionated on a number of chromatographic systems. Contrary to earlier suggestions of a homogenous unit by competition binding and Scatchard analysis, a polymorphic nature of the globulin was evident with corticosterone on Sephadex A-50 columns (components in 0.4 and 0.6 m KCl) and with synthetic steroids (triamcinolone acetonide, dexamethasone) or natural mineralocorticoids (aldosterone, 18-hydroxy-deoxy-corticosterone) on diethylaminoethyl cellulose-52 gels (species in 0.001 and 0.06 m phosphate). Besides the major component of molecular weight 55,000, a heavier shoulder in the 67,000 molecular weight region was obtained with cortisol, dexamethasone, triamcinolone, and aldosterone from Sephadex G-200 columns, on which binding was reduced in the presence of high salt (0.4 m KCl). Triamcinolone- and dexamethasone-bound components eluted at lower salt concentrations from the DE-52 column than natural steroid-corticosteroid-binding globulin complexes. The various features of serum carrier binding are discussed in terms of steroid-receptor association in hormone-specific target tissues.     

Transcortin in rat kidney tissue: distribution in microsomal fractions

During chromatography of renal tissue cytosolic proteins on DEAE-cellulose the protein specifically binding [3H]corticosterone is eluted within the potassium phosphate concentration range of 0.08-0.10 M. Analysis of kidney slices revealed the synthesis of [3H]transcortin whose electrophoretic mobility was close to that of the blood plasma protein. Using radioimmunochemical methods, it has been found that transcortin-specific [125I]IgG antibodies interact with growing polypeptide chains of membrane-bound polyribosomes. Free polyribosomes do not bind antibodies against transcortin.     

Paradoxical differences in the receptor binding of two new antimineralocorticoids

Two synthetic derivatives of spironolactone were used to examine various aspects of the mineralocorticoid receptor structure and function. Introduction of a propyl residue in the 7-position of spironolactone produced a molecule (RU 26752) that saturated the aldosterone specific receptor in the 1-10 nM range, and another, more abundant species in the 10-100 nM range which had little affinity for the natural hormone. The specificity for both sites was increased when the methoxycarbonyl group was introduced in the 7-position (ZK 91587). Neither antagonist exhibited affinity for blood serum transcortin or receptors in non-target organs like the lung and the liver. RU 26752-receptor complex was more unstable than the hormone-receptor complex at 35 degrees C but underwent comparable thermal activation as evidenced by binding to DNA cellulose and the 7 S to 4 S shift on sucrose gradients. In contrast, ZK 91587 did not permit thermal activation and greatly labilized the receptor at 35 degrees C. In ion exchange chromatography, two peaks were observed with unactivated ZK 91587-receptor complex, but RU 26752 was bound exclusively to the component eluted with high salt. Molecular filtration revealed two peaks of bound radioactivity with both antimineralocorticoids. These studies reveal important differences in the mechanism of action of two antagonists differing solely in the residue in position 7 of the spironolactone molecule. Such differences could be exploited to purify the mineralocorticoid receptor and clinically to prescribe the appropriate drug with greater precision.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Physical separation of aortic corticoid receptors with type I and type II specificities

Previous gel filtration binding assay studies indicated that rat vascular smooth muscle cells contained corticoid receptor I and corticoid receptor II sites which could be distinguished on the basis of their relative affinities for aldosterone and dexamethasone. Ion-exchange chromatography experiments were designed to separate the two sites for further studies on their physical characteristics and role in vascular smooth muscle cell physiology. Cultured aortic cells were incubated with 5-10 nM 3H steroid alone or in the presence of 10-fold non-radioactive steroid competitor for 30 min at 37 degrees C. Following cell lysis, total cellular protein-bound steroid was isolated using Sephadex G-25 and applied to a DEAE-cellulose ion-exchange column. Three peaks of radioactivity were eluted using a 1-200 mM sodium phosphate gradient: peak I (30-38 mM), peak II (52-64 mM), and peak III (92-102 mM). Peaks I and II contained 60% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor II sites (dexamethasone greater than aldosterone). Peak III contained 40% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor I sites (aldosterone greater than dexamethasone). These studies support the binding assay data on steroid specificity and relative proportion of type I and II sites. They also document the existence of type I and II corticoid receptors with different physicochemical characteristics in rat aortic smooth muscle cells.     

Characteristics of aldosterone binding in rat and human serum

Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.     

Identical properties of aldosterone and corticosterone binders and their presence in rat brain and kidney

The [3H]corticosterone binders from rat brain and kidney were characterized by binding affinity and chromatographies, and compared with the binders for [3H]aldosterone and [3H]triamicinolone acetonide. Corticosterone-binding globulin-like molecules at very high concentrations in crude extracts were completely eliminated by a DEAE-gel adsorption procedure. [3H]Aldosterone binder in the renal, DEAE-treated fraction was recovered in a single peak by gel-filtration chromatography and by ultracentrifugation in linear sucrose gradients, independent of hormone-binding and tungstate, a stabilizer of the binder. The Stokes' radius and sedimentation coefficient of the renal aldosterone binder were 6.6 nm and 9.3S, respectively, indicating an apparent molecular weight of 263,000. Corticosterone-preferring binder also existed in the DEAE-treated fraction. Both aldosterone and corticosterone binders were found in the brain and kidney preparations. Comparison among the binders showed identical values of Stokes' radius and elution pattern from DEAE-Toyopearl in a linear salt gradient regardless of the organ and the hormones. Scatchard analyses of [3H]aldosterone and [3H]corticosterone binding showed for each ligand only one group of high-affinity sites with the equivalent dissociation constants, 4-7 nM. The orders of steroids in competing for the two high-affinity sites were equivalent: corticosterone greater than or equal to aldosterone much greater than triamcinolone acetonide, and that for the triamcinolone acetonide binding was triamcinolone acetonide much greater than aldosterone greater than or equal to corticosterone. Hydroxyapatite column chromatography separated the aldosterone and corticosterone binders from the triamcinolone acetonide binder, but not the aldosterone binder from the corticosterone binder. It is concluded that aldosterone and corticosterone binders distinct from triamcinolone acetonide binder exist in rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcortin in rat kidney: subcellular distribution of transcortin-synthesizing polyribosomes

The [3H]corticosterone-transcortin complexes from kidney cytosol show elution positions on DEAE-cellulose identical to serum transcortin. The incorporation of 14C-labeled amino acids into anti-transcortin-precipitable material of kidney slices has been measured and compared with that of serum transcortin. It was established that kidney synthesized transcortin with an apparent molecular weight of 66 kDa on SDS-electrophoresis which resembles serum corticosteroid-binding globulin. Studies on the binding of [125I]anti-transcortin-IgG to membrane-bound rat kidney polyribosomes revealed an association of [125I]anti-transcortin-IgG with a discrete polyribosome fraction in the heavy polyribosome region; free polyribosomes were devoid of antigenic material able to bind antibodies to transcortin.     

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.     

Steroid-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders. Correlation with natriferic activity.

We have identified a group of proteins (Mr approximately 70 000-80 000; pI approximately 5.5-6.0) in giant-toad (Bufo marinus) urinary bladders whose synthesis appears to be related to aldosterone-stimulated Na+ transport. Spironolactone, a specific mineralocorticoid antagonist in renal epithelia, inhibits the synthesis of these proteins as well as the natriferic effect of the hormone. Since a variety of other steroids (some of which are traditionally considered to be glucocorticoids) also stimulate Na+ transport in toad urinary bladders, we examined whether their natriferic activity was expressed in a fashion similar to that of aldosterone. Short-circuit current was used to measure Na+ transport, and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. At a concentration of approximately 100 nM, dexamethasone, corticosterone and aldosterone were equinatriferic. Dexamethasone and aldosterone had identical dose-response curves, maximal and half-maximal activity being evident at concentrations of approximately 100 nM and 10 nM respectively. In contrast, at a concentration of approximately 10 nM, corticosterone had no effect on Na+ transport. The natriferic activities of these three steroids correlate with their known affinities for the putative mineralocorticoid receptor in toad urinary bladders. Natriferic concentrations of dexamethasone and corticosterone (140 nM) induced the synthesis of proteins with characteristics identical with those induced by aldosterone. Spironolactone, at an antagonist/agonist ratio of 2000:1, inhibited steroid-induced Na+ transport and the synthesis of these proteins. Thus it appears that all natriferic steroids share a common mechanism of action in toad urinary bladders. Natriferic activity can be correlated not only with relative steroid-receptor affinity but also with the induction of a specific group of epithelial-cell proteins.     

Evidence for an antagonist specific receptor that does not bind mineralocorticoid agonists

Kinetics of association--dissociation, competition and chromatography on two different resins, all revealed the presence of a new binding site which: specifically accepts 7-alpha-propyl spirolactone (3H-RU-26752), has little affinity for aldosterone, is present only in the target tissue (rat kidney), and is wanting in a non-target organ (liver). The presence of such sites could explain syndromes of mineralocorticoid excess where even trace amounts of an unusual aldosterone analogue, with little affinity for the classical mineralocorticoid receptor, can nevertheless produce hypertension through the intervention of an entirely new and abundant receptor system. This new molecule thus forms a novel tool to understand the nature and function of the soluble mineralocorticoid receptor in target organs.     

Investigation of the corticosteroid receptor system in rat hippocampus by ion exchange fast protein liquid chromatography

In order to study the receptor system for adrenocortical steroids, hippocampal cytosolic preparations--containing both type I and type II receptors--were subjected to anion exchange fast protein liquid chromatography (FPLC). With running buffer containing Tris, EDTA, and glycerol three peaks (1-3) were eluted from the column at 220, 400 and 560 mM NaCl respectively regardless of whether [3H]corticosterone or [3H]RU 28362 had been used as radiotracer. None of the peaks was caused by serum transcortin as revealed by control studies. However, the sequestering influence of transcortin on receptor binding of corticosterone could be demonstrated by the FPLC technique with mixtures containing serum and hippocampus cytosol. Competition experiments with cytosolic samples revealed that type I receptor was present only in peaks 2 and 3 while type II was found in all three peaks in variable amounts, depending on the presence of molybdate. When molybdate was added to the running buffer only two peaks (2 and 3) were eluted, both containing type I and type II receptors. Peak 1 was attributed to the activated type II receptor while peak 2 represented nonactivated receptors. The origin of peak 3 remains uncertain. The data indicate that molybdate must be present in the cytosolic preparation and in the running buffer to keep type II receptor in its nonactivated form. Type I receptor was probably not transformed into the activated form in the absence of molybdate but lost binding capacity and/or affinity for corticosterone.     

Glucocorticoid metabolism and Na+ transport in chicken intestine

The role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone and plasma concentration of aldosterone is much lower than glucocorticoids. In mammals, the aldosterone specificity is conferred on the low-selective mineralocorticoid receptors by glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase (11HSD) that converts cortisol or corticosterone into metabolites (cortisone, 11-dehydrocorticosterone) with lower affinity for these receptors. The present study examined the chicken intestine, whether changes in 11HSD activity are able to modulate the effect of corticosterone on Na+ transport, and how the metabolism of this hormone is distributed within the intestinal wall. This study shows that not only aldosterone, but also corticosterone (B), was able to increase the electrogenic Na+ transport in chicken caecum in vitro. The effect of corticosterone was higher in the presence of carbenoxolone, an inhibitor of steroid dehydrogenases, and was comparable to the effect of aldosterone. The metabolism of B in the intestine was studied; results showed oxidation of this steroid to 11-dehydrocorticosterone (A) and reduction to 11-dehydro-20beta-dihydrocorticosterone (20diA) as the main metabolic products at low nanomolar concentration of the substrate. In contrast, 20beta-dihydrocorticosterone and 20diA were the major products at micromolar concentration of B. Progesterone was converted to 20beta-dihydroprogesterone. The metabolism of corticosterone was localized predominantly in the intestinal mucosa (enterocytes). In conclusion, the oxidation at position C11 and reduction at position C20 suggest that both 11HSD and 20beta-hydroxysteroid dehydrogenase (20HSD) operate in the chicken intestine and that the mucosa of avian intestine possesses a partly different system of modulation of corticosteroid signals than mammals. This system seems to protect the aldosterone target tissue against excessive concentration of corticosterone and progesterone.     

Characterization of steroid receptors in the gut and kidney of the frog (Rana catesbeiana) and in the gut of the turtle (Chrysemys picta)

Paraglucocorticoid- and paramineralocorticoid-binding cytosolic receptors (pGR, pMR) were demonstrated in the intestine and kidney of the frog, Rana catesbeiana and in the intestine of the turtle, Chrysemys picta, in the presence of sodium molybdate. These receptors were of high affinity and low capacity with the following binding parameters: pGR:Kd:frog intestine (FI), triamcinolone acetonide (TA): 3.3 nM, corticosterone (B): 3.4 nM; frog kidney (FK), TA:4.3 nM, B: 9.3 nM; turtle intestine (TI), TA: 4.8 nM; Nmax: FI, TA: 357, B: 371; FK, TA: 301, B: 157; TI, TA: 350 fmol/mg protein. pMR:Kd: FI, aldosterone: 0.9 and 90 nM (biphasic curves); FK, aldosterone: 0.6 and 36 nM (biphasic curves); Nmax: FI, 13 and 147 fmol/mg protein; FK, 78 and 109 fmol/mg protein. The receptor had the following ligand affinities: pGR: FI and FK: triamcinolone acetonide greater than DOC greater than 11 beta-hydroxyprogesterone greater than progesterone greater than corticosterone greater than cortisol greater than aldosterone greater than 11-dehydrocorticosterone greater than 17 alpha-hydroxyprogesterone greater than cortisone; TI: triamcinolone acetonide greater than corticosterone greater than progesterone greater than DOC greater than cortisol greater than aldosterone; pMR: FI and FK: corticosterone greater than 11 beta-hydroxyprogesterone greater than aldosterone greater than triamcinoline acetonide = cortisol greater than DOC greater than 11-dehydrocorticosterone greater than progesterone greater than 17 alpha-hydroxyprogesterone greater than cortisone. Androgens, estrogens or 18-hydroxycorticosterone did not compete for binding in either tissue. The heat activated frog receptors did not bind to naked DNA, though the turtle receptor did. It was possible to show that cytosol receptor-ligand complexes from all tissues were bound by nuclear acceptor sites. On linear sucrose gradients, the FI TA-receptor complex sediments with a single peak (7.5S), the FK TA-receptor complex gave two peaks (8.0 and 4.4S) and the TI TA-receptor complex showed a single peak (9.0S). The hydrodynamic parameters of the pGR's were determined by gel exclusion on Sephacel S-300. The following results were obtained: Mr: FI, 265, 80, 40 kDa (multiple proteins); FK, 280, 60, 20 kDa (multiple proteins); TI, 366 kDa; Rs: FI, 6.9, 3.9 nm; FK, 6.9, 2.9 nm; TI, 7.6 nm; f/f0: FI, 1.6; FK, 1.6; TI, 1.6. It is suggested on the basis of the binding and hydrodynamic parameters that non-mammalian epithelia corticosterone receptors have undergone biochemical evolution from one class of vertebrates to another.     

The influence of transcortin on adrenocorticotropin-stimulated corticosterone production in monolayer cultured rat adrenal cells

To verify the influence of the protein binding status of steroids adjacent to adrenal cells on steroidogenesis, the effect of transcortin, a specific binding protein of cortisol or corticosterone, on adrenocorticotropin (ACTH)-stimulated corticosterone production in monolayer cultured rat adrenal cells was studied. The transcortin in concentration of 5 x 10(-7) M was loaded with 0, 2.5, 5 and 10 pg/ml ACTH-(1-24), and the cells were incubated for 2 and 4 hours. Since molar concentrations of corticosterone produced in the medium were below the transcortin concentration at all levels of stimulation, protein-unbound corticosterone in the medium may have been largely reduced by the addition of transcortin. However, the total corticosterone production was not influenced by the transcortin added to the medium. It was speculated that protein-unbound steroid within the concentration range modulated by transcortin in the area surrounding the adrenal cells may not affect adrenal steroidogenesis.     

Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain.

The binding of [3H]corticosterone and [3H]dexamethasone to soluble macromolecules in cytosol of the hippocampal region of the brain has been studied in adrenalectomized male rats. Unlabeled dexamethasone appears to be a less effective competitor than corticosterone in the binding of [3H]corticosterone, while both unlabeled steroids compete equally well for the binding or [3H]dexamethasone. Further investigation of macromolecular complexes with [3H]dexamethasone and [3H]corticosterone revealed that they differ from each other in their behavior during ammonium sulfate precipitation, BioRad A-5M gel permeation chromatography, DE-52 anion exchange chromatography and DNA-cellulose chromatography. (1) After exposure to a 33% ammonium sulfate solution relatively more [3H]dexamethasone complex than [3H]corticosterone complex is precipitated. (2) Treatment of the cytosol with 0.3 M KCl gives disaggregation of the supramolecular 3H-labeled corticoid complexes which are seen eluting with the void volume during gel permeation chromatography on Biorad A-5M at low ionic strength. In 0.3 M KCl, the [3H]dexamethasone complex has an elution volume somewhat smaller than that of bovine serum albumin, while the [3H]-corticosterone complex in 0.3 M KCl is too unstable to survive chromatography with A-5M. (3) Chromatography on DE-52 resolved the 3H-labeled corticoid complexes into three binding components. The complex with [3H]dexamethasone contains a higher percentage (85%) of a component less firmly attached (i.e. eluted by 0.15 M KCl) to the anion exchange resin than is observed for the complex with [3H]corticosterone (49%). (4) The complexes with 3H-labeled corticoids display an enhanced affinity for calf thymus DNA adsorbed to cellulose following "activation", warming to 25 degrees C for 15 min. Concurrently, a fraction of the [3H]dexamethasone complex becomes able to more firmly attach to the DE-52 anion exchange resin. These results with the binding of the cytosol hormone-receptor complexes to DNA-cellulose do not explain the marked in vivo preference of hippocampus for the cell nuclear uptake of [3H] corticosterone. However, the other differences in the properties of the complexes formed with the two labeled glucocorticoids support our previous inference that there may be more than one population of adrenal steroid "receptors" in brain tissue.     

Paradoxical nature of estrogen agonist and antagonist binding in rat liver

Whereas Tamoxifen exerts potent antiestrogenic action in ER dependent breast cancer, it was largely without effect on rat liver gluconeogenesis which could be dramatically diminished by estrogens and androgens. Although estradiol was preferentially bound to an ER₄ component that coeluted with CBG from DE-52 columns, ³H-tamoxifen labelled the ER₃ moiety that was clearly distinct from transcortin. Similarly, testosterone was bound to the AR₄ entity but R-1881 was eluted in the AR₃ region. All these ER and AR populations were furthermore distinct from liver GR. These, for the first time, demonstrate polymorphic nature of AR and ER and suggest that agonist and antagonist actions may be expressed via separate populations of the receptor, contrary to the established, classical view that dictates competitive antagonism between them for the one and the same site.