Ontogeny of glucocorticoid receptors in rat liver.

Specific receptors for glucocorticoids are present in liver cytosols of rat fetuses at least as early as the 18th day of gestation. The concentration of the receptor begins to decline after the 20th day reaching undetectable levels shortly before and after parturition. The receptor can be detected again 1 to 2 hours after birth, and its levels increase markedly to higher than adult values between the 2nd and 5th day. The reason for the failure to detect specific hormone binding near parturition appears to be due to occupation of binding sites by endogenous steroids rather than the absence of the receptor. This is indicated by the demonstration of both cytoplasmic and nuclear receptor sites in liver slices of newborn rats incubated with labeled dexamethasone at 37 degrees. The cytoplasmic receptors of fetal and adult liver differ in their relative affinity for cortisol and corticosterone. The fetal receptors have a higher affinity for corticosterone than cortisol while the reverse is true for the adult receptors. These observations suggest either the existence of dissimilar receptors in fetal and adult liver or the presence of more than one type of receptor sites. It is therefore possible that subtle differences in the nature of hepatic receptors may be partly responsible for the maturation-dependent qualitative differences in tissue responsiveness to glucocorticoids.     

Immunocytochemical demonstration of mineralocorticoid receptors in rat and human kidney

Using a polyclonal antiserum against the hinge region of the recently cloned human mineralocorticoid receptor (MR) and indirect peroxidase immunohistochemistry, we have shown MR-like immunoreactivity (LI) in superficial nephron segments, including distal convoluted tubule, connecting piece and initial cortical collecting duct. The absence of staining in cells tentatively identified as intercalated cells on light microscopy was confirmed by pre-embedding electron microscopy. Though the intracellular distribution of immunostaining varied with the fixative used, the cellular distribution of MR-LI is in good general agreement with earlier micropuncture and autoradiographic studies.     

Glial cells express both mineralocorticoid and glucocorticoid receptors.

In the brain, the action of glucocorticoid steroids is mediated via two intracellular receptors, the mineralocorticoid (MR), or type I receptor, and the glucocorticoid (GR), or type II receptor. These receptors are expressed in many types of neurons and are co-expressed in some neurons such as the hippocampal pyramidal cells. Although glucocorticoids are known to affect gliogenesis and glial cell differentiation, the expression of the GR in different types of glial cells throughout the brain has not been thoroughly studied and the expression of the MR in glia not previously reported. Here we review studies suggesting that both receptors are expressed in astrocytes and oligodendrocytes.     

Concanavalin A induced glucocorticoid resistance in rat thymocytes in relation to glucose metabolism and glucocorticoid receptors.

Concanavalin A stimulates glucose uptake in isolated rat thymocytes, at concentrations as low as 1.0 μg/ml. The magnitude of the response is dosedependent with maximal glucose uptake (greater than 100% over control) observed over the 50–500 μg/ml range. The response to concanavalin A occurs within 5 minutes and is maximal by 1 hour. Exposure of thymocytes to physiological concentrations of cortisol has been shown previously to lead to a 15–30% reduction in glucose uptake within 20 minutes (1). Cortisol added simultaneously to or after concanavalin A has no effect on glucose uptake, but when it is added before concanavalin A, a glucocorticoid response is observed even in the face of concanavalin A-induced glucose uptake. Unlike the cortisol effect, the concanavalin A-induced increase in thymocyte glucose uptake is not inhibited by either cycloheximide or cordycepin. The antagonism between cortisol and concanavalin A may in part exist at the specific glucocorticoid receptor level, since exposure of cells to concanavalin A rapidly and significantly reduces the number of specific, saturable glucocorticoid binding sites in isolated thymocytes.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.     

Identification and characterization of glucocorticoid receptors in liver of nude mice

Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone down-regulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and -glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction.     

Partial characterization and ontogeny of renal cytosolic glucocorticoid receptors in mouse kidney

The glucocorticoid receptor (GR) was partially characterized in mouse renal cytosol. A sensitive and reproducible [3H]dexamethasone binding assay suitable for use with small quantities of cytosolic protein, was developed. Studies defined the optimal equilibrium binding conditions, metabolism of [3H]dexamethasone in adult renal cytosol, specificity of binding of the GR, and molecular weight of the GR-[3H]dexamethasone complex by gel filtration chromatography. The assay was subsequently used to measure the renal GR during different stages of foetal and postnatal development, as well as in glomerular and renal tubular preparations from adult mice. An almost linear increase in GR occurred from day 13 to day 18 of gestation with levels rising from 100 to 201 fmol/mg cytosol protein; this was followed by a sharp rise in receptor concentration just after birth to 343 fmol/mg cytosol protein. Adult levels, 410-433 fmol/mg cytosol protein, were reached by 2 weeks after birth. The equilibrium dissociation constants (Kd) of the [3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8-11.8 nM; mean +/- SD = 6.5 +/- 2.9 nM). Specific binding was assessed to be 3- to 5-fold greater in tubular than in glomerular preparations. These data on the localization and ontogeny of GR during murine metanephric development provide a basis for study of glucocorticoid-mediated effects on various models of congenital and acquired renal disease.     

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.     

Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution

Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent.     

Size of vasopressin receptors from rat liver and kidney

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al., Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974) J. Biol. Chem. 249, 6390-6400; Butlen et al. (1978) Mol. Pharmacol. 14, 1006-1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopresin receptors. For this purpose, rat liver and kidney membranes were incubated with [3H]vasopressin and solubilized with Triton X-100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30-37 degrees C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone-receptor complexes were estimated from Utrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml x g-1; molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size.     

Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.     

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)     

Monoclonal antibodies to the rat liver glucocorticoid receptor.

Monoclonal antibodies against the 90 000 mol. wt. form of the activated rat liver glucocorticoid receptor were generated from mice immunized with a partially purified receptor preparation. The screening assay was based on the precipitation of liver cytosol, labelled with [3H]triamcinolone acetonide, with monoclonal antibodies bound to immobilized rabbit anti-mouse IgG. Out of 102 hybridomas obtained, 76 produced immunoglobulin and eight of them were found to react with the receptor molecule. Only one of the positive clones secreted IgG whereas the other seven produced IgM. The complexes of receptor and antibodies were identified by sucrose density gradient centrifugation. All seven monoclonal antibodies tested reacted with the 90 000 mol. wt. form of the receptor but not with the 40 000 mol. wt. form that contains the steroid and DNA binding domains. None of the monoclonal antibodies interfered with the binding of the receptor to DNA cellulose, thus suggesting that the antigenic determinants are located in a region of the receptor that is not directly implicated in either steroid binding or DNA binding. These antigenic determinants were common to glucocorticoid receptors from several tissues of the rat, whereas glucocorticoid receptors from other species react only with some of the antibodies.