High Affinity Glucocorticoid Receptors Research Articles

Glucocorticoid regulation of c-fos, c-jun and transcription factor AP-1 in the AtT-20 corticotrope cell.

Glucocorticoids (GC) are potent repressors of both basal and corticotropin releasing factor (CRF) stimulated transcription of the proopiomelanocortin (POMC) gene in corticotrope cells of the anterior pituitary. Despite the finding of a novel, high affinity glucocorticoid receptor (GR) binding site within the proximal region of the POMC promoter, the mechanism by which GC inhibit POMC transcription is still uncertain. Recent studies have described mechanisms whereby GC inhibit transcription of other genes via a direct interaction with components of the transcription factor AP-1. Since it has been shown that CRF stimulates c-fos in AtT-20 corticotrope cells, and that c-fos over-expression elevates POMC transcription, the current study has investigated whether GC can repress c-fos and c-jun gene expression and AP-1 DNA binding activity in AtT-20 corticotrope cells. Acute treatment with doses of dexamethasone (DEX) that markedly inhibited nuclear POMC hnRNA had no effect on basal c-fos mRNA expression, but resulted in a transient down regulation of c-jun. In addition, acute DEX pretreatment significantly lowered CRF stimulation of POMC gene expression and attenuated the CRF stimulation of c-fos mRNA by 25%. Although DEX treatment of AtT-20 cells did not affect AP-1 DNA binding capacity of nuclear extracts, DEX pretreatment blunted the stimulation of AP-1 binding in response to CRF. In further studies, nuclear extracts from CRF-treated cells were coincubated with nuclear extracts from control or DEX treated cells. High levels of DEX treated extracts led to a relative repression of CRF-induced AP-1 binding, suggesting that ligand-activated GR may lower available AP-1 levels by direct protein: protein interaction. Finally, the composition of AP-1 in AtT-20 nuclear extracts was found to be heterogeneous, with the variation dependent upon hormonal treatment. These data suggest that in the corticotrope cell relatively high levels of activated GR may influence CRF-induced AP-1 DNA binding via transient genomic actions on basal c-jun and stimulated c-fos and/or via direct protein:protein interactions.

Glucocorticoid receptors in the mammalian inner ear: RU 28362 binding sites

The effects of glucocorticoid hormones are thought to be initiated by binding of the steroid to stereospecific intracellular receptor proteins in target tissues. The synthetic glucocorticoid [ 3H]-RU 28362, which demonstrates negligible affinity for mineralocorticoid (Type I) receptors [Philibert et al., (1983) Endocrine Soc. Abstr. 65, 335], was employed to identify the high-affinity glucocorticoid (Type II) receptors in the inner ear. By Scatchard analysis, the K d of the [ 3H]-RU 28362-cytoplasmic receptor complex was 11.4 × 10 −9 M for the lateral wall of the basal turn of the cochlea and 12.7 × 10 −9 M for the ampullae of the semicircular canals. The concentration of binding sites, B max, was 240 fmol/mg dry tissue for the cochlear specimen and 89 fmol/mg dry tissue for the ampullae. Time course studies indicated that the binding of [ 3H]-RU 28362 by inner ear tissues reached equilibrium within 30 min of incubation at 25°C. Based on the total specific binding measured with [ 3H]-RU 28362, the glucocorticoid receptor concentration in the lateral wall of the basal turn of the cochlea appears to exceed that in the ampullae of the semicircular canal by a factor of 2.7. Substantial specific [ 3H]-RU 28362 binding to the cochlear lateral wall and ampullar tissue suggests the presence of glucocorticoid receptors and sites of glucocorticoid action in the inner ear.

Evidence for and against the Naples high- and low-excitability rats as genetic model to study hippocampal functions

The Naples high- (NHE) and low-excitability (NLE) are two rat lines, selectively bred for high and low activity levels in a Làt-maze, respectively. Because the activity level in a novel environment depends mainly on the integrity of the hippocampal formation, and NLE and NHE rats differ with a similar background of emotionality, arterial blood pressure, and learning ability, they have been proposed as animal model to study hippocampal functions. Our aim is to prove evidence in favor and against this hypothesis. The evidence in favor indicates that NLE/NHE rats have a defective spatial processing, and pertains to (a) Differential activity in a spatial novelty situation (selection trait), proportional to the stimulus complexity rats are exposed to (NHE are hyper- and NLE-rats hypoactive); and (b) Impaired working memory in a six-arm non-reinforced tunnel maze in both lines compared to random-bred rats, that was reversed by the introduction of a reinforcer. In addition, multiple evidence of (i) lower intra- + infrapyramidal mossy fiber terminals in both NLE/NHE vs. controls; (ii) increased sensitivity of hippocampal elements to microinjections of vasopressin (but not oxytocin) and of "delta" (but not "mu") opioids; (iii) lower number of high-affinity glucocorticoid receptors; (iv) lower number of alpha- but not beta-adrenergic receptors in the hippocampus and hypothalamus of NHE rats only; and (v) the genotype-dependent behavior of a DNA fraction with fast turnover, suggest that both NHE/NLE are "disintegrated" at the hippocampal interface. Further, neurobehavioral covariations among individual differences reveal nonlinear, complex relationships, an evidence apparently against the hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid-induced growth inhibition and differentiation of a human megakaryoblastic leukemia cell line: involvement of glucocorticoid receptor.

A human megakaryoblastic leukemia cell line, HIMeg, was established recently. Previous studies have shown that retinoic acid (RA) and 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] have potent effects on the proliferation and differentiation of HIMeg cells. Recently, the effect of dexamethasone (Dex), a synthetic glucocorticoid, on HIMeg cell growth and differentiation was examined in comparison with RA and sex steroid hormones. It was observed that in a methylcellulose culture system, Dex suppressed the clonal proliferation of HIMeg cells in a dose-dependent manner. The inhibitory effects of Dex could be reversed by RU486, a potent glucocorticoid antagonist. In contrast, sex steroid hormones had little effect on the clonal proliferation of HIMeg cells. In a liquid culture system, only 2% of HIMeg cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hormone treatment, whereas 45% and 30% of the cells expressed GPIIb/IIIa following the addition of RA and Dex, respectively. To examine the molecular basis of the hormone-induced cell differentiation, glucocorticoid receptor (GR) expression was studied by Scatchard analysis. It was shown that there existed a saturable, high-affinity GR in HIMeg cells and the GR number was altered after Dex or RA treatment of the cells, suggesting that the cellular effects of glucocorticoids on HIMeg cells were mediated by GR.

Glucocorticoid receptors

Glucocorticoid hormones are secreted uniquely from the zona fasciculata of the adrenal cortex, with marked circadian variation in basal levels and acute elevation in response to stress. Glucocorticoid receptors are almost ubiquitously distributed, and mediate a wide range of tissue-specific responses; in addition to classical, [ 3H]dexamethasone-binding GR (Type II receptors) there is excellent evidence that Type I sites (MR) act as mineralocorticoid receptors in some tissues but high affinity glucocorticoid receptors in others. Particular issues to be addressed in the presentation include: (i) the extent to which glucocoticoid receptor occupancy is modulated by extracellular (plasma-binding enzymes) or intracellular (protooncogenes) factors; (ii) whether or not there are specific response elements for Type I and II receptors; (iii) putative physiological roles for Type I, high affinity glucocorticoid receptors; (iv) evidence for glucocorticoid receptors other than classical GR and “MR”. In summary, glucocorticoid receptors appear to be a final common pathway mediating and/or modulating circadian rhythms and stress responses. Cell- and tissue-specificity of response to a whole-body signal is determined by local pre-receptor, receptor and genomic differences. On the basis of previous studies on glucocorticoid secretion, and recent information on glucocorticoid action, it would at last appear possible to begin to construct a coherent physiology for glucocorticoid hormones.

Glucocorticoid hormones prevent the induction of γ-glutamyl transpeptidase by ethanol in a rat hepatoma cell line

The increase in serum γ-glutamyi transpeptidase (GGT) is a well known marker of chronic alcoholism in man. We have previously shown that ethanol (180 mM) induces GGT activity 2–3-fold in the C 2 rat hepatoma cell line. In this study, we have analyzed the interaction of ethanol with steroid hormones and drugs in this well defined cell culture system. Dexamethasone (100 nM), a synthetic glucocorticoid agonist, completely prevented the induction of GGT by ethanol, but had no effect when added alone. This inhibitory effect was also observed with other corticosteroids, but not with sex steroids; it was prevented by RU 486, a glucocorticoid antagonist. These observations suggest that dexamethasone acts through a high affinity glucocorticoid receptor. Conversely, ethanol did not interfere with the glucocorticoid induction of alanine aminotransferase in the same cell. We have analyzed the metabolism of ethanol in the C 2 cells. These cells lack significant alcohol dehydrogenase activity as well as any cytochrome P-450 Alc immunoreactivity. Dexamethasone did not modify the disappearance of ethanol in the culture medium of those cells. We conclude that glucocorticoid hormones interact with ethanol at the cellular level, and that this interaction does not involve a modification of alcohol metabolism.

Steroid binding to cardiac type I receptors: in vivo studies.

In vitro, Type I (mineralocorticoid) receptors show equivalently high affinity for aldosterone, corticosterone and cortisol in cytosol preparations from a wide range of tissues. This steroid non-selectivity is also found for hippocampal Type I receptors in vivo; in contrast, Type I receptors in physiological mineralcorticoid target tissues are highly aldosterone-specific in vivo. In the present study we show that Type I receptors in the heart are non-selective in vivo, like those in hippocampus, and therefore presumably act as high-affinity glucocorticoid receptors rather than mineralocorticoid receptors.

Cytosolic glucocorticoid receptors in the developing rat heart

Glucocorticoid hormones alter functional cardiac responses in the rat heart. In the neonatal rat heart, glucocorticoid treatment on post-natal day 2 alters heart function for up to 3 weeks post-injection, which suggests that cardiac glucocorticoid receptors mediate cardiac function in neonates. However, glucocorticoid receptors have not been identified in neonatal rat heart. Glucocorticoid receptor binding was measured in neonatal rat heart cytosol extract using [3H] dexamethasone as ligand, and characterized by competition assays and Scatchard analysis. Saturable, specific, high affinity glucocorticoid receptor binding was found in the cytosol of the neonatal rat heart. We then examined the effects of a single, post-natal day 2 injection of hydrocortisone acetate on glucocorticoid receptor binding in 12 to 14-day-old rat heart. While this injection paradigm results in altered cardiac function in 12-day-old rat hearts, cytosol glucocorticoid receptor binding in 12 to 14-day-old heart was not altered by treatment on post-natal day 2 with hydrocortisone acetate. It is postulated that exposure to elevated glucocorticoid levels on neonatal day 2 may alter cardiac function by producing permanent organizational effects on cardiac tissue.

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 17β 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 ~200fmol/mg protein) with equal, high affinity ( K d 4°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.

Inhibition by cortisol of human natural killer (NK) cell activity

The effects of cortisol on the natural killer (NK) activity of human peripheral blood mononuclear (PBM) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target. Preincubation for 20 h of PBM cells drawn from healthy donors with 1 · 10 −8 to 1 · 10 −5 M cortisol resulted in a significant decrease of NK cell activity. The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells. Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon (IFN-γ). A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E 2 (PgE 2). The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression, whereas PgE 2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines. The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone. Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin (CBG) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects. Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity. Since the effect of cortisol was additive to that of PgE 2 and was not changed by phosphodiesterase inhibitors, it is conceivable that the hormone acts at a level different from the adenylate cyclase-phosphodiesterase system. Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity.

Characterization of glucocorticoid receptors in whole and cellular subfractions of embryonic chick spinal cord

Steroid-specific binding sites for tritiated corticosterone have been localized, via autoradiography, to motoneurons in the lateral motor columns of the adult rat spinal cord. Binding sites in adult rat spinal cord have been characterized biochemically and shown to possess the properties of a putative glucocorticoid receptor. The presence of receptors for glucocorticoids in embryonic chick spinal cord was determined and their characterization undertaken as a prelude to the study of the functions under regulation by glucocorticoids during development. Assay conditions were defined and binding of [ 3H]dexamethasone ([ 3H]Dex) to cytosols of 6- and 10-day embryonic spinal cord and cellular subfractions of 6-day spinal cord determined. Saturable, high-affinity binding of [ 3H]Dex to cytosols prepared from both whole 6- and 10-day spinal cords and cells of all 3 cellular subfractions of 6-day spinal cords was observed. The binding component in 10-day cytosols was (1) proteinaceous, as binding was eliminated by heating cytosols, and (2) a macromolecular species, as it displayed a sedimentation coefficient of 8S upon centrifugation in sucrose gradients. The putative receptor displayed binding specific for glucocorticoids in a competition assay, with the exception that some inhibition of binding by the androgen ligand, methyltrienolone (R1881) was observed. The binding affinity decreased as the values for K D increased from 3.4 ± 0.9 nM on day 6 to 15.7 ± 1.8 nM on day 10, while the values for B max increased from 270 to 855 fmol/mg protein over the same period. The neuronal fractions I and II displayed fewer sites (60 and 130 fmol/mg protein respectively) of higher affinity (2.1 and 2.5 nM respectively) than the non-neuronal fraction III (B max = 164 fmol/mg protein and K D = 4.2 nM). The number of binding sites per cord increased 4.8-fold from embryonic day 6 to 10. These results demonstrate that specific, high-affinity glucocorticoid receptors are present in chick spinal cord cells, including motoneurons, as early as embryonic day 6, when spontaneous limb movements begin.

Glucocorticoid receptors and corticosensitivity in the human monocytic cell line CM-S: Changes during phorbol ester-induced differentiation

CM-S is a line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA). Untreated CM-S cells expressed a single class of high-affinity ( K D = 4.5 ± 2.3 n M) glucocorticoid receptor sites (GCr) (27,530 ± 3752 sites/cell) as measured by a whole-cell assay at 37 °C using [ 3H]triamcinolone acetonide as tracer, while CM-S cells induced to macrophage differentiation by 10 −7 M TPA showed reduced levels of GCr (10,729 ± 2135 sites/cell). Kinetic studies indicated that this reduction was progressive, reaching about 34% of the original value 96 hr after TPA addition. The GCr in untreated and TPA-induced cells were similar in their specificity for corticosteroids. In the precursor cell population dexamethasone enhanced both the rate of protein synthesis and the production of autostimulatory growth factor(s), while in TPA-induced cells it inhibited the rate of protein synthesis in a dose-dependent manner. Our data with the CM-S cell line thus suggest that in the monocytic cell line lineage both the number of GCr and the cell response to glucocorticoids depend on the degree of cell maturation.

Catecholamines and memory retrieval: Attempts to find a role for noradrenaline in mediating reminder effects

The Naples high- (NHE) and low-excitability (NLE) are two rat lines, selectively bred for high and low activity levels in a Làt-maze, respectively. Because the activity level in a novel environment depends mainly on the integrity of the hippocampal formation, and NLE and NHE rats differ with a similar background of emotionality, arterial blood pressure, and learning ability, they have been proposed as animal model to study hippocampal functions. Our aim is to prove evidence in favor and against this hypothesis. The evidence in favor indicates that NLE/NHE rats have a defective spatial processing, and pertains to (a) Differential activity in a spatial novelty situation (selection trait), proportional to the stimulus complexity rats are exposed to (NHE are hyper- and NLE-rats hypoactive); and (b) Impaired working memory in a six-arm non-reinforced tunnel maze in both lines compared to random-bred rats, that was reversed by the introduction of a reinforcer. In addition, multiple evidence of (i) lower intra- + infrapyramidal mossy fiber terminals in both NLE/NHE vs. controls; (ii) increased sensitivity of hippocampal elements to microinjections of vasopressin (but not oxytocin) and of “δ” (but not “μ”) opioids; (iii) lower number of high-affinity glucocorticoid receptors; (iv) lower number of α- but not β-adrenergic receptors in the hippocampus and hypothalamus of NHE rats only; and (v) the genotype-dependent behavior of a DNA fraction with fast turnover, suggest that both NHE/NLE are “disintegrated” at the hippocampal interface. Further, neurobehavioral covariations among individual differences reveal nonlinear, complex relationships, an evidence apparently against the hypothesis. The data altogether suggest that NLE/NHE rats may be a useful model to test the role of the hippocampus in the integration of spatial and emotional information.

Glucocorticoid receptor studies in human leukemia

In leukocytes from peripheral blood of patients with acute lymphoblastic leukemia (ALL) we have measured glucocorticoid receptors (GRs) and glutamine synthetase activity. In addition, short-term response to steroid treatment and inducibility of complete remission were determined. Using both whole cell and cytosol binding assays with [ 3H]-triamcinolone acetonide, leukemic lymphoblasis from all the patients studied were found to contain specific high affinity GRs. However, the cytosol assay consistently underestimated the number of receptor sites with respect to the whole cell assay. There was a significant difference in the number of receptor sites, as measured by whole cell assay, between those patients who did and did not respond to treatment with glucocorticoids alone. Lymphoblasts from T- and B-cell leukemia contained fewer receptor sites than those from Null-cell leukemia. Density of receptor was closely correlated with the in vivo sensitivity to polychemotherapy. Density of receptor also correlated positively with the steroid-induced increase in glutamine synthetase activity. It is likely that the analysis of functional GR in ALL cells may be a useful adjunct to the already existing clinical and immunological prognostic indicators.

In vitro effects of glucocorticoid hormones on the synthesis of DNA in cartilage of neonatal mice

One of the most conspicuous effects of the glucocorticoid hormones is an inhibition of somatic growth in immature animals [ 11. The administration of pharmacological doses of glucocorticoids to neonatal animals was found to suppress cartilage growth and bone formation [2,3]. Glucocorticoid-induced suppression of cartilage cell proliferation is significant within 24 h and is virtually complete within 72 h of a single dose of the hormone. Recent evidence has shown that mammalian chondrocytes possess high affinity glucocorticoid receptor proteins which bind dexamethasone [4]. This study presents evidence that in organ cultures of mouse neonatal condylar cartilage the presence of glucocorticoid hormone markedly suppresses cell proliferation and that the characteristic of this suppression is similar to that observed in neonatal mice cartilage in vivo. Moreover, this growth inhibitory effect is dose-dependent, specific to fluorinated corticoids, and is dependent upon an early stimulation of specific RNA and protein synthesis.

Modulation of glucocorticoid effects and steroid receptor binding in butyrate-treated HeLa S 3 cells

Two synergistic actions of glucocorticoid hormones and sodium butyrate in HeLa S 3 cells are reported. Growth of HeLa S 3 cells in dexamethasone and sodium butyrate leads to synergistic effects on both increases in alkaline phosphatase activity and inhibition of [ 3H]thymidine incorporation into DNA. A 7- to 10-fold increase in alkaline phosphatase activity is seen in cells treated for 96 h with either 1 × 10 −7, m dexamethasone or 5 m m sodium butyrate. Simultaneous administration of the two compounds, however, elevates alkaline phosphatase activity in a synergistic manner, reaching levels 7-fold higher than those obtained with either compound alone. In addition, both agents inhibit [ 3H]thymidine incorporation into DNA, and combined administration leads to a synergistic suppression of nucleoside incorporation. These effects are dependent on the steroid hormone concentration employed, being half-maximal at 2 × 10 −8, m dexamethasone. In addition, only active glucocorticoid hormones act synergistically with sodium butyrate suggesting mediation through high affinity glucocorticoid receptors. Unexpectedly, exposure of cells to 5 m m butyrate for 20–24 h results in a significant reduction in the levels of both nuclear and cytoplasmic dexamethasone receptors (33 and 49% reduction, respectively), without altering binding site affinity. Despite decreases in total cellular receptor binding, butyrate treatment of HeLa S 3 cells does not alter absolute levels of 0.4 m KCl residual nuclear binding sites. Our results show that glucocorticoid effects on alkaline phosphatase activity and [ 3H]thymidine incorporation are augmented with butyrate treatment, despite reduced cellular binding of the hormone.

Cardiac receptors for glucocorticoids in the rat: Factors involved in [ 3H]dexamethasone binding and nuclear translocation of [ 3H]dexamethasone-receptor complexes in heart and liver

The conditions of (i) the binding of [ 3H]dexamethasone to cytoplasmic receptors and (ii) the interaction between [ 3H]dexamethasone-receptor complexes and the nucleus have been studied in heart of intact and adrenalectomized rats. This study was performed in parallel on heart and liver, the latter being take as a reference target organ. 1. (i) We hav confirmed the presence of high affinity glucocorticoid receptors in the rat heart. The concentration of cardiac receptors in intact animals (45 ± 12 fmol/mg protein) is much lower than that of liver receptors (600 ± 120 fmol/mg protein). Adrenalectomy induces an increas in receptor concentration in both organs without a change in affinity for [ 3H]dexamethasone. This increase, which is more marked in the heart (5.5-fold) than in liver (1.8-fold), can probably not be ascribed to a progressive liberation of receptors previously occupied by endogenous glucocorticoids nor to a decreased transfer of receptors to the nucleus of adrenalectomized animals. Thus adrenalectomy appears to induce a true increase in the number of receptors. 2. (ii) Heart nuclei bind both heart and liver [ 3H]dexamethasone-receptor complexes with the same efficiency, confirming the lack of organ specificity of nuclear binding for steroid hormone-receptor complexes. The apparent saturation of rat nuclei by glucocorticoid-receptor complexes is dut to a macromolecular inhibitor of nuclear binding present in heart cytosol. This factor interacts directly with activated complexes but not with nuclear receptor sites.

Detection of high-affinity glucocorticoid binding in rat bone.

Department of Medicine, Manchester Royal Infirmary, Manchester, M13 9WL (Received 17 October 1977) Recently Feldman, Dziak, Koehler & Stern (1975) have detected high-affinity gluco corticoid receptors in cytosol from isolated foetal rat calvarial bone cells. They estimated the binding constant (Kd) to be 7×10−9 mol/l for [3H]dexame[unk]hasone; however, their estimates were based on a maximum bound: free ratio of only 1%. Their data from cell cultures suggest that such binding may nevertheless be of physiological importance (Chen, Aronow & Feldman, 1977). The very small amount of binding might be due to a low initial concentration of binding sites, to their destruction during or after the trypsin–collagenase digestion, to their high dilution or to a combination of all three factors. We report here a much simpler method whereby we have greatly increased the percentage binding by setting up the incubation in intact calvaria. Calvaría were dissected from foetal rats (19–21 days

Cardiac glucocorticoid receptors: the binding of tritiated dexamethasone in rat and dog heart.

The physiological effects of steroid hormones are thought to be mediated by an initial obligatory step of binding to stereospecific intracellular receptor proteins in target tissues. Highaffinity glucocorticoid receptors have been demonstrated in adrenalectomized rat and dog heart by the specific binding of dexamethasone (DM). By Scatchard plot analysis, the equilibrium (dissociation) constant of the 3HDM-cytoplasmic receptor complex in both species was 3 × 10-9M (0 C) ; the concentration of binding sites was 1.3 × 10-13M per mg cytosol protein in the dog heart and 4.0 × 10-13M per mg cytosol protein in the rat heart. The affinity of these receptors is in the order corticosterone progesterone aldosterone testosterone estradiol. Tritiated aldosterone was also shown to be bound in rat and dog heart cytosol; this binding is specific in the sense that binding of tracer was shown displaceable by excess unlabeled aldosterone. In the heart, however, as opposed to the kidney, such binding of 3HA is better displac...

Glucocorticoid receptors in rat kidney: the binding of tritiated-dexamethasone.

The effects of glucocorticoid hormones are thought to be initiated by binding to stereospecific intracellular receptor proteins in target tissues. The kidney enjoys a variety of functions influenced by glucocorticoid hormones. High affinity glucocorticoid receptors have been demonstrated in adrenalectomized rat kidney by the specific binding of dexamethasone (DM). These receptors have been designated Type II, and can be distinguished from high affinity mineralocorticoid receptors (Type I) and the CBGlike corticosterone receptors (Type III) on the basis of differing affinities for a series of physiological and synthetic steroids. By Scatchard plot analysis, the equilibrium (dissociation) constant of the 3HDM–cytoplasmic receptor complex was 5 X 10-9M (25 C), and the concentration of binding sites 1.6 X 10-13 moles per mg protein in the cytoplasmic high speed supernatant (HSS). Similar concentrations of receptor were found in outer cortex and medulla—papilla. The affinity of the Type—II receptor for steroid...