Glucocorticoid-binding Proteins Research Articles

Discrimination exposure and DNA methylation of stress-related genes in Latina mothers

Latina mothers, who have the highest fertility rate among all ethnic groups in the US, are often exposed to discrimination. The epigenetic changes related to this discrimination are largely unknown. This study is the first to explore the relationship between discrimination and DNA methylation of stress regulatory genes in Latinas. Our sample was Latina women (n = 147) with a mean age of 27.6 years who were assessed at 24–32 weeks’ gestation (T1) and 4–6 weeks postpartum (T2) and reside in the U.S. Blood was collected at T1, and the Everyday Discrimination Scale (EDS) was administered at T1 and T2. DNA Methylation at candidate gene regions was determined by bisulphite pyrosequencing. Associations between EDS and DNA methylation were assessed via zero-inflated Poisson models, adjusting for covariates and multiple-test comparisons. Discrimination was negatively associated with methylation at CpG sites within the glucocorticoid receptor (NR3C1) and brain-derived neurotrophic factor (BDNF) genes that were consistent over time. In addition, discrimination was negatively associated with methylation of a CpG in the glucocorticoid binding protein (FKBP5) at T1 but not at T2. This study underscores associations between discrimination and epigenetic markers of DNA methylation in Latinas that warrant further investigation to better understand the biological pathways and psychopathological effects of discrimination on Latina mothers and their families.

EQUIPPED FOR LIFE: THE ADAPTIVE ROLE OF THE STRESS AXIS IN MALE MAMMALS

Habitat constrains and shapes successful ecological and physiological strategies and thus provides the context for the evolution of life-history traits. The stress axis plays a vital role in the endocrine system and is a critical component adapting mammals to particular habitat pressures. It is subject to both individual activational and organizational plasticity as well as to evolutionary modification. To illustrate, I examine the suite of traits of the stress axis associated with breeding frequency in male mammals, which varies in a continuum from semelparity to iteroparity. During the breeding season, males in species at the semelparous end of the continuum exhibit high concentrations of free corticosteroids, low concentrations of glucocorticoid-binding protein, a failure of the negative feedback system, a gonadal axis that is not inhibited by high corticosteroid concentrations, and immunosuppression. Iteroparous species exhibit the opposite traits. The evolutionary constraints selecting for the former may partially be related to phylogeny (in marsupials) as well as to an interaction of the restrictions imposed by the environment on female reproduction, the mating system, the high costs of reproduction, and the low adult survival during the nonbreeding season.

Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16β-hydroxylated stanozolol

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17α-alkylated testosterone derivative (17α-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17α-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16β-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3′-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST ( k i =30 nM) and 16β-hydroxystanozolol ( k i =13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16β-hydroxylated metabolite of ST. ST and its 16β-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Solubilization and photoaffinity labeling identification of glucocorticoid binding peptides in endoplasmic reticulum from rat liver

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic effects of steroid hormones. We have previously described that the low-affinity glucocorticoid binding protein (LAGS), present in the endoplasmic reticulum of the male rat liver, has pharmacological and biochemical properties different from those of nuclear receptors. The LAGS is under multihormonal regulation and binds glucocorticoids, progestins, and synthetic steroids but is unable to bind either estradiol, testosterone, or triamcinolone acetonide. In this study, we have solubilized the LAGS and investigated their pharmacological and hydrodynamic properties and their peptide composition. We found that LAGS is an integral protein bound to the endoplasmic reticulum. CHAPS provided its optimal solubilization without changes in its pharmacological properties. Hydrodynamic properties of LAGS showed that it has a molecular mass of at least 135 kDa. SDS-PAGE of covalently-labeled LAGS showed that [ 3 H ]dexamethasone binds two peptides of 53 and 37 kDa, respectively. Thus, the LAGS appears as an oligomeric protein under multihormonal regulation. The availability of solubilized LAGS and the fact that it can be induced in vivo represent major steps toward purification and understanding the functional significance of this unique steroid-binding protein.

Cyclic AMP potentiates glucocorticoid-induced endogenous endonuclease activation in thymocytes.

The present study was undertaken to determine whether specific interactions between cAMP and glucocorticoids regulate apoptosis in thymocytes. Incubation of murine thymocytes with agents that elevate the cAMP level resulted in enhancement of glucocorticoid-induced Ca2+ increases, DNA fragmentation, and cell death compared to levels observed in thymocytes treated with steroid alone. cAMP did not affect DNA fragmentation in thymocytes treated with Ca2+ ionophore, a compound that induces endonuclease activation via an independent mechanism. Treatment with cAMP also increased glucocorticoid potency by lowering the concentration of steroid required for induction of apoptosis. The mechanism of cAMP action appeared to involve the glucocorticoid receptor, since the glucocorticoid antagonist RU-486 abrogated the cAMP response in animals treated with the adenosine analog NECA in vivo. Analysis of cellular glucocorticoid binding and receptor protein levels revealed modest cAMP-stimulated increases that appeared insufficient to account for the effects of cAMP on endogenous endonuclease activation, suggesting the possible involvement of a posttranslational mechanism in the response. These results demonstrate that cAMP and glucocorticoids synergize to promote apoptosis in thymocytes via a mechanism that appears to involve modification of glucocorticoid receptor activity.

Seasonal changes in plasma androgens, glucocorticoids and glucocorticoid-binding proteins in the marsupial sugar glider Petaurus breviceps

An investigation spanning two breeding seasons was carried out to examine endocrine changes associated with reproduction in a wild population of the marsupial sugar glider Petaurus breviceps, a small arboreal gliding possum. Using techniques of equilibrium dialysis and polyacrylamide gel electrophoresis at steady-state conditions, a high-affinity, low-capacity glucocorticoid-binding protein was demonstrated in the plasma of Petaurus breviceps. Equilibrium dialysis at 36 degrees C using cortisol gave a high-affinity binding constant of 95 +/- 5.2 litres/mumol for a presumed corticosteroid-binding globulin (CBG) while the binding constant for the cortisol-albumin interaction was 3.5 +/- 0.4 litres/mmol. There was no difference between the sexes in the affinity of binding of cortisol to CBG; however, the cortisol-binding capacity underwent seasonal variation in both sexes. Progesterone was bound strongly to the presumed CBG while neither oestradiol nor aldosterone appeared to be bound with high affinity to P. breviceps plasma. In the males, peaks in the plasma concentration of testosterone coincided with the July-September breeding season in both years. A significant inverse relationship was shown to exist between the plasma testosterone concentration and the CBG-binding capacity. In both sexes an increase occurred in the plasma concentration of free cortisol during the first breeding season, a pattern which was not repeated in the subsequent breeding season, possibly due to a lower population density in that year.

Cytoplasmic 8 S glucocorticoid receptor binds to actin filaments through the 90-kDa heat shock protein moiety

The glucocorticoid receptor exists in the cytoplasm of hormone-untreated cells as a complex with the 90-kDa heat shock protein (HSP90). Glucocorticoids induce dissociation of the glucocorticoid binding protein from HSP90 and translocation of the receptor to the nucleus. HSP90 binds to actin filaments, and calmodulin or tropomyosin inhibits the binding. We present here evidence that the HSP90-containing glucocorticoid receptor complexes (8 S receptor) bind to filamentous actin in vitro while the HSP90-free form of the receptor does not. The binding was detectable for both the crude cytosolic fractions and the partially purified 8 S glucocorticoid receptor. Purified HSP90 or tropomyosin completely abolished the binding. Calmodulin also inhibited the binding in a Ca(2+)-dependent manner. From these results, we conclude that the glucocorticoid receptor complex is able to bind actin filaments via the HSP90 moiety. The binding may provide an anchoring mechanism for the glucocorticoid receptor in the cytoplasm.

Characteristics and biological role of steroid hormone receptors in neuroepithelial tumors

Tissue samples from 57 patients with neuroepithelial tumors (25 glioblastomas, 18 anaplastic astrocytomas, and 14 astrocytomas) were analyzed in order to evaluate the presence of estrogen, progesterone, glucocorticoid, and androgen receptors. Glucocorticoid- and androgen-specific binding proteins were present in 38.6% and 21.6% of the cases, respectively. Only a few tumors showed estrogen or progesterone receptors. A correlation was found between grade of anaplasia, patient's sex and age, and presence of glucocorticoid and androgen receptors. The biological role of these two receptors was investigated in 10 primary cell cultures derived from neuroepithelial tumors. For this purpose, dexamethasone and testosterone were added to culture medium at different concentrations (from 50 to 0.016 micrograms/ml). A significant stimulation of the cell growth was observed in four of five glucocorticoid receptor-positive cultures when dexamethasone in doses ranging from 2 to 0.016 microgram/ml was added to the culture. No modulation of the growth was observed in glucocorticoid receptor-negative cultures at the same doses. Higher dexamethasone doses induced a significant decrease of the growth index independently from the glucocorticoid receptor status. All of the cultures tested for testosterone activity were negative for androgen receptors. This hormone induced an inhibition of the growth index at doses ranging from 50 to 0.4 micrograms/ml. The data suggest that neuroepithelial tumors contain specific glucocorticoid and androgen binding proteins. Glucocorticoid receptors modulate the growth of cultured neuroepithelial tumors in the presence of different concentrations of dexamethasone.

Relationship of the 90-kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor.

Incubation of molybdate-stabilized L cell cytosol with a monoclonal antibody directed against the 100-kDa glucocorticoid-binding protein causes the immune-specific adsorption to protein A-Sepharose of both the 100-kDa glucocorticoid receptor and the 90-kDa murine heat shock protein (hsp90) (Sanchez, E. R., Toft, D. O., Schlesinger, M. J., and Pratt, W. B. (1985) J. Biol. Chem. 260, 12398-12401). When the glucocorticoid receptor in cytosol is transformed to the DNA-binding state, hsp90 dissociates. In this paper, we show that temperature-mediated dissociation of hsp90 from the receptor is a hormone-dependent event in the same manner as temperature-mediated transformation to the DNA-binding state. In contrast to temperature-mediated transformation, ammonium sulfate causes both dissociation of hsp90 from the receptor and conversion of the receptor to the DNA-binding form in a manner that does not require the presence of steroid. The untransformed form of the glucocorticoid receptor and the strongly negatively charged hsp90 protein behave similarly on DEAE-cellulose chromatography, suggesting that the hsp90 component may contribute significantly to the net negative charge behavior of the non-DNA-binding form of the receptor complex.

Tissue Differences in the Up-Regulation of Glucocorticoid-Binding Proteins in the Rat*

The magnitude of increase in glucocorticoid receptor concentration and transcortin-like binding was examined in a variety of peripheral tissues and brain structures after adrenalectomy. Glucocorticoid binding was assayed in liver, heart, kidney, pituitary, hippocampus, cerebral cortex, amygdala-entorhinal area, and hypothalamus. Glucocorticoid receptor concentration, measured using [3H]dexamethasone as ligand, increased in all eight tissues, but the magnitude of this increase varied 30-fold among tissues. The largest increase was shown by kidney cytosol, followed by amygdala-entorhinal cortex, hippocampus, liver, cerebral cortex, hypothalamus, pituitary, and heart. These increases were not due to a selective enhancement of mineralocorticoid receptors. The increase in transcortin in peripheral tissues was variable and exceeded the increase in plasma transcortin by an order of magnitude. It was concluded that up-regulation of the glucocorticoid receptor after adrenalectomy is a response common to most, if not all, glucocorticoid target tissues. However, the magnitude of this response was tissue specific and was not directly related to initial receptor density. The marked increase in tissue transcortin ([3H]corticosterone binding in the presence of excess dexamethasone) suggested that plasma transcortin is sequestered by peripheral tissues in substantial amounts in the acutely adrenalectomized rat. The increase in transcortin uptake by tissues and the increases in cytosolic receptor number are apparently subject to different regulatory control.

Physicochemical characterization of a new glucocorticoid receptor

A new glucocorticoid-binding protein (Peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography was identified previously in the rats subjected to stress or treated with glucocorticoid (100 μg/100 g body wt.), while the ‘classic’ glucocorticoid receptor (Peak B) eluted with 0.07 M NaCl was found predominantly in untreated rats. The new glucocorticoid-binding protein, Peak C, was characterized by Scatchard analysis and competition with other steroids as a glucocorticoid receptor. The saturation curve of Peak C for dexamethasone was sigmoidal, whereas that of Peak B was hyperbolic. The Hill coefficient was 1.0 for Peak B and 3.1 for Peak C. These results show that Peak C has multiple binding sites. Peak C bound specificially to only natural or synthetic glucocorticoids, whereas Peak B bound not only to glucocorticoids but also to progesterone and aldosterone. Peak C was far more labile than Peak B, its binding activity decreasing 80% when it was incubated for 30 min at 25°C. The molecular sizes of these two peaks (B and C) were similar, being about 90 000–100 000 as determined by Sepharose 6B column chromatography at high ionic strength (0.35 M KCl). The hormone-receptor complex of Peak C bound to rat liver chromatin specifically, but did not bind to calf thymus DNA. The complex of Peak B bound to not only the chromatin but also calf thymus DNA. Peak B reacted well with antiserum to the ‘classic’ glucocorticoid receptor, but Peak C did not react with this antiserum. These results indicate that Peak C is a different glucocorticoid receptor protein from Peak B, or classic glucocorticoid receptor, and plays physiologically important roles as a glucocorticoid receptor mediating the action of the hormone at a high level.

Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.

Dexamethasone 21-methanesulfonate, an affinity label for glucocorticoid-binding proteins, was incubated with rat liver cytosol preparations. The predominant covalently labeled component was identified as Yb-glutathione-S-transferase on the basis of chromatographic properties, electrophoretic mobility, and specific retention by an anti-Yb-immunoadsorbent. Affinity labeling of this protein was blocked by excess dexamethasone. Preferential reactivity of dexamethasone 21-methanesulfonate with the Yb subclass of glutathione-S-transferase (glutathione transferase, EC 2.5.1.18) was also evident with mixtures containing the multiple forms of the enzyme. Yb-glutathione-S-transferase, the nonsaturable glucocorticoid-binding component of rat liver cytosol should, therefore, be reclassified; because of its high concentration and selective interaction with steroids, this enzyme may be an intracellular glucocorticoid-binding protein and, thereby, influence transport, metabolism, and action of the steroids.

A new glucocorticoid receptor species: relation to induction of tryptophan dioxygenase by glucocorticoids.

When rats were treated with a high dose (greater than or equal to 20 micrograms/100 g BW) of dexamethasone, their liver cytosol showed a predominant peak of specific binding protein of glucocorticoid eluting with 0.13-0.14 M NaCl on diethylaminoethyl cellulose chromatography. On the other hand, when rats were treated with a low dose (approximately 2 micrograms/100 g BW) of dexamethasone, the cytosol showed only the same peak as that of untreated rats, which is widely thought to be that of the typical glucocorticoid receptor. The appearance of the new binding peak depended both on the dose of hormone and the time (from 30 min to 20 h) after dexamethasone treatment; it disappeared 40 h after treatment. A peak similar to that in the cytosol also appeared in the nuclear fraction after treatment with a high dose of hormone. The glucocorticoid-inducible enzymes, tryptophan dioxygenase [(TO) EC 1.13.11.11] and tyrosine aminotransferase (EC 2.6.1.5) were assayed as physiological markers of receptor function. TO induction correlated well with the appearance of the new binding peak in terms of dose- and time dependence on glucocorticoid, whereas tyrosine aminotransferase induction did not. The new peak of glucocorticoid-binding protein detected in the cytosol and nuclear fractions may thus represent the glucocorticoid receptor species involved in TO induction, physiological changes under stress, and pharmacological changes after therapy with high doses of glucocorticoid hormones.

Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in glucocorticoid receptor

When young rats (less than 14 days old) were treated once a day for 2 days with 100 μg/100 g body weight of dexamethasone, their liver cytosol showed a sharp new peak of glucocorticoid binding protein (peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography. When the young rats were given a single injection of the hormone, the chromatogram showed a dominant peak of binding protein (peak B), eluted with 0.07 M NaCl, which was similar to that in untreated rats. The appearance of peak C on two treatments of young rats with hormone was confirmed by both in vivo and in vitro labeling and also studies on the nuclear fraction. Peaks B and C were specific hormone-binding proteins as shown with excess unlabeled hormone. The appearance of peak C was concomitant with the precocious induction of tryptophan dioxygenase in the liver of the young rats, and pretreatment with two injections of dexamethasone were necessary for maximal enzyme induction. On the other hand, in adult rats a single injection of dexamethasone (of 20 μg/100 g body weight or more) was enough to cause the appearance of peak C and induce tryptophan dioxygenase activity maximally; an additional injection of the hormone did not change the chromatographic pattern of the specific binding or the enzyme activity. For this effect in young rats, dexamethasone could not be replaced by other hormones such as glucagon, growth hormone, thyroid hormones, insulin, sex steroids or short-acting glucocorticoid.

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis.

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.

Glucocorticoid receptors and regulation of phenylethanolamine-N- methyltransferase activity in cultured chromaffin cells

Glucocorticoids are known to regulate the enzyme phenylethanolamine-N-methyltransferase (PNMT) in the adrenal medulla of the rat and are thereby thought to control the synthesis of epinephrine. We have examined the details of this relationship in a simplified system, chromaffin cell primary cultures derived from bovine adrenal medulla. Cultured chromaffin cells were found to have a cytosolic, high affinity, saturable glucocorticoid-binding protein with the steroid specificity of a classical glucocorticoid receptor and a Kd of approximately 1 nM. Treatment of cultured cells with dexamethasone or hydrocortisone at any time up to 21 days in culture increased PNMT activity in the soluble fraction of the cell. The concentration of hormone required to produce a half-maximal response was 10 nM dexamethasone when cells were cultured in the presence of 5% fetal calf serum, or 1 nM in a defined serum-free medium. These dose-response relationships are consistent with mediation of this effect by the glucocorticoid receptor. Unexpectedly, however, the glucocorticoid-induced increment in PNMT activity was not inhibited by cycloheximide at concentrations up to 50 microM, and an acceleration of protein synthesis by insulin treatment did not augment the glucocorticoid effect on PNMT. Treatment of the cells with dexamethasone (100 microM) prevented the decline in the epinephrine-to-norepinephrine ratio seen over time in culture, an effect consistent with increased PNMT activity. However, there was no effect of dexamethasone on the ability of the cells to secrete catecholamines in response to stimulation with high KCl or 30 microM nicotine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of D- and L-thyroxine on the glucocorticoid binding capacity of adult rat liver

Administration of either D- or L-thyroxine (T 4) significantly increased the glucocorticoid binding capacity of cytosol of the livers of adrenalectomized adult rats. Administration of up to 0.5 mg/100 g body wt. of L-T 4 was more effective than that of D-T 4, but higher doses (0.8–3 mg/100 g body wt.) of D-T 4 increased the binding capacity markedly to more than that with L-T 4. T 4-administration did not alter the apparent dissociation constant of glucocorticoid binding proteins for glucocorticoid binding, or their behavior on DEAE-cellulose chromatography either before or after thermal activation (23°C for 40 min). Thus the increased binding capacity seemed to be due to increase in the level of glucocorticoid receptor in rat liver.

Appearance of new hepatic glucocorticoid binding proteins under various stressful conditions: relation to endogenous glucocorticoid secretion.

When rats were subjected to the stress of burns, tumors, or partial hepatectomy, a notable new peak of glucocorticoid binding protein appeared on DEAE-cellulose chromatography. This peak accompanied the original peak, which was the only dominant peak in intact rats. The appearance of the new binding protein was concomitant with a high rise in serum corticosterone levels. The new peak was eluted with 0.12-0.14 M NaCl and another, small new peak with 0.02-0.03 M NaCl, while the original peak of intact rats was eluted with 0.05-0.08 M NaCl. In rats adrenalectomized prior to the stress, the new peaks did not appear. To mimic these stressful conditions which provoked a burst of endogenous glucocorticoid, rats were administered with an exogenous high dose of dexamethasone (100 micrograms/100 g B.W.) in vivo. The new peak eluted with 0.12-0.14 M NaCl was again observed and was more dominant in the hormone-treated rats than the stressed rats. These three peaks eluted with 0.02-0.03 M, 0.05-0.08 M, and 0.12-0.14 M NaCl are called here Peak A, B, and C, respectively. This is the first demonstration of the effect of physiological changes in serum levels of glucocorticoid hormone on the nature of glucocorticoid binding protein by DEAE-cellulose chromatography.

Epinephrine and norepinephrine syntheses are regulated by a glucocorticoid receptor-mediated mechanism in the bovine adrenal medulla

The bovine adrenal medulla was investigated regarding the presence of glucocorticoid binding protein and the increases in ornithine decarboxylase (ODC) activity and epinephrine and norepinephrine by dexamethasone. Scatchard analysis of specific cytosol [ 3H] dexamethasone-binding study indicated a single class of high affinity (kd, 35 ± 5 nM) and limited binding sites (150 ± 26 fmoles/mg protein). Competition studies of various steroids indicated a high affinity for dexamethasone and hydrocortisone. Sedimentation in sucrose density gradients revealed a 7.3 S binding peak in the cytosol. Dexamethasone caused an increase in ornithine decarboxylase (ODC) activity within 1 to 2 hours after which the norepinephrine and epinephrine contents increased 16 hours after the peak of ODC activity in a dose dependent manner of dexamethasone in bovine adrenal medullary chromaffin cells in primary monolayer culture. These data suggest that the bovine adrenal medulla is a target organ of glucocorticoid hormone and that norepinephrine and epinephrine syntheses are regulated by a glucocorticoid receptor-mediated mechanism.

Characterization of a glucocorticoid receptor in neonatal rat mammary gland

A glucocorticoid receptor has been identified in cytosolic fractions prepared from 4-day old female Sprague-Dawley rat mammary glands at an early resting stage of mammary development. This component sedimented at 10S and 5S on respectively low and high (0.4 M KCl) ionic strength gradients. It bound dexamethasone with a high affinity ( K d t 2–6 nM) and a low capacity ( N = 300 ± 100 fmol per mg of proteins or 3.3 ± 1.3 fmol per μg, DNA), with a hierarchy of affinity by competition studies dexamethasone > corticosterone > progesterone > R 5020 ⁗ Estradiol-17β. The characteristics of this glucocorticoid-binding protein are thus very similar to the adult one isolated from adult rat mammary gland.