Glucocorticoid Receptor Binding Capacity Research Articles

Differential response of type I and type II corticosteroid receptors to changes in plasma steroid level and circadian rhythmicity.

Corticosterone (CORT) binds to two receptor systems in rat brain: the type I CORT-preferring receptor (CR) and the type II glucocorticoid receptor (GR). Discrimination between the two receptor types can be achieved with the 'pure' synthetic glucocorticoid RU 28362. In this study, we show that the binding capacity of GR in the rat hippocampus exhibits a strikingly different response from CR to adrenalectomy (ADX), chronic steroid replacement, hypophysectomy (HYPOX) and during circadian variation. Under those experimental conditions neither receptor site showed changes in binding affinity. After ADX, CR number remained relatively constant for a period of 13 days, while GR capacity increased by 133%, a level which was reached 5 days post-surgery. CR capacity showed circadian variation, since CR number was 65% higher in the evening than in the morning. GR capacities at those two time points were not significantly different. Replacement with subcutaneous CORT implants (100-mg pellets) for 7 days following ADX rats did not affect CR number, but caused a 38% decrease in GR number compared to control animals (cholesterol-treated, 7-day-ADX rats). On the other hand, dexamethasone (DEX) implants (5-, 15-, 25-mg pellets) elicited a dose-dependent increase in CR capacity (up to 99%) and a dose-dependent decrease in GR capacity (40-44%). Finally, 2 weeks after HYPOX, CR and GR numbers were increased by 60 and 38%, respectively. We conclude that the type II GR capacity responds in an autoregulatory manner to changes in circulating plasma glucocorticoid levels, while type I CR does not.

The influence of immobilization stress on cardiac protein synthesis. A possible regulatory role for glucocorticoids.

High serum corticosterone levels and transient depletion of cytosolic glucocorticoid receptor binding capacity were observed in rat cardiac muscle following immobilization stress. To evaluate the effect of this treatment on the protein synthetic capacity, biologically active polyribosomes were used to direct the in vitro synthesis of polypeptides in the rabbit reticulocyte lysate. The results of these experiments indicate that the template activity of several messenger RNAs coding for major myofibrillar proteins was increased. This change in protein synthetic activity was not observed in adrenalectomized animals. Collectively, these results suggest that glucocorticoids are involved in the regulation of cardiac protein biosynthesis.

Glucocorticoid receptors lacking hormone-binding activity are bound in nuclei of ATP-depleted cells.

The glucocorticoid receptor binding capacity of rat thymus cells disappears when the cells are depleted of ATP by anaerobiosis, and rapidly reappears when ATP levels are restored. Loss and recovery of binding capacity occurs even when protein synthesis is suppressed with cycloheximide. In view of this and similar work in other cell systems, we proposed that in cells deprived of ATP the receptor is present in a form--the 'null receptor' form, as we shall call it--that cannot bind hormone. Although many subsequent observations support this idea, no direct evidence has appeared for the existence of the null receptor. We have attempted to detect the null receptor in WEHI-7 mouse thymoma cells with a monoclonal antibody to the glucocorticoid receptor. Here we report that the null receptor is bound in the nuclei of ATP-depleted cells, and is present in amounts comparable to those of receptors in normal cells.

Hormone dependency of transformation of rat liver glucocorticoid receptor in vitro: effects of heat, salt, and nucleotides.

A majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of the cytosol at 23 degrees C, or at 0 degree C with 10 mM ATP or 0.3 M KCl caused appearance of a slower migrating (4S) form which exhibited an increased affinity toward DNA-cellulose and ATP-Sepharose. Presence of 20 mM Na2MoO4 blocked this 9S to 4S transformation of GRc. A complete conversion of the 9S to the 4S form occurred upon a 2 h incubation of GRc with 10 mM ATP at 0 degree C. Other nucleoside triphosphates (GTP, CTP, and UTP), ADP and PPi (but not AMP or cAMP) were also effective in transforming the 9S form. The heat transformation occurred in a time-dependent manner and was complete within 1 h at 23 degrees C; presence of 10 mM ATP during this 23 degrees C incubation period allowed a complete 9S to 4S alteration in 10-20 min. Addition of ATP also accelerated the rate of salt activation of the GRc; a 50% conversion to the 4S form occurred in 20 min or 3 min in the absence or the presence of 10 mM ATP during the 0 degree C incubation of GRc with 0.15 M KCl. An absolute requirement of the hormone for 9S to 4S transformation of glucocorticoid receptor (GR) was evident, as no conversion of the 9S form to the 4S form could be achieved with the ligand-free GR under any of the above conditions. Incubation of cytosol preparations at 23 degrees C or at 0 degree C with KCl or ATP caused dissociation of the GRc and reduced the steroid binding capacity of GR. Although aurintricarboxylic acid, pyridoxal 5'-phosphate, Na2MoO4, Na2WO4, o-phenanthroline, Rifamycin AF/013 and heparin inhibited the ATP-Sepharose and DNA binding of the GRc, only Na2MoO4 and Na2WO4 selectively blocked the 9S to 4S conversion. We suggest that the 9S to 4S transformation in vitro of rat liver GRc represents an acquisition of DNA and ATP-Sepharose binding ability and may involve a separation of subunits from an oligomeric receptor structure.

Relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 cell

This study tested the relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 mouse pituitary tumor cell. Four treatments were used to alter intact cell ATP levels: (1) incubation with NaF, (2) incubation with 2,4-dinitrophenol, (3) alterations in culture age, and (4) incubation with dexamethasone. Each of these treatments clearly altered glucocorticoid receptor-binding capacity. Their effects on ATP levels, however, were not the same: NaF decreased ATP levels, dinitrophenol decreased ATP levels only transiently, and the last two treatments (increasing culture age and incubation with dexamethasone) produced an inverse relationship between ATP levels and receptor-binding capacity. It is concluded that, at least in the intact AtT-20 cell, receptor-binding capacity is not always directly related to ATP levels.

Glucocorticoid receptor regulation

Glucocorticoids, like other classes of steroid hormones, must bind to cellular receptors in order to exert their effects. Because of this central role in mediating hormone action, it is important to elucidate those factors that control receptor content. The purpose of this minireview is to summarize the recent work that explores the mechanisms through which cells modulate their glucocorticoid receptor binding capacity.

Effects of metal ions and chelating agents on in vitro stability of glucocorticoid receptors in brain cytosol

In vitro studies in a variety of tissues and cell types suggest that glucocorticoid receptor binding capacity is not static and that binding sites are subject to up- and down-regulatory mechanisms. The interpretation of such studies, however, is often complicated by factors affecting the stability of the receptor. This situation is particularly acute in the absence of ligand because of the increased lability of the unoccupied receptor. Studies reported here investigate effects of various metal ions and chelating agents on the stability of unoccupied [ 3H]dexamethasone binding sites in whole mouse brain cytosol. Variation in the ionic strength of cytosol, as created by the additions of various monovalent cations (Li +, Na +, K +, Rb + and Cs +), was found to be an important factor affecting the increased stability of the receptor in vitro. Additions of divalent (Mg ++, Ca ++, Ba ++, and Mn ++) and trivalent (La +++, Cr +++ and Al +++) cations to cytosol, however, were generally found to produce a dose-dependent decrease in the stability of the unoccupied receptor. Additions of the chelating agents EDTA, EGTA and 1,10-phenanthroline to cytosol, resulted in differential, and sometimes complex, dose-dependent effects on receptor stability. The complex effects of various combinations of cations and the chelator EDTA were also investigated.

Inactivation by Na+,K+-ATPase of cytosol glucocorticoid receptors from rat brain and liver.

We have examined the effect of Na+,K+-ATPase on 3H-triamcinolone acetonide binding capacity of cytosol glucocorticoid receptors from rat brain and liver. Preincubation of the brain or liver cytosol with Na+,K+-ATPase (10 units/ml) at 30 degrees C resulted in a rapid loss of specific 3H-triamcinolone acetonide binding, with a half-life of approximately 7 min. The ATPase effect could be prevented by the addition of 10(-5) M ouabain, or substantially reduced by the omission of Na+,K+ or Mg+2. The cytosol receptor bound with 3H-triamcinolone acetonide was totally resistant to the inactivation by the ATPase. Since there is some evidence that ATP may bind to glucocorticoid receptor, our findings indicate that an ATP-receptor complex may be essential for steroid binding. The effects of the ATPase in the inactivation of the receptor are very similar to those of alkaline phosphatase reported by others. This raises doubts about the proposal based on the phosphatase inactivation that the cytosol glucocorticoid receptor may be phosphorylated.

Evidence for glucocorticoid target cells in the rat optic nerve. Hormone binding and glycerolphosphate dehydrogenase induction.

Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol-3-phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid-inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. correlative immunocytochemical studies found GPDH-positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high-affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately two fold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.

Investigation on the thermostability of steroid hormone receptors in lyophilized calf uterine tissue powder.

Powdered calf uteri were lyophilized and sealed in a vacuum. The effects of lyophilization and the thermostability of the binding activity of uterine steroid hormone receptors were studied. The lyophilized powder was analyzed for its ability to bind estrogen, progestin, androgen, and glucocorticoid steroids. Lyophilization had no deleterious effects on the binding activity. Storage of the lyophilized powder at +20 C for more than 14 days did not reduce the binding activity. No reduction of the binding capacity was also observed when the storage temperature was at +30 C for 16 hours. However, a slight loss of specific binding sites was observed for 17 beta-estradiol (-12%) at the storage temperature of +40 C, but the other steroid binding proteins were more stable. When the storage temperature was at +60 C for 16 hours, a loss of about 24% was observed for the specific estrogen binding sites, whereas the binding capacity of progestin, androgen, and glucocorticoid receptors was reduced by only less than 10%. Storage at the high temperature of +80 C for 16 hours resulted in a loss of only 36% for the estrogen binding capacity and less than 30% loss for the other binding proteins. The freeze-dried uterine powders can be useful quality control specimens for steroid receptor assays.

Aflatoxin inhibition of glucocorticoid binding capacity of rat liver nuclei

The effect of aflatoxin B 1 on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [ 3H]dexamethasonccytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B 1. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 36 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B 1 dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant. Administration of [ 3H]dexamethasone to aflatoxin B 1-treated rats produced a similar pattern of glucocortocoid binding distribution in vivo to that observed in vitro. No differences in [ 3H]dexamethasone-cytoplasmic receptor binding between control and aflatoxin B 1-treated rats were found, whereas nuclear [ 3H]dexamethasone binding was reduced 34% by aflatoxin B 1 treatment.