Dexamethasone-receptor Complexes Research Articles

Sexually dimorphic functional alterations of rat hepatic glucocorticoid receptor in response to fluoxetine

Gender-related differences in the expression and functional properties of the hepatic glucocorticoid receptor were studied before and after antidepressant fluoxetine administration to both unstressed and rats exposed to a chronic social isolation stress. Some of the receptor's functional properties, including hormone-binding capacity ( B max), hormone-binding potency ( B max/ K D ratio) and the DNA-binding ability, were found to be sexually dimorphic. Fluoxetine treatment (5 mg/kg body mass, 21 day, intraperitoneally) induced a decrease in B max and in the amount of Hsp70 co-immunoprecipitated with the glucocorticoid receptor only in males, and stimulated the association of the receptor with Hsp90 in females. When applied during the last three weeks of the 6-week isolation, fluoxetine parallelly elevated B max and the receptor protein level in female animals, while in males diminished B max and inhibited association of the receptor with Hsp70. Binding of dexamethasone-receptor complexes both to DNA-cellulose and to isolated liver nuclei did not appear to be a target for fluoxetine action. The results point to sex-related differences in the glucocorticoid receptor functioning and in its response to fluoxetine, and suggest that these differences may contribute to well known sexual dimorphism in the sensitivity to stress, to stress-related disorders and to antidepressant treatment.

Differences in temperature-sensitive receptor binding of glucocorticoids in spontaneously hypertensive and normotensive Wistar-Kyoto rats

Glucocorticoid receptor binding was compared in liver cytosol preparations from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats using homologous displacement of [ 3H]dexamethasone. At 5°C, there was no difference in receptor binding affinity or concentration between strains for dexamethasone, corticosterone or aldosterone. At 37°C, affinity for dexamethasone was lower than at 5°C for both rat strains and decreased with time. However, at this higher temperature, binding affinity in the SHR preparation was consistently higher than in the WKY preparation. The WKY preparation had a higher receptor concentration. The rate of dissociation of the [ 3H]dexamethasone-receptor complex prepared at 5°C and then incubated at 37°C was rapid but not different between strains. A possible explanation of these results is that the relationship of the heat shock proteins to the receptor heterocomplex is different between strains. Evidence exists of a genetic difference in Hsp 70 between SHR and WKY rats, although its cosegregation with blood pressure has not been established.

Interaction of antiglucocorticoid RU 486 with rat kidney glucocorticoid receptor

[3H]RU 486 competes with dexamethasone for rat kidney glucocorticoid receptor (GR) occupancy in vitro, exhibiting a higher association constant for binding to GR than [3H] dexamethasone. Unlike [3H]dexamethasone-receptor complexes which dissociate rapidly at 37 degrees C even in the presence of molybdate, [3H]RU 486-receptor complexes remained more stable both in the presence and in the absence of molybdate. Interestingly, sulfhydryl reagents such as N-ethylmaleimide, iodoacetamide and tosyllysyl chloromethane at 5mM concentration almost completely inhibited binding of [3H]dexamethasone to GR, whereas 20-30% binding to [3H]RU 486 was inhibited by these reagents. [3H]RU 486-receptor complexes readily undergo temperature-dependent activation in vitro as judged by their binding to DNA-cellulose. We propose that changes in binding affinity, stability and sulfhydryl reagent sensitivity between glucocorticoid agonist and antagonist may be due to subtle differences in the binding of the agonist and antagonist to the steroid binding domain of the receptor. This may have a direct relevance to the antiglucocorticoid properties of RU 486.

Colchicine, colcemide and cytochalasin B do not affect translocation of the glucocorticoid hormone-receptor in rat thymocytes or Ehrlich ascites cells.

The involvement of intracellular cytoskeletal elements in the translocation of the dexamethasone-receptor complex from the cytoplasm to the nucleus was studied using the cytoskeleton-disrupting agents colcemide, colchicine and cytochalasin B. These compounds did not affect the translocation of the hormone-receptor complex. We conclude that microfilaments and microtubules do not play a role in the translocation of the glucocorticoid hormone-receptor complex from the cytoplasm to the nucleus.

Regulation of glucocorticoid receptors in the kidney of immature and mature male rats

1. 1. Specific binding of [ 3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the kidney of immature (3-week) and mature (26-week) Long-Evans male rats. 2. 2. The concentration of specific binding sites was significantly higher (25%) in the kidney of immature rats as compared with mature, while dissociation constants ( K d) remain unaltered at both ages. 3. 3. Heat activation (25°C for 45 min) significantly enhanced the binding of [ 3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both ages to the same extent. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice versa) gave similar results to the non-mixed groups. 4. 4. Ca 2+ activation (0°C for 45 min with 20 mM Ca 2+) also enhanced the nuclear and DNA-cellulose binding at both ages but to a greater magnitude in immature rats. 5. 5. Differences in the number of specific binding sites and some of the physicochemical properties of kidney glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.

The nuclear binding of dexamethasone-receptor complex from fast and slow skeletal muscle in rats

1. 1. The binding to isolated muscle nuclei of the complex of dexamethasone with cytosol receptors from rat soleus (Sol) and extensor digitorum longus (EDL) muscles was measured. 2. 2. The ratio of bound to total amount of complex was higher in Sol. 3. 3. The binding of complex per mg of cytosol protein was also higher in Sol. 4. 4. These results suggest that activation and nuclear binding of the steroid-receptor complex are not the sites of the different sensitivity of the two muscle types to glucocorticoid.

Age-dependent activation of glucocorticoid receptors in the cerebral hemispheres of male rats

The binding of [ 3H]dexamethasone-receptor complexes to purified nuclei was studied in the cerebral hemispheres of immature (3-week-old) and mature (26-week-old) Long-Evans male rats to determine the age-related changes, if any, in the physicochemical properties of glucocorticoid receptors. Our data show that heat activation (for 45 min at 25 °C) significantly the nuclear binding of [ 3H]dexamethasone-receptor complexes in rats of both ages, with a greater magnitude in immature ratrs. Ca 2+ activation (20mM Ca 2+ for 45 min at 0°C) also enhances the nuclear binding of bound receptor complexes but to a similar extent at both ages. These findings indicate that some of the physicochemical properties (e.g. heat activation) of glucocorticoid receptor change, while others (e.g. Ca 2+ activation) remain unchanged at different phases of the lifespan.

Age-dependent regulation of glucocorticoid receptors in the liver of male rats

Specific binding of [ 3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the liver of immature (3 weeks old) and mature (26 weeks old) Long-Evans male rats. The concentration of specific binding sites was significantly higher (33%) in the liver of immature rats as compared to mature, while dissociation constants ( K d) remain unaltered at both ages. Heat activation (for 45 min at 25° C) significantly enhances the binding of [ 3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both the ages, with a greater magnitude in mature rats. Cross mixing experiments (i.e., binding of activated cytosol from mature rats to nuclei of immature and vice-versa) show receptor specificity. Ca 2+ activation (20 mM Ca 2+ for 45 min at 0° C) also enhances the nuclear and DNA-cellulose binding at both the ages, but to a similar extent. Differences in the number of specific binding sites and some of the physicochemical properties of glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.

Molybdate inhibits glucocorticoid-receptor complex binding to RNA

The binding of dexamethasone-receptor complexes to RNA was investigated by an assay system under cell-free conditions. By this gradient centrifugation assay, we found that, under low salt concentrations, dexamethasone-receptor complexes can bind to 18S RNA from HeLa cells. Molybdate, tungstate and methavanadate were able to inhibit dexamethasone-receptor complex binding to 18S RNA, whereas this was not the case when chloride, fluoride, or sulfate ions were present in our binding assays. Molybdate was also found to disrupt dexamethasone-receptor-18S RNA complexes once they were already formed. We concluded that interaction between dexamethasone-receptor complexes and RNA under cell-free conditions is affected by ions present in the medium.

Effects of dexamethasone on the growth of cultured rabbit articular chondrocytes:relation with the nuclear glucocorticoid-receptor complex.

This study reports that dexamethasone at a high dose (10(-4) mol/l) induced slowing of the in vitro proliferation of rabbit articular chondrocytes in both monolayer and clonal culture. This effect is consistent with an inhibition of DNA and RNA synthesis and was characterised by an accumulation of cells in the G0G1 phase of the cell cycle, as shown by flow cytometric analysis. Therefore we determined the extent of nuclear localisation of dexamethasone-receptor complexes. The results showed a discrepancy between 50% growth inhibitory dose (10(-4) mol/l) and the apparent affinity, KD (1.4 (SD 0.2) X 10(-9) mol/l). Thus the growth inhibition of rabbit articular chondrocytes by dexamethasone did not seem to be related exclusively to an interaction with the glucocorticoid-receptor complexes.

Role of lysosomotrophic reagents in glucocorticoid hormone action

Administration of (10 mg/200 g) methylamine or chloroquine to adrenalectomized rats for 2 days followed by a single injection of either cortisol (2.5 mg/200 g) or dexamethasone (0.5 mg/200 g) resulted in a significant enhancement of the tyrosine aminotransferase enzymatic activity in rat liver versus rats given a single injection only of either steroid. Lysosomotrophic reagents were unable to induce tyrosine aminotransferase when administered alone. Cytosols from rat liver treated with lysosomotrophic reagents in vivo had approx. 20–30% more specific binding to [ 3H]dexamethasone as compared to the control, untreated rats. This enhanced binding was due to an increase in the concentration of the receptor rather than change in the affinity of the hormone for the receptor. Rat livers perfused with and homogenized in 10 mM Tris-HCl/0.25 M sucrose buffer (pH 7.5) containing about 5 mM lysosomotrophic reagents showed optimum stabilization of the steroid unbound glucocorticoid receptor in vitro at both 4°C and 25°C. These reagents had no effect on in vitro transformation of [ 3H]dexamethasone-receptor complex or on the binding of the thermally transformed receptor to the nuclei. It is concluded from these studies that lysosomotrophic reagents (a) enhance tyrosine aminotransferase induction by glucocorticoids and (b) stabilize unbound glucocorticoid receptor both in vivo and in vitro without any effect on in vitro transformation of the steroid-receptor complex.

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 denned 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 ( K d) of the [ 3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8−11.8 nM; x ± 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.

Properties and Dynamics of Glucocorticoid Binding Capacity in Rat Skin

[3H]Dexamethasone binding was detected in cytosol prepared from rat skin only in the presence of dithiothreitol (DTT). Simultaneous supplement of sodium molybdate (Mo) induced synergistic enhancement of the binding. In the presence of DTT and Mo the dissociation constant was approximately 1 nM, the number of maximum binding sites was approximately 100-200 fmol/mg protein, and only steroids that possessed glucocorticoid activity competed with [3H]dexamethasone binding. [3H]Dexamethasone-receptor complexes in dermal cytosol were able to bind to DNA-cellulose after brief heating, and receptors were eluted from DEAE-cellulose with 0.2 M KCl. These observations showed that [3H]dexamethasone binding sites observed in dermal cytosols have similar binding characteristics to glucocorticoid receptors in other glucocorticoid target tissues. Binding capacity in dermal cytosols was depleted after in vivo administration of all 4 glucocorticoids used in the present study. Both the extent and the duration of depletion were dose-dependent in all instances. Non-fluorinated glucocorticoids required higher doses to induce a profound depletion than did fluorinated ones and the duration of depletion induced by the former was shorter than by the latter. Since fluorinated glucocorticoids usually have higher anti-inflammatory potency than non-fluorinated ones, we concluded that the pattern of depletion and replenishment of the dermal cytosol binding capacity was correlated with glucocorticoid biopotency.

Physicochemical and Functional Properties of Chicken Breast Muscle Proteins

Dexamethasone-receptor complexes can be extracted from HeLa cell nuclei by mild sonication. These complexes can account for 800–1000 binding sites/cell, and are indistinguishable from salt-extractable ones as judged by sucrose gradient centrifugation in the presence of 0.3 M KCl, showing a sedimentation coefficient of 3.6 S. These complexes, however, broadly sediment in the 7 to 3.6 S region of low salt sucrose gradients. Enzymatic treatment of soluble extracts from nuclear sonicates shows that RNA is associated to dexamethasone-receptor complexes.

RNase a effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25°C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment of HeLa cell cytosol at 2°C results in receptor complexes which sediment at 6.9S in low salt gradients. RNase A treatment at 25°C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2°C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25°C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25°C, but not at 2°C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating.Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25°C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2°C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25°C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A.These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.

Interaction of rat liver glucocorticoid receptor with heparin.

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin.

RNA-containing nuclear binding sites for glucocorticoid-receptor complexes

RNase A treatment of HeLa cell nuclei causes a time- and concentration-dependent release of dexamethasone-receptor complexes. If nuclei are incubated in the absence of enzyme, only 60% of RNase-releasable complexes can be detected. Sucrose density gradient analysis of nuclear extracts shows that receptor complexes released by RNase treatment sediment at 3.6 S, whereas complexes obtained from untreated nuclei sediment between 7 and 3.6 S. Our results show that a fraction of dexamethasone-receptor complexes retained by HeLa cell nuclei is located in binding sites involving RNA.

Sulfhydryl-modifying reagents reversibly inhibit binding of glucocorticoid-receptor complexes to DNA-cellulose.

Glucocorticoid -receptor complexes from intact rat thymus cells incubated with [3H]dexamethasone at 0 degree C are in the nonactivated form and do not bind to DNA-cellulose. Upon being warmed, they are transformed to activated complexes that bind to DNA-cellulose at 0 degree C. We have found that treatment of dexamethasone-receptor complexes with the sulfhydryl-modifying reagents methyl methanethiosulfonate ( MMTS ) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), either before or after the warming, inhibits subsequent binding to DNA-cellulose. The effects of these reagents can be reversed at 0 degree C by dithioerythritol and other sulfhydryl-containing compounds. These results provide the first clear evidence that sulfhydryl-modifying reagents inhibit the binding of activated dexamethasone-receptor complexes to DNA-cellulose and suggest that sulfhydryl groups may be located in or near the DNA binding domain of the rat thymus glucocorticoid-receptor complex. Furthermore, addition of dithioerythritol at 0 degree C to nonactivated receptor complexes that have been treated with MMTS or DTNB produces a substantial increase in the capacity of these complexes to bind to DNA-cellulose, raising the possibility that sulfhydryl groups may be associated with a region on the receptor that plays a critical role in the activation process.

Biochemical characterization of glucocorticoid receptors of rat testis

Rat testis cytosolic glucocorticoid receptors were characterized by DEAE-cellulose chromatography, Sephadex G-100 columns and sucrose-density gradients. The unactivated [ 3H]dexamethasone-receptor complex showed two distinct peaks of macromolecular bound radioactivity on DEAE-cellulose chromatography. Peak I eluted just after the column wash, while peak II eluted at 0.28 M KC1. Activation of the complex at 25°C for 45 min resulted in a significant increase in peak I with a concomitant decrease in peak II and the appearance of a third peak at 0.18 M KC1. Both the unactivated and activated [ 3H]dexamethasone-receptor complex, when analyzed on Sephadex G-100 columns, showed a single macromolecular bound radioactive peak having a Stokes radius of 6.5 nm. Treatment of the [ 3H]dexamethasone-receptor complex (6.5 nm holo-receptor) with trypsin (0.5 μg/ml) resulted in the appearance of a smaller (2.0 nm) fragment but no intermediate sized forms of the receptor were observed. The complexes sedimented as 7–8 S (in low salt) and as 4.6 S (in high salt) forms in sucrose gradients in the presence or absence of 10mM molybdate. Steroid unbound receptors were inactivated at 25°C and 4°C with a T 1 2 of 2 h and 24 h, respectively. Ten mM molybdate slightly protected the unbound testis receptor at 25°C. However, molybdate, dithiothreitol, and molybdate plus dithiothreitol were unable to either enhance or reactivate [ 3H]dexamethasone binding of unbound receptors at 4°C or 25°C. Activation of testis [ 3H]dexamethasone-receptor complexes resulted in a 2–3 fold enhancement in subsequent binding to testis nuclei in vitro. In addition, we observed that activated [ 3H]dexamethasone-receptor complexes were precipitated with 30–35% ammonium sulfate, while unactivated complexes were precipitated with 30–40% ammonium sulfate.

Glucocorticoid Binding Properties of Mouse Thymic Lymphosarcoma Cell Populations Selected for Resistance to the Cytolytic Effects of Glucocorticoids In Vivo<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Thirteen independently derived murine thymic lymphosarcoma lines were assayed for various aspects of sensitivity to glucocorticoid-induced cytolysis. All tumor lines were sensitive to cytolysis, as evidenced by profound tumor regression after pharmacologic doses of cortisol. All tumor lines contained about 20,000 high-affinity, dexamethasone binding sites/cell. Between 55 and 88% of these presumptive receptor sites underwent nuclear translocation during a 30-minute incubation at 37 degrees C. Dissociation constants (Kd) for the dexamethasone-receptor complex were between 1.5 and 3.6 nM in all cases. Kd for the triamcinolone acetonide-receptor complex were determined for a few tumor lines and were between 0.5 and 0.9 nM. Cytolysis-resistant subpopulations were selected by prolonged glucocorticoid treatment of BALB/c pi mice bearing tumors from seven of the lymphosarcoma lines. All seven resistant subpopulations contained about 20,000 high-affinity, dexamethasone binding sites/cell. Between 57 and 80% of these presumptive receptor sites underwent nuclear translocation under standard assay conditions. No resistant variants exhibited significantly reduced dexamethasone binding or nuclear translocation properties.