Hepatic Glucocorticoid Receptor Research Articles

Neonatal hyperoxia and cytochrome P450 imprinting in adulthood.

We hypothesized that during a critical neonatal period hyperoxia may produce alterations of sex-specific cytochrome P450 isozymes in adulthood (enzyme imprinting). To test this, newborn rats were exposed to 24 or 72 h of hyperoxia (O2 > 95%) within 24 h after birth and killed at 120 d. In males, significant negative imprinting (decrease) was found in total cytochrome P450 content and male-specific CYP2C11 in the hyperoxia groups. Positive imprinting (increase) was noted in CYP1A2 and male-specific CYP3A2 in the 72-h hyperoxia group. These alterations were essentially similar when expressed on a per microsomal protein or per liver basis. In addition, the level of hepatic glucocorticoid receptor in adult male rats was elevated after neonatal hyperoxia. In females, there was a significant body and liver weight loss after hyperoxic exposure, which resulted in a negative imprinting of CYP1A2 and female-specific 2C12 in the 72-h hyperoxia group on a per liver basis, whereas the measured parameters were unaltered when expressed per microsome. In general, the changes were more marked with longer hyperoxic exposure, suggesting that more pronounced alterations may be induced with prolonged neonatal hyperoxia. Because hyperoxic exposure in premature neonates is a common clinical practice and decreased CYP2C11 in adult males is expected to result in feminization, we believe that the scope of this work should be expanded and eventually tested for its relevance in human subjects.

Heat shock protein 70 is associated in substoichiometric amounts with the rat hepatic glucocorticoid receptor.

The 70-kDa heat shock protein (hsp70) has been shown to be an important participant in several intracellular events, including protein folding and trafficking. Hsp70 binds to many, if not all, proteins during their translation and maintains its association with some protein complexes as a subunit. We have examined the possibility that hsp70 may be associated with one or more forms of the rat hepatic glucocorticoid receptor (GR). Unliganded GR was immunoprecipitated from cytosol with the anti-GR monoclonal antibody BUGR2 and then subjected to western blotting. Both hsp70 and the 90-kDa heat shock protein (hsp90) were found to be specifically associated with the GR. Hsp70 was also bound to the liganded unactivated and activated (transformed) forms of the GR complex, while as expected, hsp90 was absent from the activated GR. Immunoprecipitation of cytosolic hsp70 with the anti-hsp70 monoclonal antibody N27 resulted in coprecipitation of GR. The components of the immunopurified GR were also analyzed by laser densitometry after polyacrylamide gel electrophoresis and Coomassie blue staining. These experiments revealed that hsp70 is bound to the GR in an approximate 1:5 ratio. Unactivated GR complexes isolated via a ligand affinity purification scheme contained hsp90 and trace amounts of hsp70. Collectively, these experiments demonstrate that hsp70 is specifically associated with several forms of the native rat hepatic GR, although its binding is substoichiometric. This is in direct contrast to hsp90, which binds as a dimeric subunit to the heteromeric unactivated GR complex.

Affinity of hepatic glucocorticoid receptors is influenced by energy/feeding status

Genetically obese animals have been shown to have a reduced number and affinity of glucocorticoid receptors. The relationship between the alterations in receptor binding and the regulation of energy balance is not known. We sought to determine the role of body energy/feeding status on the binding characteristics of glucocorticoid receptors. To accomplish this, we examined the effect of long-term food restriction on the number and affinity of hepatic glucocorticoid receptors from Sprague-Dawley rats. After 3 weeks of food restriction (40% of ad lib), animals were bilaterally adrenalectomized. Livers were removed, a crude cytosolic glucocorticoid receptor fraction was isolated, and radioreceptor assays were performed. Glucocorticoid receptors from food-restricted rats showed a significant reduction in the dissociation constant ( K d) as compared to receptors derived from free-feeding controls. No difference in receptor number was observed. These results suggest that energy or feeding status of the animal may influence the affinity of hepatic glucocorticoid receptors, while receptor number may be independent of this status.

Chronic effects of intracerebral ventricular enterostatin in Osborne-Mendel rats fed a high-fat diet

Enterostatin, a pentapeptide found in the procolipase molecule of the pancreas, has been infused chronically into the lateral ventricle of Osborne-Mendel rats for 11 days. Treatment with enterostatin acutely lowered food intake more in the treated animals than in the vehicle-treated controls. Weight gain and serum insulin were significantly reduced by enterostatin. Corticosterone was significantly increased in rats receiving chronic infusions of enterostatin into the lateral ventricle and was also increased following an acute ICV injection of enterostatin. CRH antagonist did not block the acute feeding response to enterostatin in Sprague-Dawley rats. The elevation of corticosterone during chronic infusion of enterostatin reduced the level of hepatic glucocorticoid receptor as measured by Western blot. We conclude that body weight gain of OM rats is reduced by centrally administered enterostatin, and that the acute effects of enterostatin on food inkake are not mediated through stimulation of CRH secretion.

Dehydroepiandrosterone regulation of the hepatic glucocorticoid receptor in the zucker rat. The obesity research program

Dehydroepiandrosterone (DHEA) decreases the activity of hepatic tyrosine aminotransferase (TAT), a glucocorticoid-inducible enzyme, in the obese, hypercorticosteronemic Zucker rat. To investigate the mechanism of this antiglucocorticoid action, the effect of exogenous DHEA on hepatic glucocorticoid receptor (GC) number and affinity was quantitated. Food supplementation with DHEA (0.6% w/w) for 1 or 7 days had no effect on either receptor number or affinity in obese Zucker rats. After 28 days, however, DHEA treatment resulted in a nearly 40% decrease in cytosolic hepatic receptor content (B max; fmol/mg cytosolic protein) without any change in affinity ( K d ) in both lean and obese rats. DHEA treatment for 28 days also resulted in an increased liver size and cytosolic protein content. When the hepatic GC receptor content was normalized based on the change in liver size and protein content, the apparent number of GC binding sites per liver was not affected by DHEA treatment. This observation suggests that DHEA's effect on GC receptor content may not be a specific action and that downregulation of the GC receptor is not the mechanism of DHEA action on GC induced TAT activity. This is supported by the effect of DHEA on obese rat TAT activity in the same experiment where the greatest inhibition occurred after only 1 day of treatment. From these experiments it is concluded that although long-term DHEA treatment may decrease the relative concentration of GC receptors in rat liver, this change is not the mechanism through which DHEA mediates its acute antiglucocorticoid action.

Glucocorticoid regulation of splanchnic glutamine, alanine, glutamate, ammonia, and glutathione fluxes.

Interorgan glutamine and associated metabolite fluxes were measured across the gut and liver to delineate splanchnic bed fluxes secondary to enhanced arterial loads mobilized in the periphery by glucocorticoid. Experiments were performed on adrenalectomized rats since adrenalectomy doubled the hepatic glucocorticoid receptor population compared with intact animals. Under these conditions, triamcinolone supplement (40 micrograms.day-1.100 g body wt-1) enhanced the combined net glutamine uptake by gut and liver eightfold, whereas combined gut and liver unidirectional breakdown and synthesis fluxes both increased (3.4- and 7.4-fold, respectively). Triamcinolone supplement also altered the pattern of metabolite released; gut released predominantly ammonium and some alanine, whereas the liver removed more alanine along with glutamine and released more urea, glutamate, and glutathione. Mechanistically, enhanced cellular glutamine uptake could be attributed to a three- to fourfold acceleration of glutamine transport associated with a rise in intracellular glutamine content. However, uptake by isolated membrane vesicles revealed only a small (27%) increase in System N activity, whereas extraction and reconstitution of the transporter into proteoliposomes failed to demonstrate increased transporter activity. Similarly, activity of phosphate-dependent glutaminase and glutamate dehydrogenase increased in crude homogenates (2-fold), but the former disappears in completely disrupted preparations. Furthermore, whereas messenger RNA and assayable enzymic activity for glutamate dehydrogenase clearly increased with glucocorticoid, glutaminase message was less significantly increased. Thus glucocorticoid appears directly capable of accelerating hepatic glutamine extraction primarily by modulating transporter activity that is closely coupled to glutamine utilization.

Epidermal and hepatic glucocorticoid receptors in cats and dogs

Low capacity, high affinity [ 3H] dexamethasone binding receptors were identified in cytosolic preparations of liver (mean number 45±10·1 fmol mg −1 protein, apparent dissociation constant 0·4±0·1 nM) and skin (mean number 46·4±23·8 fmol mg −1 protein, apparent dissociation constant 1 ± 0·2 nM) of clinically normal dogs. For clinically normal cats, approximately half these numbers of receptors with a lower affinity, were detected in liver (mean number 23·1±10·4 fmol mg −1 protein, apparent dissociation constant 3·2±0·9 nM) and skin (mean number 23·90±10·9 fmol mg −1 protein, apparent dissociation constant 2·2±1·5 nM). This difference between dogs and cats in [ 3H] dexamethasone binding receptors may contribute to the relative glucocorticoid resistance observed in cats.

Vitamin A deficiency and glucocorticoid receptor activity in rat liver.

The influence of vitamin A on the binding properties of hepatic glucocorticoid receptors (GR) was studied in young rats 7 weeks after they had been given a diet with or without vitamin A. Scatchard analysis showed an increased capacity of cytosolic GRs to bind dexamethasone in vitamin A deficiency. In these rats, an increase in tyrosine aminotransferase also occurred, and this could be related to the increased formation of hormone-GR complexes. Measurement of protein kinase C activity showed an increase which might be related to the functional activity of GRs.

Phorone (diisopropylidene acetone), a glutathione depletor, decreases rat glucocorticoid receptor binding in vivo.

The exact mechanism by which carcinogens and tumor promoters act on the glucocorticoid receptor system in vivo is not known. Based on earlier studies that sulfhydryl-reducing agents stabilize glucocorticoid receptor binding in vitro, some workers have postulated that endogenous reducing factors may be important for glucocorticoid receptor function in vivo. To test whether glutathione (GSH) may serve this purpose, we investigated the effects of phorone, an agent that partially depletes intracellular GSH, on the hepatic cytosolic glucocorticoid receptor (GRc) binding characteristics in intact and 7-10 day adrenalectomized (ADX) adult female Sprague-Dawley rats. Biochemical analysis revealed that a single treatment of phorone (300 mg/kg) to both intact and ADX rats significantly decreased the liver GSH concentration (70-90% of control levels) as well as the GRc maximum binding concentration (30% of control levels). The decrease in GSH levels preceded the reduction in GRc maximum binding concentrations; both effects were reversible after 24 h of treatment. The phorone-mediated decrease of GSH levels was maximum at doses greater than 75 mg/kg, whereas GRc maximum binding concentrations in vivo appeared dose dependent up to 400 mg/kg. Pretreatment with phorone or the carcinogens mirex and 3-methylcholanthrene significantly decreases GRc binding and nuclear uptake in vivo, as well as diminishes intracellular cytosolic GSH levels. Although a temporal relationship between the GSH levels and the GRc maximum binding concentrations in vivo was observed, there was no quantitative relationship between these two parameters based on our phorone dose-response and the carcinogen pretreatment data. Our findings suggest that during the early phases of carcinogenesis, the hepatocellular GSH does not play a direct role upon the biochemical action of certain carcinogens and tumor promoters on the glucocorticoid receptor binding in the liver.

Regulation of glucocorticoid receptors during adaptive liver growth.

Mirex, an organochlorine pesticide, is a potent inducer of liver hyperplasia and hypertrophy in intact (INT) rats. In contrast, mirex elicits predominantly hyperplastic liver growth in adrenalectomized (ADX) rats and hypertrophic liver growth in thyroidectomized (THX) rats. Supplements of glucocorticoids restore liver hypertrophy and inhibit DNA synthesis in ADX mirex-dosed rats. Because responsiveness to glucocorticoids is in part dependent on the number and affinity of glucocorticoid receptors (GR) we have measured hepatic GR levels and dexamethasone inducibility of tyrosine aminotransferase (TAT) in mirex-dosed INT, ADX, and THX rats. Specific [3H]dexamethasone binding sites decreased to 48, 49, and 59% of control values in mirex-dosed INT, ADX, and THX rats, respectively, with no changes in the apparent equilibrium dissociation constant at 48 h postdose. The significant depletion of hepatic cytosolic GR in all experimental groups dosed with mirex failed to impair both uninduced and dexamethasone-induced TAT levels in these groups. It appears that the decrease in steroid binding is neither a result of an interaction between mirex and hepatic GR nor a simple "dilution" of receptors due to the accompanying liver hypertrophy. Taken collectively, these data suggest that despite the ligand-independent downregulation of hepatic GR, responsiveness of hepatocytes from mixed-dosed rats to glucocorticoids is not altered. Therefore, glucocorticoid-mediated liver hypertrophy in mirex-dosed rats probably involves either 1) modulation of steps subsequent to ligand binding and activation translocation of hepatic GR, 2) glucocorticoid effects at extrahepatic sites that release factors that are the actual effectors of parenchymal cell enlargement, or 3) an unconventional receptor-mediated mechanism.

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-β-(4-dimethylaminophenyl)-17a-hydroxy-17ß-(3-hydroxypropyl)-13α-methyl-4,9-gonadien-3-onel). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [ 3H]ZK98299 with affinity approximately equal to that of [ 3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0°C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [ 3H]ZK98299 and [ 3H]triamcinolone acetonide binding. While [ 3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide [ 3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [ 3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 °C) and receptor transforming (23 °C) conditions, and exhibited a faster rate of dissociation at 23°C when compared with [ 3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [ 3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.

The effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the hepatic estrogen and glucocorticoid receptors in congenic strains of Ah responsive and Ah nonresponsive C57BL/6J mice

The present study examines the role of the Ah receptor in the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the binding capacity of the hepatic glucocorticoid (GC) and estrogen (E) receptors in female congenic C57BL/6J mice differing only at the Ah (aromatic hydrocarbon responsiveness) locus. The Ah locus is thought to encode the Ah receptor, which regulates the effects of TCDD and related compounds on cytochrome P450IA1 and appears to mediate most of the toxic effects of TCDD. The differences between Ah responsive (Ah b/b) and nonresponsive ( Ah d d ) mice appear to reflect differences in the affinity of the Ah receptor in the two strains for ligands such as TCDD. Administration of a single oral dose of TCDD (30 μg/kg) to Ah b/b mice produced approximately a 30% decrease in the maximum binding capacities of both the hepatic GC and E receptors, as well as a 50-fold induction of a P450IA1-mediated enzymatic activity, ethoxyresorufin- O-deethylase (EROD). Tyrosine aminotransferase (TAT) activity, which is mediated by the GC receptor, was also decreased ∼30% by TCDD. Dose-response curves indicated that Ah responsive mice are 10-fold more sensitive to induction of EROD than Ah nonresponsive mice (ED50 1.6 vs 15 μg/kg), as would be expected for an effect mediated by the Ah receptor. Dose-response curves also indicated that there was a statistical difference in the responsiveness of the hepatic E receptor to TCDD in the two congenic strains of mice ( p < 0.01). Surprisingly, no significant differences in the dose-response curves for the effect of TCDD on hepatic GC receptor binding or TAT activity were observed in the two strains of mice in two separate experiments. These results indicate that the Ah receptor regulates the effects of TCDD on the binding of estrogen to the hepatic estrogen receptor, but suggest that the decrease in the binding capacity of the hepatic GC receptor does not appear to be mediated directly by the Ah locus.

Hepatic glucocorticoid receptor behaves differently when its hormone binding site is occupied by agonist (triamcinolone acetonide) or antagonist (RU486) steroid ligands

We have examined the influence of sulfhydryl (SH)-group modifying agents on the interaction of the rat liver glucocorticoid receptor (GR) with its known agonist triamcinolone acetonide (TA) and the newly synthesized antagonist mifepristone (RU486). In the freshly prepared cytosol, [ 3H]TA or [ 3H]RU486 bound to macromolecule(s) which sediment as 8–9 moieties: the binding of either ligand can be competed with radioinert TA or RU486. The presence of 2–10 mM dithiothreitol (DTT), β-mercaptoethanol (β-MER), and monothioglycerol (MTG) caused a 2–3 fold increase in the [ 3H]TA and [ 3H]RU486 binding to GR. Iodoacetamide (IA) and N-ethylmaleimide (NEM) decreased the agonist binding significantly. In contrast, the [ 3H]RU486 binding to GR increased by 50 percent in the presence of IA. IA and NEM inhibited the binding of the heat-transformed [ 3H]TA-receptor complex to DNA-cellulose by 70–90 percent whereas DNA binding of [ 3H]RU486-bound GR was inhibited only slightly. These results indicate that either a) the interaction of GR with the agonist or antagonist steroid ligands causes differential structural alterations, which are more readily detectable in the presence of SH-modifying agents or b) the agonist and the antagonist interact with distinct steroid binding sites.

A Study on the Circadian Rhythm of Glucocorticoid Receptor

Circadian rhythm in glucocorticoid receptor (GR) was studied in the rat liver and human peripheral leukocytes. For rats exposed to a natural environmental photic cycle or a 12L:12D artificial light regime, peak values of hepatic GR were detected between 23:00 and 02:00 h. Except for a 4-hour advancement of the peak, a similar circadian rhythm of hepatic GR was detected in rats reared under a reversed lighting regimen (12D:12L; lights on between 18:30 and 06:30 h). In human leukocytes, the peak value of GR was found to parallel that of plasma cortisol with high and low values detected at 04:00-08:00 h and 23:00-24:00 h, respectively. In patients suffering from Cushing's syndrome, the circadian rhythm of plasma cortisol either disappeared or was inverted while that of GR did not significantly deviate from the normal subjects. For apoplexic patients with lesions localized to the base of the brain as indicated by computerized tomography, the diurnal variation of GR was abolished. Conversely, diurnal rhythmicity persisted in apoplexy patients whose lesions were in the cerebral cortex. Thus, we postulated that the circadian modification of GR was independent of the diurnal fluctuations in plasma cortisol level or the circadian variations in environmental lighting and that the rhythmicity might be regulated by the 'circadian pacemaker' located in the human basal brain. These diurnal variations in GR might serve to coordinate the reactivity of the target cells to cortisol because the diurnal rhythms of a GR-mediated response, the fractional inhibition of chemotactic migration rate of polymorphonuclear leukocytes by cortisol, were found to be synchronous with those of GR.

Alterations in the binding characteristics of glucocorticoid receptors from obese zucker rats

Obese Zucker rats appear to lack a circadian rhythm of serum corticosterone and maintain relatively high concentrations throughout the 24-h day. The binding characteristics of glucocorticoid receptors in lean and obese Zucker rats were examined in three tissues suggested to be involved in the feedback inhibition of corticosterone: the anterior pituitary, hypothalamus and hippocampus. Hepatic glucocorticoid receptors were also examined to determine if receptor alterations exist in a peripheral tissue. The dissociation constant (Kd) of glucocorticoid receptors in the anterior pituitary of obese rats was 50% greater than the Kd of receptors derived from lean rats. This suggests a decrease in the affinity of these receptors and could indicate a reduced feedback inhibition of corticosterone at the anterior pituitary. Hepatic glucocorticoid receptors of obese rats also showed an increase (150%) in the Kd of binding and a reduction (40%) in the number of receptors. No difference was observed in the Kd or maximal binding of receptors from the hypothalamus or hippocampus of lean and obese rats. It appears that glucocorticoid receptor alterations exist in obese Zucker rats and that these alterations may affect the drive of the pituitary-adrenal axis and possibly the expression of obesity.

Receptor-mediated prednisolone pharmacodynamics in rats: Model verification using a dose-sparing regimen

Our receptor/gene-mediated model of corticosteroid action was tested and extended by examining the pharmacokinetics/dynamics of multiple low doses vs. a single higher dose of intravenously administered prednisolone in adrenalectomized male Wistar rats. Low-dose rats received 3 bolus doses (5 mg/kg) of prednisolone at 0, 0.5 and 1.0 hr. High-dose animals were given a single 25 mg/kg dose of prednisolone. Both regimens were expected to produce equivalent net responses based on model predictions. Control rats were not dosed. The profiles of free hepatic cytosolic glucocorticoid receptors and the hepatic tyrosine aminotransferase (TAT) enzyme were examined. Plasma prednisolone concentrations showed bi-exponential decline for both doses using pooled animal data. Clearance of total plasma prednisolone was 4.16 and 3.21 L/hr per kg in low- and high-dose groups. Volume of distribution at steady state (approximately 1.50 L/kg) and central volume (approximately 0.6 L/kg) were similar for both groups. Receptor levels from 5-16 hr stabilized at 64% of the 0-hr control value. Receptor and TAT profiles were essentially superimposable for both dosing groups. Our previous model was used to simultaneously describe prednisolone plasma concentrations, hepatic receptors, and TAT activity. The ability of total plasma prednisolone (Cp), corticosteroid binding globulin (CBG)-free plasma prednisolone (CCBG), and free plasma prednisolone (CF) to describe the kinetics/dynamics were examined. The CF values produced optimum fitting of all receptor data. The similarity of the two dosing groups supports the view that appropriately timed doses of a steroid can be used in an optimally efficacious manner by first filling all receptor sites and then replacing steroid as receptors are expected to recycle from nuclear/DNA binding sites as the steroid is eliminated.

The effect of exercise, diet restriction, and aging on the pituitary - adrenal axis in the rat

The effects of calorie restriction, exercise, and aging on the pituitary-adrenal axis were studied in male Wistar rats from 12 to 28 months of age. There were 4 experimental groups: sedentary, ad libitum fed (A); sedentary, diet restricted by feeding on alternate days (R); exercised by swimming on alternate days, ad libitum fed (AE); exercised as AE, diet restricted as R (RE). Pituitary-adrenal function was assessed by measuring serum ACTH and corticosterone concentrations, adrenal weight, hepatic glucocorticoid receptor concentration, and hepatic tyrosine aminotransferase (TAT) activity. Serum corticosterone concentration increased with age while serum ACTH decreased from 12 to 20 months of age and then increased thereafter. TAT activity decreased and receptor concentration remained constant with age. Adrenal weights increased with age; those of AE rats increased dramatically. Analyses for relationships between variables revealed a quadratic relationship between serum ACTH and corticosterone concentrations. There tended to be an inverse relationship between TAT activity and corticosterone concentration. These observations may be indicative of a loss of feedback loop integrity with aging. Neither calorie restriction nor exercise were able to maintain the integrity of pituitary-adrenal function during aging, though dietary restriction did slow age-associated decrements of TAT activity.

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius R s ∼ 7.5 nm, expected value for heteromeric complexes containing a dimer of the M r ∼ 90,000 heat shock protein, hsp90 (R s ∼ 7.0 nm). The oxysterol receptor represented a much smaller entity (R s ∼ 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at ∼0.30 M and 0.26–0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11–0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10–0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.

Glucocorticoid receptor regulation: The effects of adrenalectomy, exogenous glucocorticoid, and stress on hepatic receptor number in male and female mice

Although glucocorticoids are known to regulate their own receptor number, the physiologic significance of this process is not known. In order to assess this process in intact animals the effects of adrenalectomy, stress, and exogenous glucocorticoid on the number of hepatic glucocorticoid receptors in Swiss-Webster mice were evaluated. In males 24 hr after adrenalectomy there was a clear 2- to 2.5-fold increase (upregulation) in glucocorticoid receptor number. Conversely, 24 hr after the ip administration of exogenous corticosterone there was a clear downregulation of receptor number. In each case (upregulation and downregulation) female mice were much less responsive than males. Three stressors were used to evaluate the effect of the endogenous secretion of glucocorticoids on downregulation. Male mice were exposed to ether, vibration, and confinement either once or daily for periods up to 3 days. Animals were sacrificed 24 hr after the last stress and hepatic receptor number was compared to an unstressed control. Cytosolic receptor number was not influenced by any of these stimuli. It is concluded that although glucocorticoids clearly regulate glucocorticoid receptor number, as demonstrated by adrenalectomy and the administration of steroids to adrenalectomized animals, the physiologic significance of this process is uncertain as receptor number does not appear to be changed by stimuli of adrenal glucocorticoids in the intact animal.

Decreased ligand binding to the hepatic glucocorticoid and epidermal growth factor receptors after 2,3,4,7,8-pentachlorodibenzofuran and 1,2,3,4,7,8-hexachlorodibenzofuran treatment of pregnant mice

2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) are environmental contaminants which mimic many of the toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Like TCDD, these polychlorinated dibenzofurans (PCDFs) induce hepatic benzo[ a]pyrene hydroxylase activity (BPH) and possess high affinity for the Ah receptor. Another similarity of these PCDFs to TCDD is their ability to induce teratogenic effects such as cleft palate and hydronephrosis in mice. Recent studies have shown that TCDD modifies the equilibrium binding kinetics of the rat liver cytosolic glucocorticoid receptor (GRc) and the hepatic plasma membrane epidermal growth factor (EGF) receptor. To gain a better understanding of the action of halogenated hydrocarbons on these cytosolic and membrane-bound receptor systems during pregnancy, we investigated the biochemical effects of PeCDF and HCDF on the binding kinetics of maternal mouse liver GRc and EGF receptors and the induction of BPH activities. Pregnant C57BL/6N mice were treated once daily on gestation Days 10 through 13 with PeCDF (0–30 μg/kg) or HCDF (0–300 μg/kg). Hepatic [ 3H]dexamethasone and [ 125I]EGF equilibrium binding studies indicated that all doses of PeCDF tested (10, 20, and 30 μg/kg) significantly reduced the GRc and EGF receptor maximum binding capacities but did not affect the binding affinities of these receptors when compared to corn oil-treated control pregnant mice. Similar effects were observed for doses of HCDF ≥ 100 μg/kg. These data suggest that the dibenzofuran-mediated decreases in GRc and EGF receptor binding capacities are similar to those caused by TCDD. Although the mechanism of action is not yet clear, our results indicate that halogenated aromatic compounds in addition to TCDD have profound effects on both steroid and growth factor receptor systems.