Liver glucocorticoid receptor and heat shock protein 70 levels in rats exposed to different stress models

The aim of the present study was to define the stress-induced pattern of cytosolic glucocorticoid receptor (GR) and Hsp70 protein in the liver of male Wistar rats exposed to different stress models: acute (2 h/day) immobilization or cold (4 degrees C); chronic (21 days) isolation, crowding, swimming or isolation plus swimming and combined (chronic plus acute stress). Changes in plasma levels of corticosterone were studied by radioimmunoassay (RIA). The results obtained by Western immunoblotting showed that both acute stressors led to a significant decrease in cytosolic GR and Hsp70 levels. Compared to acute stress effects, only a weak decrease in the levels of GR and Hsp70 was demonstrated in chronic stress models. Chronically stressed rats, which were subsequently exposed to novel acute stressors (immobilization or cold), showed a lower extent of GR down-regulation when compared to acute stress. The exception was swimming, which partially restores this down-regulation. The observed changes in the levels of these major stress-related cellular proteins in liver cytosol lead to the conclusion that chronic stressors compromise intracellular GR down-regulation in the liver.     

Repeated immobilization stress increases total cytosolic glucocorticoid receptor in rat liver

Glucocorticoids are well-known mediators of stress-related endocrine, autonomic, and behavioral responses in mammals and human beings. However, our understanding of the mechanisms of glucocorticoid action in response to stress remains elusive. Therefore, in the present study, an effort has been made to systematically examine glucocorticoid action during acute (2 h) and repeated (2 h daily for 7, 15, and 30 days) immobilization stress in male Sprague-Dawley rats. Prolonged 30-day stress resulted in reduced total body weight gain. There was a dramatic 3- to 4-fold increase in plasma corticosterone levels after single acute stress paradigm, which remained augmented 2- to 3-fold higher than basal control levels during the repeated 30-day immobilization conditions. There was good relationship between increased plasma corticosterone levels and elevation of tyrosine aminotransferase activity in the liver during 30 days of stress. Because repeated immobilization stress animals showed increased levels of both plasma corticosterone and tyrosine aminotransferase activity, the regulation of cytosolic glucocorticoid receptor (GR) in rat liver, a major target tissue for glucocorticoid, was carried out during repeated stress by using GR binding assay, exchange assay, and Western blotting techniques. Exposure of animals to acute and repeated stress resulted in decreased free cytosolic GR. Interestingly, the bound cytosolic GR increased remarkably in liver during prolonged stress of 7-30 days. Overall, results obtained by using both binding assays and Western blotting for the first time showed that repeated stress animals had higher levels of total hepatic cytosolic GR as compared to control animals. These novel results suggest that repeated stress influences the hypothalamic-pituitary-adrenal axis in rats by elevating both the level of plasma corticosterone and total hepatic cytosolic GR.     

Comparison of the biopotency of corticosterone and dexamethasone acetate in glucocorticoid receptor down regulation in rat liver

The effects of long treatment with dexamethasone 21-acetate and corticosterone on the glucocorticoid receptor in rat liver cytosol were compared. Dexamethasone acetate (5 micrograms/ml or 10 micrograms/ml water) or corticosterone (100 micrograms/ml water) was given to adrenalectomized animals as drinking solution for 6 days, and glucocorticoid receptor concentration was determined at 0, 12, 24, 48 and 72 h after steroid withdrawal. Dexamethasone acetate caused a dose dependent depletion of cytosol receptor. There was no measurable binding at time 0; the values of Bmax for the glucocorticoid receptor with decreased at 12, 24 and 48 h after the steroid withdrawal. Increased dissociation constant (Kd) were calculated for 12 and 24 h samples. The effect of corticosterone on receptor depletion was less pronounced. Bmax for the receptor was decreased at 0, 12, 24 h after steroid withdrawal with no change in Kd. The extent of steroids-induced receptor depletion showed good correlation with the induction of tyrosine aminotransferase (TAT), however, maximum TAT activity measured immediately after withdrawal of dexamethasone acetate was lower than that found after a single injection of dexamethasone acetate. We conclude that both steroids cause down regulation of the glucocorticoid receptor in rat liver cytosol, with both the extent and the duration of depletion being dependent on the biopotency of the glucocorticoid.     

Hyperthermic stress modulates the functions of rat liver glucocorticoid receptor

A mild whole body hyperthermic stress causes a rapid and reversible reduction of rat liver glucocorticoid receptor (GR) binding capacity and affects the stability of the GR-DNA complexes formed after thermal transformation of the receptor. These changes appear to be physiologically relevant, since they are accompanied by a decrease in dexamethasone induction of hepatic tyrosine aminotransferase (TAT). In spite of the decreased rate of the GR degradation in liver cytosol of hyperthermic as compared to control rats, the total amount of the GR and its proteolytic products recognized by BuGR2 monoclonal antibody was found to be lower in the former cytosol, but higher in the respective nuclei.     

Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradiography and computerized image analysis

The rat brain contains two receptor systems for corticosterone (CORT): the glucocorticoid (GR) and corticosterone or mineralocorticoid-like (CR) receptor sites. We have studied the localization of these receptors by in vitro autoradiography and by in vitro cytosol binding assays in microdissected brain areas. In vitro autoradiography revealed that CR receptor sites are almost entirely restricted to the septal-hippocampal complex, whereas the presence of GR extends throughout the brain. Highest levels of GR are present in the lateral septum, hippocampal, cortical and thalamic regions and the paraventricular nucleus. In vitro determination of binding of 3H-labelled steroids to CR and GR in cytosol of "punched out" brain tissue revealed a similar neuroanatomical distribution as observed with the autoradiographic analysis. In addition, it was found that CORT binds to CR (KD approximately 0.5 nM) with 5-10-fold higher affinity than to GR (KD approximately 2.5-5 nM).     

Steroid induction of low-affinity glucocorticoid binding sites in rat liver microsomes

Rat liver contains two glucocorticoid binding sites: the high-affinity or glucocorticoid receptor (GR) and the low-affinity glucocorticoid binding sites, or LAGS. The Kd of LAGS predicts that they can be half-saturated by plasma corticosteroids in some physiological circumstances and, therefore, that they can play relevant roles in the rat liver. [3H]dexamethasone was used as a ligand in exchange assays, to study the relative abundance of GR and LAGS in cell fractions of rat liver. GR were found in the cytosol, but not in the purified nuclei, the mitochondria, or the microsomes. LAGS were found in all the particulate fractions, being more abundant in the smooth-surfaced microsomes, but they were not found in the cytosol. The LAGS of microsomes and purified nuclei showed the same Kd and also the same broad range of steroid competition with [3H]dexamethasone (cortisol = progesterone greater than dexamethasone greater than or equal to corticosterone greater than R5020 greater than DHEA greater than testosterone = estradiol). LAGS were found in liver, placenta and kidney, but not in other GR-containing organs. This suggests that the LAGS could be involved in physiological functions related to the metabolism of steroid hormones. The liver microsome LAGS were undetectable at rat birth, and became present in the 25-day-old rat. The level of LAGS then increased progressively, reaching its maximum level in the 2-3-month-old rats (10 pmol/mg protein), and declining afterwards to reach the adulthood level (5 pmol/mg protein) in 6-month-old rats. LAGS are mainly controlled by the corticoadrenal steroids, which is shown by their dramatic decrease after adrenalectomy, and especially after hypophysectomy. Many steroid hormones, like estradiol, testosterone, and corticosterone (but not progesterone) induce LAGS, estradiol being the most effective. A combination of T4 and corticosterone was more effective in inducing LAGS than when the two hormones were injected separately. It is possible to conclude that rat liver LAGS are mainly microsomal proteins, whose concentration is regulated by a multihormone system under pituitary control.     

Interactions of corticosterone, 5alpha-dihydrocorticosterone and dexamethasone with proteins in rat-liver cytosol.

The intracellular binding of [3H]corticosterone and [3H]dexamethasone and their metabolites to macromolecules in rat liver cytosol was studied in vivo and in vitro. The macromolecules binding corticosterone and its metabolites were characterized as (a) a steroid conjugate-binding (Stokes radius 2.5 nm and sedimentation coefficient 4.1 S in high ionic strength; pI 8.7, (b) transcortin and (c) a glucocorticoid "receptor". Competition experiments indicate that corticosterone and dexamethasone bind to the same site of the glucocorticoid receptor molecule. Different Stokes radii between the corticosterone-receptor and the dexamethasone-receptor complexes (6.9 and 6.3 nm, respectively, in high ionic strength) indicate that the two ligands induce different conformations of the receptor protein. This may be of importance when explaining the qualitative differences between the cellular effects of natural and synthetic glucocorticoids. 5alpha-Dihydrocorticosterone, on the other hand, competed to a very limited extent with dexamethasone for binding sites on the receptor. An assay of the inductive effect on liver tyrosine aminotransferase and tryptophan oxygenase indicated that 5alpha-dihydrocorticosterone was practically devoid of glucocorticoid activity. It is concluded that 5alpha-dihydrocorticosterone probably does not act as the mediator of corticosterone action in rat liver.     

Maternal glucocorticoid hormones as a factor mediating the effect of prenatal stress on offspring anxiety

The plus-maze behavior was studied in offsprings of female rats subjected to immobilization stress on the 15-18 days of pregnancy. Prenatal stress decreased the level of anxiety in males and increased in females. The blockade of the mother's stress-induced glucocorticoid secretion by prior adrenalectomy and subsequent corticosterone injection during immobilization in a low dose (0.3 mg/kg) prevented the behavioral disorders in offsprings. In case of a higher dose of corticosterone (3 mg/kg) injection, the behavior of offsprings was the same as that of offsprings of the intact mothers subjected to immobilization. The results suggest that the stress-induced increase in maternal glucocorticoid level may be the mechanism by which prenatal stress impairs the development of sex differences in rat anxiety behavior.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Corticosterone regulates the level of hepatic glucocorticoid receptors in mice

The effect of exogenous corticosterone on the level of mouse hepatic glucocorticoid receptor was monitored to ascertain whether agonist-induced glucocorticoid receptor regulation takes place in living animals as it does in isolated cell systems. Adrenalectomized male Swiss-Webster mice were given 1 mg of corticosterone ip and 24 hr later the glucocorticoid receptor binding capacity of a high-speed cytosolic extract of liver was measured. It was shown that at this time point the administered steroid had been totally cleared and thus, the decrease in binding capacity was a reflection of downregulation. Receptor binding capacity was decreased by 25%. Downregulation was not permanent; 48-72 hr after the injection receptor content returned to baseline. Multiple daily injections of corticosterone were no more effective at causing downregulation than a single injection. It is concluded that glucocorticoid agonists downregulate their own receptors in the glucocorticoid target organs of intact animals as they do in cloned cell models.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70

The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes.     

Differential involvement of amygdala corticosteroid receptors in visceral hyperalgesia following acute or repeated stress

Symptoms of irritable bowel syndrome (IBS) are exacerbated by stress. Previously, we demonstrated that the stress hormone corticosterone applied directly to the amygdala induced visceral hypersensitivity through the actions of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). However, the involvement of amygdaloid GR and MR in the regulation of visceral sensitivity following psychological stress is unknown; therefore, the goal of the present study was to determine the relative importance of amygdaloid GR and MR in the regulation of visceral sensitivity in a rodent model of behavioral stress. Male F-344 rats were stereotaxically implanted with micropellets bilaterally on the dorsal margin of the amygdala containing the GR antagonist mifepristone, the MR antagonist spironolactone, or cholesterol as a control. Animals were then exposed to 1 h of water-avoidance stress (WAS) or sham stress for 1 day (acute) or 7 days (repeated). Visceral sensitivity was assessed either 1 h or 24 h after the final session of WAS and quantified as the number of contractions of the external abdominal oblique, a visceromotor response, in response to colorectal distension at pressures of 0-60 mmHg. Acute stress induced transient visceral hyperalgesia, which was absent 24 h after WAS and independent of GR and MR. Conversely, repeated WAS induced sustained visceral hyperalgesia that was abolished by specifically targeting the amygdala with GR and MR antagonists. These results demonstrate that the amygdala corticosteroid system plays an essential role in mediating the effects of repeated WAS on visceral sensitivity. Furthermore, our findings suggest that amygdaloid GR and MR may be involved in IBS symptomatology.     

Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes

Dexamethasone-treated L6 (a rat cell line) and C2C12 (a mouse cell line) myotubes are frequently used as in vitro models of muscle wasting. We compared the effects of different concentrations of dexamethasone and corticosterone (the naturally occurring glucocorticoid in rodents) on protein breakdown rates, myotube size, and atrogin-1 and MuRF1 mRNA levels in the two cell lines. In addition, the expression of the glucocorticoid receptor (GR) and its role in glucocorticoid-induced metabolic changes were determined. Treatment with dexamethasone or corticosterone resulted in dose-dependent increases in protein degradation rates in both L6 and C2C12 myotubes accompanied by 25-30% reduction of myotube diameter. The same treatments increased atrogin-1 mRNA levels in L6 and C2C12 myotubes but, surprisingly, upregulated the expression of MuRF1 in L6 myotubes only. Both cell types expressed the GR and treatment with dexamethasone or corticosterone downregulated total cellular GR levels while increasing nuclear translocation of the GR in both L6 and C2C12 myotubes. The GR antagonist RU38486 inhibited the dexamethasone- and corticosterone-induced increases in atrogin-1 and MuRF1 expression in L6 myotubes but not in C2C12 myotubes. Interestingly, RU38486 exerted agonist effects in the C2C12, but not in the L6 myotubes. The present results suggest that muscle wasting-related responses to dexamethasone and corticosterone are similar, but not identical, in L6 and C2C12 myotubes. Most notably, the regulation by glucocorticoids of MuRF1 and the role of the GR may be different in the two cell lines. These differences need to be taken into account when cultured myotubes are used in future studies to further explore mechanisms of muscle wasting.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

Levels and subcellular distribution of endogenous corticosterone in rat liver during development

The concentration of corticosterone in liver homogenates, liver cytosol and purified nuclear fractions, and in plasma of fetal, newborn, immature and adult rats has been measured by radioimmunoassay.Highest plasma corticosterone levels were found in fetal rats, decreasing close to the levels observed in the adrenalectomized rat by the 6th day of postnatal life followed by a rise in the adult rat. The concentration of corticosterone in liver during development paralleled the plasma levels, the liver to plasma corticosterone ratio ranging between 0.09 and 0.17 suggesting that the corticosterone retained by the tissue is related to the unbound fraction of the hormone in plasma.Both plasma and tissue corticosterone levels declined after adrenalectomy and they were elevated after ether stress.Fractionation of liver homogenates showed that the major fraction of liver corticosterone is localized in the cytosol. Purified liver nuclei contained between 9 and 16% of the total liver corticosterone. The amount of corticosterone in the nuclei seems to be related to the plasma and tissue hormone levels rather than the concentration of cytoplasmic glucocorticoid receptors. Since most of the nuclear corticosterone appears to be bound to receptors, it has been calculated that close to 60% of the cellular receptors in fetal liver are localized in the nucleus. In adult rat liver, only about 10% of the cellular receptors appear to be associated with nuclei. Changes in the concentration of glucocorticoid receptors in liver during development and after adrenalectomy are inversely related to changes in plasma corticosterone levels. It is suggested that corticosterone may regulate the levels of its own receptors in liver.     

The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test

Pre-clinical and clinical studies have employed treatment with glucocorticoid receptor (GR) antagonists in an attempt to limit the deleterious behavioral and physiological effects of excess glucocorticoids. Here, we examined the effects of GR antagonists on neuroendocrine and behavioral stress responses, using two compounds: mifepristone, a GR antagonist that is also a progesterone receptor antagonist, and CORT 108297, a specific GR antagonist lacking anti-progestin activity. Given its well-documented impact on neuroendocrine and behavioral stress responses, imipramine (tricyclic antidepressant) served as a positive control. Male rats were treated for five days with mifepristone (10 mg/kg), CORT 108297 (30 mg/kg and 60 mg/kg), imipramine (10 mg/kg) or vehicle and exposed to forced swim test (FST) or restraint stress. Relative to vehicle, imipramine potently suppressed adrenocorticotropin hormone (ACTH) responses to FST and restraint exposure. Imipramine also decreased immobility in the FST, consistent with antidepressant actions. Both doses of CORT 108297 potently suppressed peak corticosterone responses to FST and restraint stress. However, only the higher dose of CORT 108297 (60 mg/kg) significantly decreased immobility in the FST. In contrast, mifepristone induced protracted secretion of corticosterone in response to both stressors, and modestly decreased immobility in the FST. Taken together, the data indicate distinct effects of each compound on neuroendocrine stress responses and also highlight dissociation between corticosterone responses and immobility in the FST. Within the context of the present study, our data suggest that CORT 108297 may be an attractive alternative for mitigating neuroendocrine and behavioral states associated with excess glucocorticoid secretion.     

Evidence of a gene-dosage effect for the glucocorticoid receptor in trisomy 18 mice

The amount of cytosolic glucocorticoid receptor in liver of Ts18, Ts16, and Ts19 vs euploid mouse fetuses was studied after incubation of [3H]dexamethasone with cytosol followed by isoelectric focusing on polyacrylamide gels. In addition, corticosterone concentrations and enzyme activities of alanine aminotransferase and tyrosine aminotransferase were measured in the cytosol of the livers. The amount of glucocorticoid receptor in the cytosol fractions of the livers was always higher in the Ts18 than in the euploid fetuses of the same litter. It was also significantly (P less than 0.0005) higher if pooled data from different litters were analyzed. The ratio of the glucocorticoid receptor in Ts18 vs euploid mice varied between 1.3 and 4.7, with a mean of 2.1. In contrast, the glucocorticoid receptor levels in Ts16 and Ts19 fetuses were not different from the corresponding euploid controls. Comparing the corticosterone levels of the three trisomies tested with the corresponding euploid fetuses, no significant differences were found, indicating that the markedly elevated cytosolic glucocorticoid receptor concentrations in Ts18 were not due to different corticosterone levels. This finding is consistent with the assignment of the glucocorticoid receptor gene to chromosome 18 in the mouse. There was no correlation between glucocorticoid receptor levels and the activity of the two glucocorticoid inducible enzymes tested in the liver of mouse fetuses.