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 (Kd) remain unaltered at both ages. Heat activation (for 45 min at 25 degrees 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. Ca2+ activation (20 mM Ca2+ for 45 min at 0 degree 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 physiochemical properties of glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.     

Decreased liver cytosol glucocorticoid receptors in protein-deficient rats

A low protein diet (5% as compared to a control 21% protein diet, $\text{ad libitum}$) caused a significant decrease in the concentration of liver cytoplasmic glucocorticoid receptors; the equilibrium dissociation constant (Kd) did not change. The maximum decrease occurred in two weeks and was reversible upon substitution of the low protein by a control diet. This influence of protein deficiency could not be attributed to elevated plasma corticosterone levels since a comparable increase in plasma corticosterone of calorie-deficient rats (21% protein diet in restricted quantity) did not decrease glucocorticoid receptors and the difference in receptor levels of control and protein deficient animals persisted following adrenalectomy. These results suggest that glucocorticoids might not exert their usual biologic effects in the presence of protein malnutrition.     

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

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. The concentration of specific binding sites was significantly higher (25%) in the kidney of immature rats as compared with mature, while dissociation constants (Kd) remain unaltered at both ages. 3. Heat activation (25 degrees 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. Ca2+ activation (0 degree C for 45 min with 20 mM Ca2+) also enhanced the nuclear and DNA-cellulose binding at both ages but to a greater magnitude in immature rats. 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.     

Changes in glucocorticoid receptors in different regions of brain of immature and mature male rats

Specific cytosolic binding for synthetic glucocorticoid dexamethasone was studied in several brain regions (hypothalamus, hippocampus, caudate nucleus, cerebellum, cerebral cortex) of immature (3-week) and mature (26-week) male rats, intact and adrenalectomized. A significant regional difference was observed in the concentration of in vitro [3H] dexamethasone binding in the brain of adrenalectomized rats at both ages, with the highest levels in the hippocampus. A marked decrease in specific binding was observed in all brain regions of adrenalectomized mature rats as compared to immature. The dexamethasone binding was significantly lower in all brain regions of normal intact animals as compared to adrenalectomized rats in both ages.     

Rapid spinogenesis of pyramidal neurons induced by activation of glucocorticoid receptors in adult male rat hippocampus

Modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. Dendritic spines are essential for memory storage processes. Here, we investigated the effect of dexamethasone (DEX), a specific agonist of glucocorticoid receptor (GR), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of Lucifer Yellow-injected spines in slices. The application of 100 nM DEX (stressful high concentration) induced rapid modulation of the density and morphology of dendritic spines in CA1 pyramidal neurons within 1h. The total spine density increased from 0.88 spines/microm (control) to 1.36 spines/microm (DEX-treated). DEX significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. Because the presence of 10 microM cycloheximide, an inhibitor of protein synthesis, did not suppress the DEX effect, these responses are probably non-genomic. Western immunoblot analysis demonstrated the localization of classical type GR in Triton-insoluble synaptosomal fractions (enriched in postsynaptic membranes) from hippocampal slices, suggesting a possible action site of DEX at spines.     

Ontogeny of glucocorticoid receptors in rat liver.

Specific receptors for glucocorticoids are present in liver cytosols of rat fetuses at least as early as the 18th day of gestation. The concentration of the receptor begins to decline after the 20th day reaching undetectable levels shortly before and after parturition. The receptor can be detected again 1 to 2 hours after birth, and its levels increase markedly to higher than adult values between the 2nd and 5th day. The reason for the failure to detect specific hormone binding near parturition appears to be due to occupation of binding sites by endogenous steroids rather than the absence of the receptor. This is indicated by the demonstration of both cytoplasmic and nuclear receptor sites in liver slices of newborn rats incubated with labeled dexamethasone at 37 degrees. The cytoplasmic receptors of fetal and adult liver differ in their relative affinity for cortisol and corticosterone. The fetal receptors have a higher affinity for corticosterone than cortisol while the reverse is true for the adult receptors. These observations suggest either the existence of dissimilar receptors in fetal and adult liver or the presence of more than one type of receptor sites. It is therefore possible that subtle differences in the nature of hepatic receptors may be partly responsible for the maturation-dependent qualitative differences in tissue responsiveness to glucocorticoids.     

ATP-induced activation of purified rat hepatic glucocorticoid receptors

We have utilized unactivated rat hepatic glucocorticoid receptor complexes purified to near homogeneity by a three-step scheme which includes affinity chromatography, gel filtration and anion exchange chromatography, to demonstrate for the first time that ATP can interact directly with the receptor protein in stimulating activation. This stimulation is reflected by an increase in DNA-cellulose binding as well as by a shift in the elution profile of the purified receptor complexes from DEAE-cellulose. A concentration of 10 mM Na2MoO4 is able to block both of these effects. ATP stimulates activation in a dose-dependent manner (maximally at 10 mM), and elicits maximal activation within 30 min at 15 degrees C. There appears to be no nucleotide specificity since GTP, CTP and UTP, as well as ADP and GDP also stimulate activation. All of these observations closely parallel data obtained from similar activation experiments performed with crude rat hepatic receptors. ATP does not appear to stimulate activation of receptors (crude or purified) by initiating a phosphorylation reaction since hydrolysis-resistant analogues of ATP are also effective. Pyrophosphate (PPi) is as effective as ATP in promoting receptor activation, since it elicits similar increases in DNA-cellulose binding, shifts in elution patterns from DEAE-cellulose, and dose-response relationships. None of the compounds tested stimulate activation indirectly by pH or ionic strength effects. Despite the fact that high ATP concentrations (3-4-fold higher than those present in vivo) are necessary to stimulate maximal activation, a physiological role of ATP in directly regulating in vivo activation of glucocorticoid receptors cannot be ruled out.     

Age-dependent loss of dimeric immunoglobulin A receptors in the liver of the Fischer 344 rat

Characterization of the receptor for dimeric immunoglobulin A (dIgA) on an isolated rat liver plasma membrane-enriched fraction showed a single class of binding sites specific for dIgA. Both the rat and human immunoglobulins competed for the binding site, but human IgA bound much less strongly to the receptor. Other proteins did not compete for the dIgA receptor, including asialoorosomucoid. Scatchard analysis of binding data from young adult, mature, and senescent animals indicated an age-dependent decrease in the number of receptors present on liver plasma membranes. This loss of receptors with increasing animal age may be responsible for the concomitant reduction in hepatobiliary secretion of dIgA.     

Comparative characteristics of cytosol glucocorticoid receptors from normal liver, the liver of tumor-bearing rats and hormone unresponsive Zajdela hepatoma]

Specific dexametasone (D) and cortisol (F) receptors have been found both in liver and Zajdela hepatoma. Rat liver cytosol receptors are characterized by the association constant (Kas) = 3,8 X 10(8) M-1 for D and 0,57 X 10(8) M-1 for F as well as by a number of binding sites (NBS)=4,9 X 10(-13) moles/mg protein and 4,06 X 10(-13) moles/mg protein, respectively. The receptors show stric specificity to glucocorticoids. Cytosol glucocorticoid-receptor complexes from liver and hepatoma sediment at 6-7S, when centrifuged in the buffer of a low ionic strength, and at 3-4S in the buffer of a high ionic strength (0,4 M KCl). The properties of cytosol receptors in the course of in vivo hepatoma growth were found to be gradually altering: Kas for D dropped whereas that for F increased; the NBS is decreased 3-4 fold as compared to normal liver cytosol--which may partially be accounted for by the unresponsiveness of the tumour to the hormones.     

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.     

Identification and characterization of glucocorticoid receptors in liver of nude mice

Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone down-regulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and -glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction.     

Glucocorticoid negative feedback and glucocorticoid receptors after hippocampectomy in rats

In rats with dorsal hippocampectomy, glucocorticoid receptors in the hypothalamus and anterior pituitary, as well as the pituitary transcortin-like compound, are preserved, in spite of a 60% depletion of glucocorticoid receptors in the hippocampus. In the hippocampectomized group, basal levels of serum corticosterone (CORT) were increased, although there was a normal response to ether stress. Inhibition of the response to ether with dexamethasone (DEX) was dose-dependent: whereas 100 micrograms/kg completely suppressed serum CORT, 10 micrograms/kg were ineffective. However, we observed a reduced sensitivity to DEX inhibition with 25 micrograms/kg in hippocampectomized animals. These results indicate that the hippocampus is involved in negative feedback mechanisms, although different doses of DEX are needed for this demonstration. The inhibition of serum CORT due to 100 micrograms/kg DEX suggests that negative feedback at sites other than the hippocampus was still operative, in agreement with normal levels of glucocorticoid receptors in the anterior pituitary and hypothalamus of hippocampectomized rats.     

Aggravation of nonsteroidal antiinflammatory drug gastropathy by glucocorticoid deficiency or blockade of glucocorticoid receptors in rats

Our previous investigations suggest that the reduction of stress-induced corticosterone release, or inhibition of corticosterone actions, promotes stress-induced gastric erosions in rats. In this study the effect of glucocorticoid deficiency on susceptibility to gastric mucosal injury by nonsteroidal antiinflammatory drugs (NSAIDs) was evaluated in rats. Gastric erosions induced in male rats by indomethacin (25 mg/kg sc) or acidified aspirin (40 mM po) were studied one week after adrenalectomy with or without corticosterone replacement or after occupation of glucocorticoid receptors by the antagonist RU-38486 during the period of erosion formation. Corticosterone for replacement (4 mg/kg sc) was injected 15 min before the administration of indomethacin or acidified aspirin to adrenalectomized rats. The antagonist RU-38486 (10 mg/kg po) was administered twice, 20 min before and 60 min after NSAID administration. Plasma corticosterone levels were measured by fluorometry. Gastric erosions were quantitated by measuring the area of damage. Indomethacin or acidified aspirin induced both plasma corticosterone rise and gastric erosions. Adrenalectomy decreased both basal and NSAID-induced corticosterone levels and markedly promoted gastric erosion formation caused by the NSAID. An acute corticosterone replacement mimicking indomethacin-and aspirin-induced corticosterone rise prevented the effect of adrenalectomy on the gastric erosions. The administration of the glucocorticoid/progesterone antagonist RU-38486 significantly potentiated the formation of gastric erosions induced by indomethacin as well as aspirin. These observations suggest a gastroprotective action of glucocorticoids released in response to NSAID treatment against NSAID-induced injury.     

Tissue-specific programming expression of glucocorticoid receptors and 11 beta-HSDs by maternal perinatal undernutrition in the HPA axis of adult male rats.

Maternal undernutrition leads to intrauterine growth retardation and predisposes to the development of pathologies in adulthood. The hypothalamo-pituitary-adrenal axis is a major target of early-life programming. We showed previously that perinatal maternal 50% food restriction leads to hypothalamo-pituitary-adrenal axis hyperactivity and disturbs glucocorticoid feedback in adult male rats. To try to better understand these alterations, we studied several factors involved in corticosterone sensitivity. We showed that unlike the restricted expression of 11 beta-HSD2 mRNA, the 11 beta-HSD1, glucocorticoid, and mineralocorticoid receptor genes are widely distributed in rat. In contrast to the hypothalamus, we confirmed that maternal undernutrition modulates hippocampal corticosterone receptor balance and leads to increased 11 beta-HSD1 gene expression. In the pituitary, rats exhibited a huge increase in both mRNA and mineralocorticoid receptor binding capacities as well as decreased 11 beta-HSD1/11 beta-HSD2 gene expression. Using IN SITU hybridization, we showed that the mineralocorticoid receptor gene was expressed in rat corticotroph cells and by other adenopituitary cells. In the adrenal gland, maternal food restriction decreased 11beta-HSD2 mRNA. This study demonstrated that maternal food restriction has both long-term and tissue-specific effects on gene expression of factors involved in glucocorticoid sensitivity and that it could contribute, via glucocorticoid excess, to the development of adult diseases.     

ATP-dependent activation of L cell glucocorticoid receptors to the steroid binding form.

The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.     

ATP-dependent activation of glucocorticoid receptor from rat liver cytosol.

The glucocorticoid--receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5--10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an 'activated' transformed form, which readily bound to nuclei, ATP--Sepharose, phosphocellulose and DNA--cellulose. This transformation was comparable with the activation achieved by warming the steroid--receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid--receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.     

Receptor recycling: liberation of glucocorticoid receptors from liver nuclei in vitro

Dynamics of the steroid receptors seems to be the consequence of receptor recycling. In the present study, as a clue to elucidate the mechanism of receptor recycling, factors which affect the rate of liberation of nuclear bound 3H-glucocorticoids were examined in vitro. Among the factors examined, NAD, NADPH, cAMP and p-nitrophenyl phosphate accelerated the liberation of radioactivity from nuclei in a temperature-dependent manner when added to the incubation mixture. The presence of a large amount of unlabeled dexamethasone (Dex) did not modify the rate of liberation. From these results, it was concluded that the metabolism of ligand bound to the receptor is not a necessary step in the liberation of receptor from nuclei. These agents did not influence the binding process of 3H-Dex-receptor complex to DNA-cellulose. Therefore the stimulation of receptor release does not seem to be mediated by reducing the binding affinity between nuclei and receptor complexes. The liberated radioactivity was eluted on a Sephadex G-100 column in the void volume and in macromolecule-unbound fractions. In both fractions, the majority of the radioactivity comigrated with authentic glucocorticoids on thin-layer chromatography.     

Age-dependent increase of etheno-DNA-adducts in liver and brain of ROS overproducing OXYS rats

Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) by immunoaffinity/32P-postlabelling. While epsilondA and epsilondC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for epsilondA by immunostaining of nuclei. Brains from old OXYS rats accumulated epsilondA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.