Effects of adrenal steroid agonists on food intake and macronutrient selection.

These experiments tested the effects of subcutaneous (SC) and paraventricular nucleus (PVN) administration of the steroid receptor agonists, corticosterone (CORT), aldosterone (ALDO), RU28362, and dexamethasone (DEX), on food intake and macronutrient selection during the first h of the dark feeding period in the rat. Results indicate that CORT and the selective type II receptor agonist RU28362 specifically stimulate carbohydrate ingestion after SC or PVN administration, while DEX has no effect on feeding. This selective effect of SC CORT on carbohydrate ingestion is dose dependent, seen at doses ranging from 0.125 to 2.0 mg/kg. Moreover, the stimulatory effects of CORT and RU28362 on carbohydrate intake are observed in ADX rats but not in sham rats. This is in contrast to SC and PVN administration of the type I receptor agonist ALDO, which specifically enhances fat ingestion in both sham and ADX rats. These results, with both peripheral and central steroid administration, reveal selective effects of type I and type II receptor stimulation on fat and carbohydrate intake, respectively.     

Colocalization of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and hypothalamus

We investigated the distribution and colocalization pattern of the two corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the hippocampus and hypothalamus, the main target regions of corticosterone in the rat brain, using double immunofluorescence histochemistry in conjunction with specific polyclonal antibodies against MR and GR. In the hippocampus, MR- and GR-immunoreactivity (ir) were colocalized in CA1 and CA2 pyramidal neurons and granule cells of the dentate gyrus, while only MR-ir was seen in the CA3 pyramidal neurons. Colocalization of MR- and GR-ir was also observed in the parvocellular region, but not in the magnocellular region of the paraventricular nucleus (PVN). Subcellular distribution of MR-ir was more cytoplasmic in comparison with that of GR-ir, while the ratio of cytoplasmic to nuclear distribution of these receptors was different among the regions. After adrenalectomy (ADX), the distribution pattern of both receptors was changed to cytoplasmic, although the degree of the change of distribution was different among each region. Replacement of corticosterone after ADX recovered the distribution pattern to that of the sham-operated animals. These results suggest that the balance of MR and GR in the cell underlies the potential regulation of corticosteroid through the hippocampus and hypothalamus.     

Acute differential regulation by corticosteroids of epithelial sodium channel subunit and Nedd4 mRNA levels in the distal colon

The molecular mechanisms by which corticosteroids affect fluid and electrolyte balance have yet to be fully elucidated. The apical amiloride-sensitive electrogenic epithelial sodium channel (ENaC) has been shown to have a central role in corticosteroid-mediated sodium transport in the distal colon. The acute response of the alpha-, beta- and gammaENaC subunit genes to a single parenteral dose of aldosterone or dexamethasone was examined in the rat distal colon in vivo. The response of the Nedd4 gene, whose product is involved in channel turnover, was also examined. Whilst the alphaENaC and Nedd4 genes showed no significant response to either steroid, both the beta- and gammaENaC mRNA levels were increased acutely by both aldosterone and dexamethasone. The gammaENaC mRNA appears to have a very short half-life. Use of the highly selective glucocorticoid receptor agonist RU28362 confirmed that the response was mediated by both the mineralocorticoid and glucocorticoid receptors.     

Characterization of human trophoblast as a mineralocorticoid target tissue

In mineralocorticoid target tissues, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) confers mineralocorticoid receptor selectivity by metabolizing hormonally active cortisol to inactive cortisone, allowing aldosterone access to the receptor. This enzyme is also expressed in high abundance in fetal tissues, particularly in placental trophoblast, where a role has been proposed in regulating fetal growth and development by protecting the fetus from maternal hypercortisolaemia and modulating local glucocorticoid receptor (GR), rather than mineralocorticoid receptor-mediated responses. As such the placenta has not been considered a mineralocorticoid target tissue. We have used conventional RT-PCR and real-time quantitative RT-PCR to demonstrate that primary cultures of term human cytotrophoblast express the mineralocorticoid-responsive genes Na/K-ATPase (alpha1 and beta1 subunits), epithelial sodium channel (ENaC, alpha and gamma subunits) and the serum and glucocorticoid-inducible kinase (SGK). SGK expression was found to be rapidly and strongly induced by corticosteroids (24- and 38-fold by 10(-7) mol/l aldosterone and 10(-7) mol/l dexamethasone respectively after 1 h). Dexamethasone-, but not aldosterone-stimulated SGK induction was inhibited by GR antagonist (RU38486), confirming the presence of a functional mineralocorticoid receptor and suggesting that placental trophoblast expresses a functional mineralocorticoid receptor, which is in part responsible for the corticosteroid regulation of SGK expression. Placental 11beta-HSD2 may protect the MR in a fashion analogous to classical mineralocorticoid tissues to modulate trophoblast sodium transport.     

Acute, chronic, and interactive effects of type I and II corticosteroid receptor stimulation on feeding and weight gain

Type I (aldosterone) and/or type II (dexamethasone or RU28362) corticosterone receptor agonists were continuously infused in adrenalectomized Sprague-Dawley rats for 28 days at doses of 3.4, 17.2, or 86.2 nmol/day. Additional groups received combined agonist infusions, blank infusions, or sham operations. The type I agonist stimulated body weight gain, and the type II agonists were both suppressive, differing mainly in degree. Although there were a few early effects of these hormones (usually a stage of exaggerated activity), once passed, chronic stimulation was marked by steady or slightly increasing steroid influence on body weight. Throughout the chronic phase of this study there was no departure from a simple opponent model of type I and II ligand actions, and their combination approximated an arithmetic summation of the two separate agonists. This was generally true of feeding as well, although steroid effects on intake were always less pronounced. In contrast to chronic administration, acute combinations of these agonists were highly interactive, producing slight losses than large gains for the aldosterone and RU28362 combinations, but a large gain then small loss for the aldosterone and dexamethasone combination. These results imply that RU28362 and dexamethasone differ in more respects than potency. Because normal endogenous type II stimulation is acute and occurs against a background of type I receptor occupation, mixed agonist interactions are probably the rule for everyday physiological activity, not the exception.     

Development of specific antibody andin vivo response to antigen in different rat strains: Effect of dexamethasone and importance of endogenous corticosteroids

Endogenous glucocorticoids undoubtedly play a role in the control of immune responses: their contribution to inter-strain variation is unknown. The development of specific IgG and IgE was measured following inoculation with ovalbumin in Lewis, Fischer, Wistar and Brown Norway rats. The Lewis gives a smaller IgG and IgE response than the other strains and the responsein vivo to antigen injected into the paw correlates with the titre of specific antibody. Treatment with the steroid receptor antagonist RU486 (mifepristone) following inoculation reveals that in the Lewis, and to a lesser extent in the Brown Norway, the development of a specific IgG response is limited by endogenous corticosteroids. The IgG response in different strains is differently sensitive to treatment with the synthetic glucocorticoid dexamethasone, the Lewis being particularly resistant. The importance of control by endogenous corticosteroids should not be overlooked in contributing to strain differences in immune response.     

Induction of an appetite for sodium in rats that show no spontaneous preference for sodium chloride solution--the Fischer 344 strain

Fischer 344 rats show no spontaneous preference for isotonic sodium chloride (NaCl) solution. These experiments indicate, however, that a strong appetite for this solution may be induced by various methods, including adrenalectomy, administration of a mineralocorticoid hormone, acute depletion of sodium, and treatment with inhibitors of the angiotensin I converting enzyme (ACE). These treatments were also shown to produce the expected changes in the renin-angiotensin-aldosterone system, which thus appears to be involved in the induction of an appetite for NaCl solution in this strain of rat. The intakes of NaCl induced in the Fischer 344 rats by these experimental paradigms are less than those that have been reported in either Sprague-Dawley or Wistar strains in similar paradigms. In the case of sodium depletion, the intake of NaCl solution by Fischer 344 rats appears to be more closely related to the deficit than in the other two strains. Thus, the Fischer 344 strain of rats may be a particularly good model for studies of need-related sodium appetite.     

Angiotensin II and fluid ingestion in old rats

Fluid ingestion was studied in Fischer 344/Brown Norway F1 rats aged 3, 12, 20, and 24 months of age. There was an age-related decrease in fluid ingestion when fluid intake was measured over 24 h. After water deprivation, 24- and 20-month-old rats drank less than 3- and 12-month-old rats. Twelve, 20-, and 24-month-old rats had less fluid intake associated with food deprivation than did 3-month-old rats. Three month old rats drank more fluid after angiotensin II than did 12-, 20-, and 24-month-old rats when expressed as fluid intake per kg body weight. These studies confirm that the rat is a reasonable model to study age-related hypodipsia.     

Involvement of corticosterone in cardiovascular responses to an open-field novelty stressor in freely moving rats

The aim of the present study was to investigate the modulatory action of different concentrations of circulating corticosterone occupying either predominantly mineralocorticoid receptors (MR) or both MR and glucocorticoid receptors (GR) in control of cardiovascular responses to a novelty stressor. Six groups of rats were instrumented with radiotelemetry transmitters: sham-operated controls, adrenalectomised (ADX) controls, ADX with chronic implantation of a 20-mg corticosterone pellet, ADX with chronic implantation of a 100-mg corticosterone pellet, ADX receiving acute bolus injection of 0.25 mg/kg of corticosterone, and ADX with both implantation of a 20-mg corticosterone pellet and bolus treatment. Exposure to the novelty of an open field caused an increase in blood pressure, heart rate, body temperature and exploratory locomotor activity. The pressor response was dose-dependently increased in ADX rats implanted with a corticosterone pellet. Bolus injection of corticosterone at 10 min prior to novelty had no effect. The tachycardia was reduced in ADX rats compared to sham-operated controls, and this effect was restored by implantation of a 20-mg, but not 100-mg, corticosterone pellet. Bolus injection of corticosterone facilitated the return of heart rate towards baseline levels. The increase in body temperature was reduced in ADX rats, a deficit that was normalised by implantation of either corticosterone dose but not by acute bolus treatment. Locomotor activity was not different between the groups except for a slightly more rapid decline of locomotor activity in both groups treated with a bolus injection of corticosterone. These data show an important role of putative brain MR in maintaining adequate cardiovascular and behavioural responsiveness to a mild psychological stressor, while additional acute or chronic occupation of GR has further differential and sometimes opposing effects.     

Opposing roles of glucocorticoid receptor and mineralocorticoid receptor in trimethyltin-induced cytotoxicity in the mouse hippocampus

The organotin trimethyltin (TMT) is known to cause neuronal degeneration in the murine brain. Earlier studies indicate that TMT-induced neuronal degeneration is enhanced by adrenalectomy and prevented by exogenous glucocorticoid. The aim of this study was to investigate the regulation of TMT neuroxicity by corticosterone receptors including type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in adult mice. The systemic injection of TMT at the dose of 2.0 or 2.8 mg/kg produced a marked elevation in the level of plasma corticosterone that was both dose and time dependent. The MR agonist aldosterone had the ability to exacerbate TMT cytotoxicity in the dentate granule cell layer, whereas its antagonist spironolactone protected neurons from TMT cytotoxicity there. In contrast, the GR antagonist mifepristone exacerbated the TMT cytotoxicity. Taken together, our data suggest TMT cytotoxicity is oppositely regulated by GR and MR signals, being exacerbated by MR activation in adult mice.     

Limited brain diffusion of the glucocorticoid receptor agonist RU28362 following i.c.v. administration: implications for i.c.v. drug delivery and glucocorticoid negative feedback in the hypothalamic-pituitary-adrenal axis

The experiments described herein present a method for tracking diffusion of the glucocorticoid receptor agonist RU28362 in brain following i.c.v. drug administration. A useful property of glucocorticoid receptor is that it is primarily cytoplasmic when unbound and rapidly translocates to the nucleus when bound by ligand. Thus, removal of endogenous glucocorticoids by adrenalectomy allows us to identify brain regions with activated glucocorticoid receptor after i.c.v. glucocorticoid receptor agonist treatment by examining the presence or absence of nuclear glucocorticoid receptor immunostaining. We have previously demonstrated that an i.p. injection of 150 microg/kg RU28362 1 h prior to restraint stress is sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis hormone secretion [Ginsberg AB, Campeau S, Day HE, Spencer RL (2003) Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus. J Neuroendocrinol 15:1075-1083]. We report here, however, that in rats i.c.v. treatment with a high-dose of RU28362 (1 microg) 1 h prior to stressor onset does not suppress stress-induced hypothalamic-pituitary-adrenal axis activity. We then performed a series of experiments to examine the possible differences in glucocorticoid receptor activation patterns in brain and pituitary after i.c.v. or i.p. treatment with RU28362. In a dose-response study we found that 1 h after i.c.v. injection of RU28362 (0.001, 0.1 and 1.0 microg) glucocorticoid receptor nuclear immunoreactivity was only evident in brain tissue immediately adjacent to the lateral or third ventricle, including the medial but not more lateral portion of the medial parvocellular paraventricular nucleus of the hypothalamus. In contrast, i.p. injection of RU28362 produced a uniform predominantly nuclear glucocorticoid receptor immunostaining pattern throughout all brain tissue. I.c.v. injection of the endogenous glucocorticoid receptor agonist, corticosterone (1 microg) also had limited diffusion into brain tissue. Time-course studies indicated that there was not a greater extent of nuclear glucocorticoid receptor immunostaining present in brain after shorter (10 or 30 min) or longer (2 or 3 h) intervals of time after i.c.v. RU28362 injection. Importantly, time-course studies found that i.c.v. RU28362 produced significant increases in nuclear glucocorticoid receptor immunostaining in the anterior pituitary that were evident within 10 min after injection and maximal after 1 h. These studies support an extensive literature indicating that drugs have very limited ability to diffuse out of the ventricles into brain tissue after i.c.v. injection, while at the same time reaching peripheral tissue sites. In addition, these studies indicate that significant occupancy of some glucocorticoid receptor within the paraventricular nucleus of the hypothalamus and pituitary is not necessarily sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis activity.     

Age-related changes in glucocorticoid receptor binding and mRNA levels in the rat brain and pituitary.

Hypothalamic-pituitary-adrenal (HPA) activity under both basal and poststress conditions is often increased in the aged rat. This change has been associated with the loss of corticosteroid receptors in specific regions that mediate glucocorticoid negative feedback. In order to study the cellular basis for the loss of receptors, we measured glucocorticoid (type II corticosteroid) receptor binding and mRNA levels in pituitary and selected brain regions in rats at 6, 12, and 24 months of age. Receptor binding, measured using [3H]RU 28362, was stable in all regions (pituitary, hypothalamus, amygdala, and frontal cortex) except the hippocampus, where there was about a 40% decrease in binding capacity, with no change in the affinity of the receptor for RU 28362. The loss of receptors in the hippocampus was apparent in animals at 12 months of age, and binding was further decreased at 24 months. Glucocorticoid receptor mRNA levels were significantly higher in all regions at 12 months of age than at 6 months. By 24 months, however, receptor mRNA levels in most regions had returned to levels that were similar to those at 6 months of age. In contrast, glucocorticoid receptor mRNA levels in the hippocampus were significantly decreased at 24 months of age compared to levels at both 6 and 12 months of age. Thus, in general, variations in receptor mRNA levels parallel those in receptor binding in animals 6 and 24 months of age, with the hippocampus as the only region showing a significant loss of receptors and a decrease in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal and spatial dynamics of corticosteroid receptor down-regulation in rat brain following social defeat

The experiments explored the nature and time course of changes in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) binding in homogenates of various brain regions and pituitary of male Wistar rats following social defeat stress. One week after defeat, the binding capacity of GRs was decreased in the hippocampus and the hypothalamus while no changes were observed in the parietal cortex and the pituitary. The number of MRs remained at the same level as in undefeated rats. Three weeks postdefeat, the initially down-regulated GR returned to baseline level in the hippocampus and the hypothalamus. However, GR binding was now decreased in the parietal cortex. Severe down-regulation of MRs was detected in the hippocampal and septal tissue. The results show that brief but intense stress like social defeat induces a long-lasting down-regulation of corticosteroid receptors and that the temporal dynamics of these changes are not only differential for GRs and MRs but also for brain sites.     

Blockage of glucocorticoid, but not mineralocorticoid receptors prevents the persistent increase in circulating basal corticosterone concentrations following stress in the rat

Exposure to a single session of intense inescapable stressors results in elevations of plasma corticosterone levels selective to the trough of the circadian rhythm that last for several days after stressor cessation. In the present study, we examined whether this stress-induced alteration in the regulation of the circadian trough is dependent upon glucocorticoid and/or mineralocorticoid receptor activation during stress. Pre-treatment with the mineralocorticoid receptor (MR) antagonist, spironolactone (RU-28318; 50 mg/kg, s.c.), and/or the glucocorticoid receptor (GR) antagonist, mifepristone (RU-38486; 50 mg/kg, s.c.) 1 h before inescapable stress (40, 2.0-mA tail-shocks delivered over a 1 h period) had no effect on the acute plasma corticosterone response to inescapable stress. Treatment with the MR antagonist alone did not affect the appearance of basal corticosterone elevations in stressed rats. However, the elevated trough plasma corticosterone levels were no longer evident in rats treated previously with the GR antagonist either alone or in combination with the MR antagonist. GR activation during stressor exposure appears to be necessary for the development of subsequent basal corticosterone elevations.     

Seizure-induced changes of mineralocorticoid and glucocorticoid receptors in the hippocampus in seizure sensitive gerbils

Abnormal corticosteroid hormone levels during stress and resultant mineralocorticoid receptor (MR)/glucocorticoid receptor (GR) imbalance enhance the vulnerability of specific hippocampal neurons. In the present study, we investigated the distribution of MR and GR in seizure resistant (SR) and seizure sensitive (SS) gerbils, and observed the seizure-induced changes of MR and GR in the hippocampus of SS gerbils using immunohistochemistry and western blot analysis. MR and GR immunoreactivities were higher in the SS pre-seizure gerbils than that in SR gerbils. In the SR gerbils, the immunodensity of GR was high compared to that of MR. The changes of MR and GR immunoreactivities were significant in the stratum pyramidale of the hippocampal CA1 region and the infrablade of the dentate gyrus after seizure on-set. MR immunoreactivity in the CA1 region was significantly increased at 12h after seizure on-set, thereafter MR immunoreactivity was decreased. MR immunoreactivity in the dentate gyrus was decreased time-dependently after seizure on-set. GR immunoreactivity was decreased in the CA1 region and dentate gyrus time-dependently after seizure on-set. At 12h after seizure on-set, differences in MR and GR immunodensity diminished in the CA1 region and dentate gyrus. This imbalance of MR and GR immunoreactivity in these regions may be associated with seizure generation in the Mongolian gerbil, which is a hereditary seizure model.     

Central mineralocorticoid receptors are indispensable for corticosterone-induced impairment of memory retrieval in rats

Previous studies indicated that stress levels of glucocorticoid hormones (cortisol in humans, and corticosterone in rodents) induce impairment of long-term memory retrieval, but the underlying mechanisms (genomic or nongenomic) are not clear. To clarify this issue, we investigated the involvement of brain corticosteroid receptors and protein synthesis in the corticosterone-induced impairment of memory retrieval. Young rats were trained in the water maze task with six trials per day for 6 consecutive days. Retention of the spatial training was assessed 24 h after the last training session with a 60-s probe trial. Experiments included intraventricular injections of anisomycin, a specific protein synthesis inhibitor or specific antagonists for both types of corticocosteroid receptors (mineralocorticoid receptor, MR, and glucocorticoids receptor, GR) before corticosterone administration shortly before retention testing. The results showed that administration of anisomycin did not change the corticosterone response. Administration of the MR, but not GR, antagonist blocked the corticosterone-induced response dose dependently. These findings provide evidence for the view that glucocorticoids impair memory retrieval through nongenomic mechanisms involving an interaction with central MRs.     

Chronic lithium chloride injection increases glucocorticoid receptor but not mineralocorticoid receptor mRNA expression in rat brain

Lithium has been used clinically for the treatment of bipolar disorders. However, the brain mechanisms, by which lithium acts, are still unclear. An impaired hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathogenesis of mood disorders. In this study, we investigated the effects of chronic lithium on the corticosteroid receptors in the brain. Male Wistar rats were injected with LiCl (1.5 mEq/kg) or saline intraperitoneally (i.p.) once a day for 14 days. Twenty-four hours after the last injection, the expressions of mRNA for glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in the brain were determined by non-radioactive in situ hybridization. Chronic administration of LiCl increased the expression of GR mRNA in the hippocampus and paraventricular nucleus of the hypothalamus (PVN). However, no significant changes were observed in the expression of either MR mRNA in the hippocampus or GR mRNA in the locus ceruleus. Since the hippocampus and PVN mediate negative feedback regulation of the HPA axis, an increased expression of GR mRNA in these regions may normalize HPA axis activity in mood disorders. Thus, the effect of chronic lithium on GR function may be involved in its antimanic and/or prophylactic activity in bipolar disorders.     

Aldosterone induces elastin production in cardiac fibroblasts through activation of insulin-like growth factor-I receptors in a mineralocorticoid receptor-independent manner

Aldosterone is known to regulate electrolyte homeostasis, but it may also contribute to other processes, including the maladaptive remodeling of postinfarct hearts. Because aldosterone has been implicated in the stimulation of collagen production in the heart, we investigated whether it would also affect elastin deposition in cultures of human cardiac fibroblasts. We first demonstrated that treatment with 1 to 50 nmol/L aldosterone leads to a significant increase in collagen type I mRNA levels and in subsequent collagen fiber deposition. Pretreatment of cells with the mineralocorticoid receptor antagonist spironolactone, but not with the glucocorticoid receptor antagonist RU 486, inhibited collagen synthesis in aldosterone-treated cultures. Most importantly, we demonstrated that aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition in a dose-dependent manner. Strikingly, neither spironolactone nor RU 486 eliminated aldosterone-induced increases in elastin production. We further discovered that the proelastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor and that the insulin-like growth factor-I receptor kinase inhibitor AG1024 or an anti-insulin-like growth factor-I receptor-neutralizing antibody inhibits both insulin-like growth factor-I and aldosterone-induced elastogenesis. Thus, we have demonstrated for the first time that aldosterone, which stimulates collagen production through the mineralocorticoid receptor-dependent pathway, also increases elastogenesis via a parallel mineralocorticoid receptor-independent pathway involving I insulin-like growth factor-I receptor signaling.