Corticosterone regulation of brain and lymphoid corticosteroid receptors

Circulating lymphocytes are often used as a model for brain corticosteroid receptor regulation in clinical disease states, although it is not known if lymphoid receptors are regulated in a similar manner as brain receptors. In the present study the regulation of brain (hippocampus, frontal cortex, hypothalamus and striatum), lymphoid (circulating lymphocytes, spleen and thymus) and pituitary glucocorticoid receptors in response to alterations in circulating corticosterone levels was examined. Seven days following adrenalectomy, type II corticosteroid receptors (i.e. glucocorticoid receptors) were significantly increased in the hippocampus, frontal cortex and hypothalamus, but not in any other tissues. Administration of corticosterone (10 mg/kg) for 7 days significantly decreased type II as well as type I (i.e. mineralocorticoid receptors) receptors in the hippocampus. Type II receptors in the frontal cortex, circulating lymphocytes and spleen were also significantly decreased by chronic corticosterone treatment. Immobilization stress (2 h a day for 5 days) failed to alter receptor density in any of the tissues. These results demonstrate that homologous regulation of corticosteroid receptors by corticosterone does not invariably occur in all tissues and emphasize the complex degree of regulation of these receptors. However, the simultaneous downregulation of both hippocampal and lymphocyte glucocorticoid receptors by corticosterone provides support for the hypothesis that circulating lymphocytes do reflect some aspects of brain glucocorticoid receptor regulation.     

The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response

The cloning of the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) cDNAs provides a basis for understanding the actions of glucocorticoids in the central nervous system. Structural evidence is presented for the identity of the type I corticosteroid binding site as the MR expressed in the brain. This identification is supported by the anatomical distribution of MR mRNA, determined by in situ hybridization histochemistry, which parallels the steroid autoradiographic localization of the type I sites. An in vitro assay for MR and GR function demonstrates that these receptors respond to different levels of glucocorticoid, suggesting that together they confer a larger dynamic range of sensitivity to this hormone. These studies lead to a new hypothesis for glucocorticoid action in the central nervous system.     

Corticosteroid receptors in lymphocytes: a possible marker of brain involution?

A similarity has recently been found between the regulation of corticosteroid receptors in brain and in lymphoid tissue. We have studied the regulation of corticosteroid receptors in human mononuclear leukocytes as a possible marker of brain involution. Type I corticosteroid receptors are down regulated by excess of mineralocorticoids (primary and secondary hyperaldosteronism, pseudohyperaldosteronism) and of glucocorticoids (Cushing's syndrome). Type II corticosteroid receptors are not reduced by excess of endogenous corticostiroids (Cushing's syndrome). In normal adults there is a direct significant correlation between plasma cortisol and Type I and between plasma cortisol and Type II receptors in mononuclear leukocytes, while in Cushing's syndrome the correlation is inverse between plasma cortisol at 8 a.m. and Type II receptors. In an aged population the mean numbers of Type I and of Type II receptors are lower and plasma cortisol is higher than in adult controls, but the increase of plasma cortisol is not followed by a clinical picture of hypercorticism. Corticosteroid Type I and Type II receptors are inversely correlated with age. After dexamethasone suppression (1 mg at 11 p.m.) Type I receptors always decrease in controls while the response of Type II is not homogeneous. In an aged group of patients, both receptors are reduced by dexamethasone. We conclude that the decrease with age of corticosteroid receptors is possibly related to a physiological involution of corticosteroid receptors and that this reduction does increase plasma cortisol concentration, without affecting the glucocorticoid effector mechanism.     

Nuclear receptors. II. Intestinal corticosteroid receptors

Two corticosteroid receptors have been cloned; they are the glucocorticoid receptor and the mineralocorticoid receptor. These receptors are members of the steroid/thyroid/retinoid receptor family of nuclear transactivating factors, which are characterized by two highly conserved zinc fingers in the central DNA binding domain, a COOH-terminal domain that encompasses the ligand binding site, and a variable NH(2)-terminal domain. In addition to these cloned receptors, other corticosteroid receptors have recently been identified in intestine. Steroid binding studies have identified two novel putative corticosteroid receptors in intestinal epithelia, and molecular cloning studies have detected two low-affinity receptors in small intestine that are activated by corticosteroids and induce CYP3A gene expression. This article focuses on the identification of these novel corticosteroid receptors and the potential role they may play in intestinal physiology.     

Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB

Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.     

Adrenocorticoid receptors in C6 glioma cells: Effects on cell growth

Rat C6 glioma cells contain two receptors for adrenocorticoids—the predominant glucocorticoid receptor and low densities of the Type I corticosteroid (mineralocorticoid) receptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosteceptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosterone, which fully occupy Type I receptors, produced a slight stimulatory effect on C6 cell growth in serum-free media. However, spironolactone, a Type I receptor antagonist, and pregnenolone, which does not bind to Type I receptors, had similar effects. Therefore, the slight growth stimulation produced by low steroid concentrations is not mediated by Type I or glucocorticoid receptors, but may be due to an effect on cell membrane properties or other receptor-independent action. Occupation of glucocorticoid receptors by higher concentrations of corticosteroids inhibited C6 cell growth.     

Stimulation of both type I and type II corticosteroid receptors blunts counterregulatory responses to subsequent hypoglycemia in healthy man

Antecedent increases of corticosteroids can blunt counterregulatory responses to subsequent stress. Our aim was to determine whether prior activation of type I corticosteroid (mineralocorticoid) or type II corticosteroid (glucocorticoid) receptors blunts counterregulatory responses to subsequent hypoglycemia. Healthy volunteers participated in five randomized 2-day protocols. Day 1 involved morning and afternoon 2-h hyperinsulinemic (9 pmol.kg(-1).min(-1)) euglycemic clamps (PE; n = 14), hypoglycemic clamps (PH; n = 14), or euglycemic clamps with oral fludrocortisone (PE + F; type I agonist, 0.2 mg, n = 14), oral dexamethasone (PE + D; type II agonist, 0.75 mg, n = 13), or both (PE + F + D; n = 14). Day 2 was identical in all protocols and consisted of a 2-h hyperinsulinemic hypoglycemic clamp. Day 2 insulin (625 +/- 40 pmol/l) and glucose (2.9 +/- 0.1 mmol/l) levels were similar among groups. Levels of epinephrine, norepinephrine, glucagon, growth hormone, and MSNA were significantly blunted by prior activation of both type I and type II corticosteroid receptors to PE. Prior activation of both corticosteroid receptors also significantly blunted NEFA during subsequent hypoglycemia. Thus, levels of a wide spectrum of key counterregulatory mechanisms (neuroendocrine, ANS, and metabolic) were blunted by antecedent pharmacological stimulation of either type I or type II corticosteroid receptors in healthy man. These data suggest that activation of type I corticosteroid receptors in man can have acute and profound regulating effects on physiological stress in man. Both type I and type II corticosteroid receptors may be involved in the multiple mechanisms controlling counterregulatory responses to hypoglycemia in healthy man.     

Long-term control of neuronal excitability by corticosteroid hormones

Hippocampal CA1 neurons express both mineralocorticoid and glucocorticoid receptors. Due to the difference in affinity of the two receptor types for corticosterone and variations in endogenous steroid levels, occupation of the receptors will range between a situation of predominant mineralocorticoid receptor activation and conditions where both receptor types are occupied. It was observed that local signal transduction is regulated by activation of the corticosteroid receptors. Particularly, transmission mediated by biogenic amines appears to be sensitive to steroid control. The data indicate that cholinergic and serotonergic responses are small with predominant mineralocorticoid receptor activation, while additional glucocorticoid receptor activation results in large responses; the reverse has been found for noradrenalin. The steroid-dependent control over transmission by biogenic amines will influence local excitability and therefore functional processes in which the hippocampal system is involved.     

Specific hydroxylations determine selective corticosteroid recognition by human glucocorticoid and mineralocorticoid receptors

The ligand binding domains of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR) display a high sequence homology. Aldosterone and cortisol, the major mineralocorticoid and glucocorticoid hormones, are very closely related, leading to the cross-binding of these hormones to both receptors. The present study reports on the mechanism by which hMR and hGR are activated preferentially by their cognate hormones. We found that the ability of corticosteroids to stimulate the receptor's transactivation function is depending on the stability of the steroid-receptor complexes. In the light of a hMR structural model we propose that contacts through the corticosteroid C21 hydroxyl group are sufficient to stabilize hMR but not hGR and that additional contacts through the C11- and C17-hydroxyl groups are required for hGR.     

Kainic Acid-Induced Decrease in Hippocampal Corticosteroid Receptors

The potential role of excitatory amino acids in the regulation of brain corticosteroid receptors was examined using systemic administration of kainic acid. Administration of kainic acid (5, 10, and 15 mg/kg) to 24-h adrenalectomized rats that were killed 3 h later produced large, dose-related decreases in glucocorticoid receptors (GR) in hippocampus (23-63%), frontal cortex (22-76%), and striatum (41-49%). Kainic acid did not decrease hypothalamic GR. Hippocampal mineralocorticoid receptors (MR) were also markedly decreased (50-71%) by kainic acid. Significant decreases in corticosteroid receptors could be detected as soon as 1 h after kainic acid (10 mg/kg) administration. Decreases in hippocampal, cortical, and hypothalamic GR as well as hippocampal MR were observed 24 h after administration of kainic acid (10 mg/kg) to adrenalectomized rats. Kainic acid (10 mg/kg) also significantly decreased hippocampal GR and MR as well as GR in the other three brain regions when administered to adrenal-intact rats that were subsequently adrenalectomized and killed 48 h after drug administration. The kainic acid-induced decreases in hippocampal GR and MR binding were due to decreases in the maximum number of binding sites (Bmax) with no change in the apparent affinity (KD). Kainic acid when added in vitro did not displace the GR and MR radioligands from their respective receptors. These studies demonstrate that excitatory amino acids play a prominent role in the regulation of hippocampal corticosteroid receptors. In addition, the data indicate that noncorticosterone factors are involved in corticosteroid receptor plasticity.     

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.     

Cyclic AMP-dependent protein kinase modulation of the glucocorticoid-induced cytolytic response in murine T-lymphoma cells

The antiproliferative effect of glucocorticoid hormones on lymphoid tissue serves as the basis for their use in chemotherapy of lymphomas and leukemias. The effectiveness of the steroid-mediated response is potentially contingent upon a variety of factors, including the cellular level of glucocorticoid receptors. This report demonstrates that differences in the expression of the glucocorticoid receptor gene can modulate steroid sensitivity of individuals within a population of lymphoma cells. We have also found that loss of cAMP-dependent protein kinase activity caused a measurable decrease of steroid sensitivity in the murine T-lymphoma WEHI-7 without producing a significant change in steroid binding capacity. However, the extent of this change in sensitivity was dependent upon the level of glucocorticoid receptor expression. Lymphoma cells containing few spare steroid receptors became significantly resistant to glucocorticoids through loss of cAMP-dependent kinase function. On the other hand, elevated levels of cAMP were found to cause an increase in glucocorticoid receptor mRNA concentrations. Thus, cAMP-dependent protein kinase activity has the potential to modulate a lymphoma cell's steroid sensitivity by affecting the level of glucocorticoid receptor expression as well as the receptor's efficiency in producing a cytolytic response.     

Cortisol controls the pubertal development of agonistic behavior in male golden hamsters via type II corticosteroid receptors

In male golden hamsters, agonistic behavior undergoes a pubertal transition from play fighting to adult aggression. Previous studies have shown that this aspect of behavioral development is associated with pubertal increases in glucocorticoids and that daily social stress or injections of a synthetic glucocorticoid accelerate the transition. The goals of this study were to confirm the effects of cortisol on the development of agonistic behavior and to investigate the role of type II corticosteroid receptors in this process. First, animals treated with cortisol during early puberty [from postnatal days 31 (P-31) to P-36] showed an accelerated transition from play fighting to adult aggression. In a second experiment, the behavioral effects of cortisol were blocked by a co-treatment with a type II corticosteroid receptor antagonist. These findings are the first to show a facilitating role for type II corticosteroid receptors in the pubertal development of a social behavior. As such, these findings provide new insights into the neuroendocrine mechanisms controlling behavioral development during puberty.     

Regulation of intracellular cholesterol synthesis in hypercholesterolemia by glucocorticoids]

The rate of endogenous cholesterol synthesis in blood lymphocytes and skin fibroblasts from patients with type IIa hyperlipidemia was found to be increased in comparison with healthy donors. The cells of hyperlipidemic patients had lowered levels of glucocorticoid receptors concomitantly with a partial loss of their sensitivity to glucocorticoids. In fibroblasts from patients with hereditary hypercholesteremia of homozygous type the number of glucocorticoid receptors did not exceed 10% of their content in normal cells. The decrease of the number of glucocorticoid receptors in patients with type IIa hyperlipidemia seems to be a compensatory response of cells culminating in activation of endogenous cholesterol synthesis.     

Brain corticosteroid receptor gene expression and neuroendocrine dynamics during aging

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary in old (30 months) and young (3 months) male Brown Norway rats. The data demonstrate that circulating ACTH rather than the corticosteroid plasma level was elevated under basal conditions and following stress. Furthermore, a reduction of mineralocorticoid receptor (MR) number in the hippocampus and of glucocorticoid receptor (GR) number in the hypothalamus and the pituitary correspond to increased neuroendocrine responsiveness and negative feedback following stress. The changes in receptor binding do not parallel the changes in the amount of MR and GR mRNA measured with in situ hybridization. This suggests that the processing rather than the receptor gene expression is affected in senescence.     

A factor which modifies the interaction of steroids with glucocorticoid receptors

A mechanism which determines the difference in the ability of deoxycorticosterone (DOC) to inhibit the binding of 3H-triamcinolone acetonide (3H-TA) to glucocorticoid receptors of rat heart and liver cytosols was investigated. DOC was found to strongly inhibit the binding of 3H-TA by heart cytosol, but to exert only a slight inhibitory effect towards the live cytosol binding. This difference was not due to the influence of the enzymes sensitive to molybdate ions, the presence of DOC-degrading enzymes or contamination of liver cytosol by blood serum. The liver cytosol devoid of the glucocorticoid receptor activity by heating was found to contain a factor modifying the "in vitro" interaction of DOC with the heart cytosol glucocorticoid receptors (receptor modifying factor, RMF). This factor is coeluted with the high molecular weight fraction during gel filtration, is precipitated at 50-70% ammonium sulphate saturation, can be absorbed by DEAE-Sephacel from cytosol at pH 7.4 under hypotonic conditions and extracted at about 0.06 M KC1. The sensitivity to proteases and the lack of sensitivity to nucleases point to the proteinic nature of the factor. It was assumed that in terms of the interaction of some steroids with glucocorticoid receptors, the tissue specificity can, at least partly, be explained by the differences in RMF concentration.     

Modular design of non-viral vectors with bioactive components

Inefficient gene delivery continues to limit gene therapy applications to both basic and applied sciences. Approaches for engineering vectors increasingly include bioactive components that bind cellular receptors, disrupt membranes, or enhance nuclear transport. Recently, a novel cationic lipid was developed by modifying the glucocorticoid dexamethasone. This cationic corticosteroid condenses plasmids for gene delivery, while also modulating inflammation. Modular vectors containing bioactive components that target various cellular processes can overcome the barriers limiting gene transfer.