Pharmacological characterization of type II glucocorticoid binding sites in AtT20 pituitary cell culture

Recent evidence indicates that at least two functional glucocorticoid receptors (Type I and Type II) are present in many tissues. It has also become increasingly recognized that, as in other systems, stimulus-response relationships for steroid hormones are often nonlinear. Thus, precise pharmacological parameters are required to establish a functional relationship(s) between binding site and response characteristics. We therefore pharmacologically characterized a glucocorticoid binding site present in AtT20 mouse pituitary cells, a cell line extensively used in studying Type II glucocorticoid receptor function. By several different criteria, glucocorticoids were shown to bind to a single class of binding sites, which, in comparison to available literature, correspond to classical Type II glucocorticoid receptors. No evidence for Type I adrenal steroid binding sites was observed, under the experimental conditions used. Unambiguous Kb values for both glucocorticoid agonists and antagonists were therefore calculated. These parameters should prove of use in elucidating the relationships between glucocorticoid receptor activation and different responses in both AtT20 cells and other glucocorticoid responsive tissues.     

Adrenocorticoid action in the spinal cord: Some unique molecular properties of glucocorticoid receptors

1. Glucocorticoid hormones affect several functions of the spinal cord, such as synaptic transmission, biogenic amine content, lipid metabolism, and the activity of some enzymes (ornithine decarboxylase, glycerolphosphate dehydrogenase), indicating that this tissue is a target of adrenal hormones. 2. Corticosterone, the main glucocorticoid of the rat, is detected at all regional levels of the spinal cord, and cold stress increases this steroid, predominantly in the cervical regions. 3. Intracellular glucocorticoid receptors have been found in the spinal cord, with higher concentrations in the cervical and lumbar enlargements. Prima facie, these receptors presented biochemical, stereospecifical, and physicochemical properties similar to those of receptors found in other regions of the nervous system. The prevalent form in the spinal cord is the type II receptor, although type I is also present in small amounts. 4. The type II glucocorticoid receptor of the spinal cord shows an affinity lower (Kd 3.5 nM) than that of the hippocampal type II site (Kd 0.7 nM) when incubated with [3H]dexamethasone. This condition may impair the nuclear translocation of the spinal cord receptor. 5. Another peculiar property of spinal cord type II site is a greater affinity for DNA-cellulose binding than the hippocampal receptor during heat-induced transformation. Also, the spinal cord receptor shows resistance to the action of RNAse A, an enzyme which increases DNA-cellulose binding of the hippocampal receptor, indicating that both receptors may be structurally different. 6. Therefore, it is possible that a different subclass of type II, or "classical glucocorticoid receptor," is present in the spinal cord. This possibility makes the cord a useful system for studying diversity of glucocorticoid receptors of the nervous system, especially the relationship between receptor structure and function.     

Mechanisms of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants

The highly selective Type II glucocorticoid ligand RU28362 showed a clear biphasic effect on alpha-lactalbumin (alpha-LA) production in rat mammary gland explants, with a peak at 1 nM and a return to basal levels at 30-300 nM; dexamethasone showed a similar profile. Corticosterone, which has a higher affinity for Type I than Type II receptors, produced a variable response. In six out of eleven studies this was biphasic, with a maximum at 300 nM; in five no increase above baseline was seen. Classical Type I receptor ligands--aldosterone and deoxycorticosterone--showed responses parallel to their Type II agonist activity. We interpret these data as follows occupancy of Type I receptors does not increase alpha-LA production the response to selective Type II receptor ligands is truly biphasic and one explanation of this pattern may be the existence of both "turn-on" and "turn-off" acceptor sites in the nucleus.     

Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration

We have used 32P-labeled cRNA probes directed against Type I (mineralocorticoid, high affinity glucocorticoid) and Type II (classical glucocorticoid) receptor mRNA to screen various tissues, and have investigated the effect of adrenalectomy (ADX) and dexamethasone (DM) administration on their levels in hippocampus. Both Northern blot and S1 nuclease analysis showed Type I mRNA to be high in hippocampus, colon, and heart; low in liver; and undetectable in thymus. Type II mRNA was high in liver, thymus, and brain; and low in testis and parotid. A transient increase in both hippocampal Type I and Type II mRNA was noted at 1-3 days post ADX. DM similarly elicited a rise in hippocampal Type I mRNA at 2-4 days after ADX, but prevented the ADX-induced increment in Type II mRNA. In contrast to the transient increase in Type I receptor mRNA levels, hippocampal levels of Type I receptors measured by [3H]aldosterone binding were constant 1-16 days post ADX. DM administration caused a doubling in Type I receptor levels over 4 days, with plateau levels at 4-16 days; previously, DM has been shown to lower Type II receptor levels in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroids and electrical activity in the brain

Corticosteroid hormones can enter the brain and bind to two receptor subtypes: the high affinity mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) with approximately 10-fold lower affinity. Under physiological conditions the degree of receptor occupation will range from a predominant MR occupation (at the beginning of the inactive period, under rest) to concurrent activation of MRs and GRs (at the circadian peak and after stress). With in vitro electrophysiological recording techniques we observed that neuronal excitability in the CA1 hippocampal field is under a long-term control of MR- and GR-mediated events. The predominant occupation of MRs is associated with a stable amino acid-carried synaptic transmission; calcium- and potassium-currents are small, as are the responses to biogenic amines. Occupation of GRs in addition to MRs results in a gradual failure of CA1 neurons to respond to repeated stimulation of amino acid-mediated input; ionic conductances and responses to biogenic amines are large. In general, electrical properties recorded when both MRs and GRs are unoccupied (i.e. after adrenalectomy) resemble the responses observed when both receptor types are activated. The corticosterone dependency of electrical properties is thus U-shaped. We conclude that MR occupation may be responsible for the maintenance of information processing in the CA1 field and the stability of the circuit. Additional activation of GRs will initially suppress synaptic activity, but may eventually result in an increased instability and even vulnerability of the neuronal networks.     

Glial cells express both mineralocorticoid and glucocorticoid receptors.

In the brain, the action of glucocorticoid steroids is mediated via two intracellular receptors, the mineralocorticoid (MR), or type I receptor, and the glucocorticoid (GR), or type II receptor. These receptors are expressed in many types of neurons and are co-expressed in some neurons such as the hippocampal pyramidal cells. Although glucocorticoids are known to affect gliogenesis and glial cell differentiation, the expression of the GR in different types of glial cells throughout the brain has not been thoroughly studied and the expression of the MR in glia not previously reported. Here we review studies suggesting that both receptors are expressed in astrocytes and oligodendrocytes.     

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.     

Differential regulation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene by human mineralocorticoid hormone-receptor complexes.

The brain tissues of the rat and mouse express two types of corticosteroid binding proteins, the glucocorticoid (GR) and aldosterone (MR) receptors. Unlike the type II (GR) receptor, type I receptor has a high affinity for aldosterone (ALDO) and corticosterone and is structurally similar to the kidney mineralocorticoid receptor (MR). The results reported in this study provide direct evidence for the interaction of dexamethasone (DEX), triamcinolone acetonide (TA), dexamethasone-21-mesylate (DXM) and 11-deoxycorticosterone (DOC) with human MR expressed in cells by transient co-transfection of a hMR expression vector. The interactions of hMR with DEX, TA, DXM, DOC, promegestone (R5020) and methyltrienelone (R1881) were measured by trans-activation of mouse mammary tumor virus long terminal repeat fused to bacterial chloramphenicol acetyltransferase (MMTV-tk-CAT) in gene co-transfection experiments and by cell free hormone binding assay. The incubation of various steroid hormones in the presence of [3H]ALDO in a competition assay with extracts prepared from HeLa cells co-transfected with hMR expression vector, showed that hMR expressed under these conditions has a high relative affinity for DEX which is similar to ALDO, TA and DOC. Incubation with DXM under these conditions showed very little competition, as was observed with R1881 and R5020. Incubation of the co-transfected cells with DEX, ALDO, DOC, R5020, TA, R1881 and DXM demonstrated that the level of trans-activation did not reflect the previously observed order of binding affinity for the hMR. The level of transactivation was always higher with DEX and TA compared to ALDO and DOC. Analysis of the binding of labeled glucocorticoid regulatory element (GRE) and hMR incubated with DEX, ALDO and DXM by gel shift analysis demonstrated that the trans-activation of MMTV-tk-CAT by hMR is a result of the interaction of hMR with GRE in the MMTV-LTR.     

Regulation of basal ACTH secretion by corticosterone is mediated by both type I (MR) and type II (GR) receptors in rat brain.

The mechanisms involved in the physiology of the secretion of ACTH are reviewed. The secretion is regulated by the biological consequences of the occupancy of high affinity mineralocorticoid (MR) and lower affinity glucocorticoid receptors (GR) for corticosterone at specific sites of the rat brain. The regulation by this mechanism of basal secretion during the circadian rhythm, the effect of adrenalectomy and of corticosterone replacement is discussed. Experiments with RU486, a specific glucocorticoid antagonist, suggest that occupancy of both MR and GR is required for normal control of ACTH at the time of peak activity. The occupancy of the GR for a few hours per day apparently suffices to maintain steady levels of the products of GR-responsive genes throughout the body.     

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.     

Effect of adrenocorticoid receptors on potassium and sodium flux in rat C6 glioma cells

C6 glioma cells contain two types of receptors for adrenocorticoids. Glucocorticoid (Type II) receptors are present at higher density and mediate increases in glycerol phosphate dehydrogenase and glutamine synthetase activity. The function of mineralocorticoid (Type I) receptors present at low density in C6 cells is unknown. Since mineralocorticoid (Type I) receptors in renal epithelial cells regulate cation transport, we sought to determine whether adrenocorticoid receptors located in glioma cells are similarly linked to electrolyte transporting activity. Occupation of mineralocorticoid receptors in C6 glioma by adrenocorticoids did not alter Na+ or K+ transport, in contrast to their effects on renal epithelial and vascular smooth muscle cells. Occupation of glucocorticoid receptors produced a 20-25% decrease in K+ uptake into C6 cells, but did not alter Na+ influx. Stimulation of Na+ influx with the ionophore monensin produced a large ouabain-sensitive increase in glucose utilization, as measured by 2-deoxyglucose uptake. However, mineralocorticoid receptor occupation did not alter glucose utilization, providing further evidence that these receptors do not influence Na+ transport in C6 cells. These studies provide evidence that mineralocorticoid receptors in glioma cells do not regulate Na+ or K+ transport. Glial glucocorticoid receptors have an inhibitory effect on glial K+ influx, which may contribute to glucocorticoid hormone effects on brain excitability.     

Brain mineralocorticoid receptors in cognition and cardiovascular homeostasis

Mineralocorticoid receptors (MR) mediate diverse functions supporting osmotic and hemodynamic homeostasis, response to injury and inflammation, and neuronal changes required for learning and memory. Inappropriate MR activation in kidneys, heart, vessels, and brain hemodynamic control centers results in cardiovascular and renal pathology and hypertension. MR binds aldosterone, cortisol and corticosterone with similar affinity, while the glucocorticoid receptor (GR) has less affinity for cortisol and corticosterone. As glucocorticoids are more abundant than aldosterone, aldosterone activates MR in cells co-expressing enzymes with 11β-hydroxydehydrogenase activity to inactivate them. MR and GR co-expressed in the same cell interact at the molecular and functional level and these functions may be complementary or opposing depending on the cell type. Thus the balance between MR and GR expression and activation is crucial for normal function. Where 11β-hydroxydehydrogenase 2 (11β-HSD2) that inactivates cortisol and corticosterone in aldosterone target cells of the kidney and nucleus tractus solitarius (NTS) is not expressed, as in most neurons, MR are activated at basal glucocorticoid concentrations, GR at stress concentrations. An exception may be pre-autonomic neurons of the PVN which express MR and 11β-HSD1 in the absence of hexose-6-phosphate dehydrogenase required to generate the requisite cofactor for reductase activity, thus it acts as a dehydrogenase. MR antagonists, valuable adjuncts to the treatment of cardiovascular disease, also inhibit MR in the brain that are crucial for memory formation and exacerbate detrimental effects of excessive GR activation on cognition and mood. 11β-HSD1 inhibitors combat metabolic and cognitive diseases related to glucocorticoid excess, but may exacerbate MR action where 11β-HSD1 acts as a dehydrogenase, while non-selective 11β-HSD1&2 inhibitors cause injurious disruption of MR hemodynamic control. MR functions in the brain are multifaceted and optimal MR:GR activity is crucial. Therefore selectively targeting down-stream effectors of MR specific actions may be a better therapeutic goal.