Rat Liver Glucocorticoid Receptor Research Articles

Aminosulfhydryl and aminodisulfide compounds enhance binding of the glucocorticoid receptor complex to deoxyribonucleic acid-coated cellulose and to chromatin

In the presence of amine-containing sulfhydryl compounds, binding of heat-transformed cytosolic rat liver glucocorticoid receptor complex (GRC) to double-stranded calf thymus DNA-coated cellulose and to rat liver chromatin was enhanced up to 10-fold. These observations were made under conditions when a maximum of 8% of the total GRC bound to DNA in the absence of test compound. Compounds which did not contain both a sulfhydryl and amine group were inactive. Phosphorothioate derivatives of the active sulfhydryl compounds were also inactive. However, pretreatment of the phosphorothioate compounds with alkaline phosphatase restored activity. Upon centrifugation at 8800 g , amine-containing disulfide compounds at millimolar concentrations caused considerable sedimentation of the GRC in the absence of DNA-coated cellulose or chromatin and no apparent increase in GRC binding to DNA or chromatin. Amine-containing disulfide compounds at micromolar concentrations did not cause heavy sedimentation of the GRC and enhanced binding of the GRC to DNA-coated cellulose up to 9.5-fold. Thus, diaminosulfhydryl compounds and the disulfide 1,18-diamino-6,13-diaza-9,10-dithiaoctadecane (WR 149,024) possess both the ability to restore and preserve the steroid binding capacity of the glucocorticoid receptor and to enhance binding of the GRC to DNA and chromatin.

Binding of RU486 and deacylcortivazol to the glucocorticoid receptor is insensitive to sulfhydryl-modifying agents

The differential sensitivity of the rat liver glucocorticoid receptor (GR) to sulfhydryl group modifying agents when bound to various agonist and antagonist ligands was studied. [ 3H]Triamcinolone acetonide (TA) binding was completely abolished by previous treatment of the unbound receptor with various N-alkylmaleimides. On the contrary, [ 3H]RU486 binding was only slightly affected by treatment with N-ethylmaleimide (NEM) and more significantly decreased with maleimides bearing bulky substituents. Ligand exchange experiments demonstrated that, unlike the agonist TA, the antiglucocorticoid RU486 was unable to protect the GR binding site from the effect of NEM. This lack of protection would seem to be due to the presence of the bulky 11β-substituent in RU486 since RU26988 and RU28362, two 11β hydroxylated glucocorticoids bearing the same 17α-propynyl side chain as RU486 but lacking the 11β-substituent could protect GR against NEM. The ability of a GR ligand to prevent NEM inactivation of TA binding appeared unrelated to its agonist or antagonist nature: deacylcortivazol, a potent agonist, afforded no protection whereas antagonists of the 17β-carboxamide series did. These data strongly suggest that compounds bearing bulky substituents on the steroid A and/or C rings, like deacylcortivazol and RU486, are positioned differently from canonical glucocorticoids in the steroid binding groove of the GR.

Prenatal development of glucocorticoid receptor gene expression and immunoreactivity in the rat brain and pituitary gland: a combined in situ hybridization and immunocytochemical analysis.

By means of in situ hybridization and immunocytochemical techniques it has been possible to follow the prenatal development of glucocorticoid receptor (GR) messenger RNA (mRNA) expression and GR immunoreactivity (IR) in the rat brain from embryonic day (E) 15 to 22. A 700-base-pair GR cDNA fragment was used for RNA probe generation. In the immunocytochemical analysis a mouse monoclonal antibody (IgG2a) against the rat liver GR was used in combination with the indirect fluorescence technique or the avidin-biotin immunoperoxidase method. At E15 till E22 a moderate to strong GR mRNA signal was observed within the neuro-epithelium from the medulla oblongata to the telencephalon. A moderate to strong labelling was also present within the paraventricular hypothalamic nucleus, the arcuate nucleus, the nucleus raphe magnus, the nucleus raphe obscurus and the locus coeruleus. In these areas a weak to moderate nuclear GR IR developed in nerve cells 1 or 2 days after the appearance of the GR mRNA signal. From E15 the adenohypophysis showed the strongest expression of GR mRNA. At E17 a strong GR IR was especially demonstrated in the nuclei of many pituitary cells, some exhibiting adrenocorticotropin IR. The results open up the possibility that there exist active GR in embryonic life capable of regulating proliferation events within the adenohypophysis and the neuro-epithelia of the brain. This embryonic GR may modulate the development of inter alia neuro-endocrine areas such as the paraventricular and arcuate nuclei and arousal-related areas such as the central 5-hydroxytryptamine and noradrenaline neuronal systems. Provided that this embryonic GR is capable of becoming activated by glucocorticoids in fetal life, it may mediate several neurochemical and behavioural impairments caused by prenatal stress.

Modulation of glucocorticoid binding to rat liver cytosol receptor by lipid-soluble extracts from the serum of AIDS patients.

The total liposoluble extract of sera from AIDS patients, IVC1 and IVD stages, containing cortisol and free fatty acids (FFA) inhibited [3H]dexamethasone binding to a lesser extent than did the same quantity of total liposoluble extract of sera from healthy men. FFA isolated from extracts of AIDS sera by Sephadex LH20 chromatography had less effect on [3H]dexamethasone binding to rat liver glucocorticoid receptor than those extracted from sera of healthy men. These results suggest the presence in sera of AIDS patients of a liposoluble substance which could be limiting the inhibitory effect of FFA on [3H]dexamethasone binding to glucocorticoid receptor by inducing a conformational change in glucocorticoid receptor that could alter the biological action of glucocorticoids. The pathological consequence could be the apparent contradiction of high cortisolemia and clinical symptoms of adrenal insufficiency that have been observed in AIDS patients.

Stress-induced changes of glucocorticoid receptor in rat liver

The effect of corticosterone injection and of acute and repeated stress on rat liver cytosol glucocorticoid receptor was studied to ascertain whether corticosterone-induced glucocorticoid receptor (GR) regulation also takes place in intact animals as it does in adrenalectomized ones. Adult male rats were exposed to six different stressors (swimming, 10 mg/kg histamine i.p., 500 mU/kg vasopressin s.c., heat, immobilization and cold) acutely or three times daily for 18 days (repeated stress). Each of the stressors applied acutely provoked a pronounced increase of plasma corticosterone with subsequent induction of hepatic tyrosine aminotransferase activity. Depletion of cytosol receptor was however only noticed after swimming and histamine injection. On the other hand, sustained hypersecretion of corticosterone evoked by repeated stress significantly reduced the number of GR in rat liver cytosol without any change in K d . It is concluded that in the presence of intact adrenal glands cytosol receptors are more resistant to corticosterone-induced depletion than in their absence. Further, repeated stress causes down-regulation of GR in the liver, most probably by sustained corticosterone secretion, yet the effect of other stress factors cannot be excluded.

Duration of antagonizing effect of RU486 on the agonist induction of tyrosine aminotransferase via glucocorticoid receptor

The duration of the antagonizing activity of RU486 on tyrosine aminotransferase (TAT) induction and the glucocorticoid receptor in rat liver was studied. A single dose of RU486 (10 mg/kg) caused occupation of the cytosol glucocorticoid receptor in rat liver at 1h. During this time no nuclear binding of [ 3H]dexamethasone ([ 3H]Dex) receptor complex was recorded, and TAT induction was completely blocked. TAT inducibility recovery parallelled receptor binding in both the cytosol and the nuclei, reaching maximum at 12 h. In contrast, nuclear binding recovered in 24 h, and [ 3H]Dex receptor binding in cytosol 48 h after RU486 application. It is concluded that the inhibitory effect of a single dose of RU486 on TAT induction is of rather short duration. At concomitant presence of agonist and antagonist in vivo, no direct correlation between agonist receptor occupancy and TAT induction could be observed.

Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60°C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cellls transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.

In vivo effects of cadmium on rat liver glucocorticoid receptor functional properties

1. 1. Cadmium (Cd 2+) administered in vivo induced a 40% reduction of rat liver glucocorticoid receptor (GR) capacity and inhibition of glucocorticoid-receptor complexes binding to mouse mammary tumor virus (MMTV) DNA fragment containing GR consensus sequence. 2. 2. The effect of Cd 2+ on the GR binding activity can be reversed with DTT, suggesting Cd 2+ interaction with thiol groups. 3. 3. Cd 2+-related GR modification seems to be mediated by Cd 2+ binding to cytoplasmic components included in the regulation of the receptor function, although the direct binding of the metal to the receptor thiols could not be ruled out.

Immunocytochemical Localization of the Glucocorticoid Receptor in Rat Brain, Pituitary, Liver, and Thymus with Two New Polyclonal Antipeptide Antibodies*

The intracellular localization of the glucocorticoid receptor (GR) was studied in male rat brain, pituitary, liver, and thymus. Two new polyclonal anti-GR antibodies, GR 57 and GR 59, raised against two synthetic peptides (346-357 and 245-259) that correspond to unique regions of the amino-terminus of human GR were used. Vibratome sections (30-50 microns) of perfused brain and frozen sections (6-8 microns) of pituitary, liver, and thymus fixed in paraformaldehyde were incubated in preimmune serum, immunoserum, epitope-purified immunoserum, or peptide-absorbed immunoserum of either GR 57 or GR 59 and immunostained by the avidin-biotin peroxidase method. GR immunoreactivity (GR-ir) was primarily nuclear in brain, pituitary, liver, and thymus sections from intact rats. Adrenalectomy caused nuclear GR-ir to decrease and cytoplasmic GR-ir to increase. When adrenalectomized rats were treated with corticosterone (100 micrograms and 1 mg) or dexamethasone (1 microgram, 100 micrograms, and 1 mg), GR-ir was again predominantly nuclear. One microgram of corticosterone failed to cause nuclear GR-ir when administered to adrenalectomized rats. Immunoreactive neurons and glial cells were extensively distributed, with varied intensity, throughout the rat forebrain. The areas include cortex, septum, hippocampus, amygdala, thalamus, and hypothalamus. Cells with the strongest GR-ir were located in the caudate putamen, paraventricular, arcuate, and central amygdala nuclei, areas CA1-CA2 of the hippocampus, and laminae 4 and 5 of the cortex. In the pituitary, cells of the anterior and posterior lobes were GR immunoreactive, while those in the intermediate lobe were not. Hepatocytes of the liver and thymocytes and reticuloepithelial cells of the thymus were GR immunoreactive. The results show that GR can be localized immunocytochemically in numerous rat tissues using antipeptide polyclonal antibodies and correlated with the results of biochemical and ligand receptor studies.

Modulatory Effects of Unsaturated Fatty Acids on the Binding of Glucocorticoids to Rat Liver Glucocorticoid Receptors*

Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.

Postnatal ontogeny of mineralocorticoid and glucocorticoid receptor gene expression in regions of the rat tel- and diencephalon

In situ hybridization was used to study the neuroanatomical distribution of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression during development in the rat. This study was based on incubation of adjacent sections of brains from 2-, 8-, 12-, 16-day-old and adult (3 months) rats with 35S-labelled cRNA probes. These probes are transcribed from 513 and 500 basepair cDNA fragments with little homology from rat brain MR and rat liver GR respectively. Different patters of expression were found in the brain of MR and GR during ontogeny. At postnatal day (pnd) 2, a high density of labelled MR mRNA was found in all pyramidal (CA1–4) and granular (dentate gyrus) cell fields of the hippocampal structure, the anterior hippocampus and indusium griseum, and cortex layer II. Modest to high labelling of MR mRNA was observed in the subfornical organ and the anterior hypothalamus. A variety of other telencephalic regions anterior and posterior of bregma exhibited modest to weak intensity of labelled MR mRNA. The diencephalon virtually lacked labelled MR mRNA. At older postnatal ages including the adult age, this regional distribution of radiolabelled MR mRNA did not change. At pnd 2, abundant radiolabelled GR mRNA was found widespread over the tel- and diencephalon, with the highest density observed in cell field CA1 and CA2 of the hippocampus and the parvocellular division of the hypothalamic paraventricular nucleus. Modestly labelled GR mRNA was observed in various hypothalamic and thalamic nuclei, basal ganglia, the lateral septum and the amygdala. At older postnatal ages and in adulthood, the intensity of labelled GR mRNA became progressively stronger in the hippocampus. Moreover, we observed a trend towards a more condensed and narrow band of cell bodies in the hippocampus for both MR and GR mRNA during ontogeny. A semi-quantitative comparison of the intensity of both labelled mRNA's performed at each age revealed a significantly lower expression of GR than MR mRNA in the CA3 cell field at pnd 2. At pnd 8 and 12, the amount of GR mRNA was significantly lower in the dentate gyrus and the CA3, whereas in adulthood, less GR mRNA was measured in all pyramidal and granular cell fields. The present study demonstrates that MR and GR genes are expressed in early postnatal development in a pattern resembling that in adulthood. As is the case in the adult brain, there is more MR than GR mRNA in the hippocampus during ontogeny, especially in the CA3 cell field and the DG.

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-β-(4-dimethylaminophenyl)-17a-hydroxy-17ß-(3-hydroxypropyl)-13α-methyl-4,9-gonadien-3-onel). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [ 3H]ZK98299 with affinity approximately equal to that of [ 3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0°C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [ 3H]ZK98299 and [ 3H]triamcinolone acetonide binding. While [ 3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide [ 3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [ 3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 °C) and receptor transforming (23 °C) conditions, and exhibited a faster rate of dissociation at 23°C when compared with [ 3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [ 3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.

Hepatic glucocorticoid receptor behaves differently when its hormone binding site is occupied by agonist (triamcinolone acetonide) or antagonist (RU486) steroid ligands

We have examined the influence of sulfhydryl (SH)-group modifying agents on the interaction of the rat liver glucocorticoid receptor (GR) with its known agonist triamcinolone acetonide (TA) and the newly synthesized antagonist mifepristone (RU486). In the freshly prepared cytosol, [ 3H]TA or [ 3H]RU486 bound to macromolecule(s) which sediment as 8–9 moieties: the binding of either ligand can be competed with radioinert TA or RU486. The presence of 2–10 mM dithiothreitol (DTT), β-mercaptoethanol (β-MER), and monothioglycerol (MTG) caused a 2–3 fold increase in the [ 3H]TA and [ 3H]RU486 binding to GR. Iodoacetamide (IA) and N-ethylmaleimide (NEM) decreased the agonist binding significantly. In contrast, the [ 3H]RU486 binding to GR increased by 50 percent in the presence of IA. IA and NEM inhibited the binding of the heat-transformed [ 3H]TA-receptor complex to DNA-cellulose by 70–90 percent whereas DNA binding of [ 3H]RU486-bound GR was inhibited only slightly. These results indicate that either a) the interaction of GR with the agonist or antagonist steroid ligands causes differential structural alterations, which are more readily detectable in the presence of SH-modifying agents or b) the agonist and the antagonist interact with distinct steroid binding sites.

Depletion of rat liver glucocorticoid receptor hormone-binding and its mRNA in sepsis

Glucocorticoid receptor (GR) hormone-binding activity, its physical characteristics, and GR mRNA levels were studied in the liver, brain and muscle of normal (saline-injected) and hypermetabolic septic rats 24 h after the subcutaneous injections of E. coli . The GR levels (hormone-binding activity) declined by about 40%, 56%, and 40% in septic liver, brain, and muscle cytosol, respectively. The mechanism of the decrease in the GR levels in sepsis was studied in liver. The GR levels remained low (45% of control hormone-binding) even after 48 h of E. coli administration. The decrease in the liver GR occurred in the 9S untransformed GR. The 9S GR from septic liver transformed to the 4S form in proportions comparable to the control liver GR. In addition, the 4S GR from control and septic liver was capable of binding to DNA-cellulose to a similar extent. The GR mRNA level in septic liver declined by about 30%. Thus, a decrease in GR hormone-binding activity in sepsis appears to be due to a decline in the steady-state GR mRNA level and not from a change in the qualitative properties of the GR protein.

Type II glucocorticoid receptors are expressed in oligodendrocytes and astrocytes

Glucocorticoid hormones affect gene expression directly at the level of transcription via intracellular receptors that translocate to the nucleus in the presence of steroid. In the brain, two types of high-affinity receptors bind glucocorticoids, the type I, mineralocorticoid receptor and the type II, glucocorticoid receptor (GR). Both receptor types are expressed by many types of neurons. Although binding studies have suggested that glial cells may also express receptors, the expression of these receptors in specific classes of glia has not been studied previously. This immunocytochemical study was undertaken to determine which of the different classes of glial cells express type II GR. Primary cultures of mixed glial cells from rat cerebrum and cerebellum, purified oligodendrocytes and astrocytes, as well as two glial tumor cell lines were screened for the expression of glucocorticoid receptors using a mouse monoclonal antibody directed against rat liver GR (BuGR-2). Glial cell types were identified by morphology and immunoreactivity (IR) with antibodies directed against glial fibrillary acidic protein (GFAP), cyclic nucleotide phosphodiesterase (CNP), or myelin basic protein (MBP). Double immunofluorescence microscopy revealed that all GFAP-IR cells (type 1 and type 2 astrocytes), all CNP- or MBP-IR cells (oligodendrocytes), as well as immature and intermediate cell types expressed GR, although at different levels. C6 glioma and JScl1 Schwannoma cells were observed to express moderate to high levels of GR. Furthermore, cells grown in the absence of glucocorticoids had diffuse GR staining over the cytoplasm, whereas cells grown in the presence of the synthetic glucocorticoid dexamethasone had strong nuclear staining. These results demonstrate that, in vitro, all classes of glial cells express glucocorticoid receptors that can translocate to the nucleus in the presence of hormone. These observations suggest that glial cells are major targets for glucocorticoid-directed control of gene transcription in the nervous system.

Modifications of rat liver glucocorticoid receptor by insulin-induced hypoglycemia

Stability-, equilibrium- and kinetic binding parameters, transformation rate and sedimentation properties of liver cytosol glucocorticoid receptor from insulin-treated rats were studied. 40% elevation of cytosolic glucocorticoid binding and a lower affinity of the receptor for ligand were observed in hypoglycemic rats as compared to the controls. A small but significant decrease of [ 3H] triamcinolone acetonide-receptor complexes association rate and an increase of dissociation rate were also found. The rate and the extent of activation of the complexes from insulin-treated rats were somewhat higher compared to the controls, and the complexes from both groups showed higher affinity for the nuclei isolated from insulin-treated animals. Mixing experiments suggested that insulin treatment lead to alterations at the level of both the receptor protein and the nuclear binding sites. Sedimentation properties of transformed and untransformed receptor remained unchanged upon insulin treatment. The physiological relevance of the data was confirmed by hypoglycemia-related stimulation of tyrosine aminotransferase induction by dexamethasone.

Distribution of glucocorticoid receptor-like immunoreactivity in the brain, and its relation to CRF and ACTH immunoreactivity in the hypothalamus of the japanese quail, Coturnix coturnix japonica

Monoclonal antibody (MAb49) against the rat liver glucocorticoid receptor was used to evaluate glucocorticoid receptor (GR) immunoreactive structures in the brain of the japanese quail, Coturnix coturnix japonica. Using the avidin-biotin technique, the immunoreaction was present in the nerve cell nuclei in intact male birds. High density of glucocorticoid receptor-like immunoreactivity was found in the tubero-infundibular area, nucleus paraventricularis, posteromedialis and lateralis hypothalami, lateral septum and in some brainstem nuclei. Cerebellar cortex was also immunopositive. In contrast to mammals, no immunoreactive cell nuclei were found in the hippocampal region. The glucocorticoid receptors were colocalized with adrenocorticotropin (ACTH) immunoreactivity in the tubero-infundibular region, while corticotropin releasing factor (CRF) positive cells in the paraventricular nucleus did not contain glucocorticoid receptor-like immunoreactivity in their cell nuclei.

Glucocorticoid-receptor complexes are associated with small RNA in vitro

Identification of RNA associated with soluble glucocorticoid-receptor complexes of HeLa cells was performed by immunoprecipitation of receptor complexes with a monoclonal antibody raised against rat liver glucocorticoid receptor. Polyacrylamide gel electrophoresis of RNA extracted from immunoprecipitates of cytosolic complexes revealed the presence of eight RNA bands, consisting of 28S, 18S, and small RNAs, including 5.8S, 5S and tRNA. A comparison of RNA species immunoprecipitated by monoclonal anti-glucorcorticoid receptor antibody and IgG purified from normal mouse serum showed that four small RNAs were preferentially recovered after immunoprecipitation with anti-glucocorticoid receptor antibody. When these species were analyzed on sequencing gels, their nucleotide lengths coincided with those of 7-3, 7S, U2, and U1 RNA. Immunoprecipitation of nuclear extracts containing glucocorticoid-receptor-RNA complexes showed that the same set of small RNAs was preferentially immunoprecipitated by anti-glucocorticoid receptor antibody. The four small RNAs we detected represented minor species in whole extracts, and their preferential immunoprecipitation by anti-glucorcorticoid receptor antibody was prevented by removal of glucocorticoid-receptor complexes from HeLa cell extracts. We conclude that 7-3, 7S, U2, and U1 RNA are associated with glucocorticoid-receptor complexes in vitro, and hypothesize that post-transcriptional effects of glucocorticoids may in part be mediated through interaction of receptor complexes with these small RNAs.

Effect of selenite on glucocorticoid receptor.

Rat liver glucocorticoid receptor was partially purified and characterized for its hormone binding using selenite. Selenite at very low concentrations irreversibly inhibited the hormone binding. The concentration for half maximal inhibition was approximately 2.8 microM. The inhibition was restored by dithiothreitol. The receptor-hormone complex became considerably insensitive to the selenite inhibition. The receptor inhibited by selenite was eluted at the same position as the native receptor from DEAE ion exchange and gel filtration columns. The results suggest that at least four sulfhydryl groups are located in the hormone binding domain of the receptor making a cluster.

In SituHybridization Mapping of Glucocorticoid Receptor Messenger Ribonucleic Acid in Rat Brain

We have mapped the distribution of glucocorticoid receptor (GR) mRNA in the male adult rat brain using T7 RNA polymerase transcripts of a 1155 base pair rat GR cDNA clone comprising the coding region for amino acids 140-525. Strong expression of GR mRNA was found in the neurons of the CA1 and CA2 fields of the hippocampus and in the paraventricular and periventricular hypothalamic nuclei. Moderate to strong hybridization was found in the dorsal thalamic nuclei, layers II and VI of the cerebral cortex, the anterior olfactory nucleus and primary olfactory cortex, the hypothalamic mammillary nuclei, the subthalamus, and the granule and mitral cells of the olfactory bulb. Weak to moderate hybridization was found in many other regions of the tel- and diencephalon. Mes- and rhomboencephalic neurons displayed very low levels of GR mRNA relative to the levels observed in the tel- and diencephalon. In the cerebellum, moderate to strong levels of mRNA were detected in the granule and Purkinje cell layers with very low levels elsewhere. Nonneuronal brain elements, such as glial cells, the pia mater, and the choroid plexus, were found to express low to moderate amounts of GR mRNA. These results confirm and extend mapping studies of steroid receptors in the brain using radiolabeled steroids or monoclonal antibodies against rat liver GR and demonstrate that the relative distribution of GR protein in different brain nuclei reflects differences in GR mRNA levels. The rat GR cDNA clone is also shown to provide suitable probes for mapping GR gene expression in the mouse brain.