Two receptor systems for corticosterone (CORT) can be distinguished in rat brain: mineralocorticoid-like or CORT receptors (CR) and glucocorticoid receptors (GR). The microdistribution and extent of occupation of each receptor population by CORT were studied. The CR system is restricted predominantly to the lateral septum and hippocampus. Within the hippocampus, the highest density occurs in the subiculum +/- CA1 cell field (144 fmol/mg protein) and the dentate gyrus (104 fmol/mg protein). Affinity of CR for CORT was very high (Kd, approximately 0.5 nM). The GR system has a more widespread distribution in the brain. The highest density for GR is in the lateral septum (195 fmol/mg protein), the dentate gyrus (133 fmol/mg protein), the nucleus tractus solitarii and central amygdala. Substantial amounts of GR are present in the paraventricular nucleus and locus coeruleus and low amounts in the raphe area and the subiculum + CA1 cell field. The affinity of GR for CORT (Kd, approximately 2.5-5 nM) was 6- to 10-fold lower than that of CR. Occupation of CR by endogenous ligand was 89.5% during morning trough levels of pituitary-adrenal activity (plasma CORT, 1.4 micrograms/100 ml). Similar levels of occupation (88.7% and 97.6%) were observed at the diurnal peak (plasma CORT, 27 micrograms/100 ml) and after 1 h of restraint stress (plasma CORT, 25 micrograms/100 ml), respectively. Furthermore, a dose of 1 microgram CORT/100 g BW, sc, resulted in 80% CORT receptor occupation, whereas GR were not occupied. For 50% occupation of GR, doses needed to be increased to 50-100 micrograms/100 g BW, and for 95% occupation, a dose of 1 mg CORT was required. The plasma CORT level at the time of half-maximal GR occupation was about 25 micrograms/100 ml, which is in the range of levels attained after stress or during the diurnal peak of pituitary-adrenal activity. Thus, CR are extensively filled (greater than 90%) with endogenous CORT under most circumstances, while GR become occupied concurrent with increasing plasma CORT concentrations due to stress or diurnal rhythm. We conclude that CORT action via CR may be involved in a tonic (permissive) influence on brain function with the septohippocampal complex as a primary target. In view of the almost complete occupation of CR by endogenous hormones, the regulation of the CORT signal via CR will, most likely, be by alterations in the number of such receptors. In contrast, CORT action via GR is involved in its feedback action on stress-activated brain mechanisms, and GR occur widely in the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

In the studies reported here we have examined the role of the medial prefrontal cortex (MpFC) in regulating hypothalamic-pituitary-adrenal (HPA) activity under basal and stressful conditions. In preliminary studies we characterized corticosteroid receptor binding in the rat MpFC. The results revealed high-affinity (Kd approximately 1 nM) binding with a moderate capacity (42.9 +/- 3 fmol/mg) for 3H-aldosterone (with a 50-fold excess of cold RU28362; mineralocorticoid receptor) and high-affinity (Kd approximately 0.5-1.0 nM) binding with higher capacity (183.2 +/- 22 fmol/mg) for 3H-RU 28362 (glucocorticoid receptor). Lesions of the MpFC (cingulate gyrus) significantly increased plasma levels of both adrenocorticotropin (ACTH) and corticosterone (CORT) in response to a 20 min restraint stress. The same lesions had no effect on hormone levels following a 2.5 min exposure to ether. Implants of crystalline CORT into the same region of the MpFC produced a significant decrease in plasma levels of both ACTH and CORT with restraint stress, but again, there was no effect with ether stress. Neither MpFC lesions nor CORT implants had any consistent effect on A.M. or P.M. levels of plasma ACTH or CORT. Manipulations of MpFC function were not associated with changes in the clearance rate for CORT or in corticosteroid receptor densities in the pituitary, hypothalamus, hippocampus, or amygdala. Taken together, these findings suggest that MpFC is a target site for the negative-feedback effects of glucocorticoids on stress-induced HPA activity, and that this effect is dependent upon the nature of the stress.

The orphan receptor CAR-beta binds DNA as a heterodimer with the retinoid-X receptor and activates gene transcription in a constitutive manner. Here we show that, in contrast to the classical nuclear receptors, the constitutive activity of CAR-beta results from a ligand-independent recruitment of transcriptional co-activators. While searching for potential ligands of CAR-beta, we found that the steroids androstanol and androstenol inhibit the constitutive activity of CAR-beta. This effect is stereospecific: only 3alpha-hydroxy, 5alpha-reduced androstanes are active. These androstanes do not interfere with heterodimerization or DNA binding of CAR-beta; instead, they promote co-activator release from the ligand-binding domain. These androstane ligands are examples of naturally occurring inverse agonists that reverse transcriptional activation by nuclear receptors. CAR-beta (constitutive androstane receptor-beta), therefore, defines an unanticipated steroidal signalling pathway that functions in a manner opposite to that of the conventional nuclear receptor pathways.

Development of prostatic hyperplasia is an almost universal feature of the aging man and dog, and in both species the process develops only in males with intact testes. As the result of studies of plasma hormone levels as a function of age, measurements of the concentration of androgen and of androgen receptor proteins within the prostate, and studies of the effects of the administration of various hormones on growth of the prostate in the castrated dog, it is possible to provide a working hypothesis as to the pathogenesis. Dihydrotestosterone accumulation within the gland serves as the hormonal mediator for the hyperplasia in both species; the accumulation probably occurs in part because of decreased catabolism of the molecule and in part because of enhanced intracellular binding of the molecule. The process is accelerated by estrogen, which enhances the level of the androgen receptor in the gland; increase in the androgen receptor allows for androgen-mediated growth even in the face of declining androgen production in advanced age. On theoretic grounds the therapeutic implications of this model are exciting; several potential medical treatments may be feasible that do not involve a chemical castration.

The rat brain contains two receptor systems for corticosterone (CORT): the glucocorticoid (GR) and corticosterone or mineralocorticoid-like (CR) receptor sites. We have studied the localization of these receptors by in vitro autoradiography and by in vitro cytosol binding assays in microdissected brain areas. In vitro autoradiography revealed that CR receptor sites are almost entirely restricted to the septal-hippocampal complex, whereas the presence of GR extends throughout the brain. Highest levels of GR are present in the lateral septum, hippocampal, cortical and thalamic regions and the paraventricular nucleus. In vitro determination of binding of 3H-labelled steroids to CR and GR in cytosol of "punched out" brain tissue revealed a similar neuroanatomical distribution as observed with the autoradiographic analysis. In addition, it was found that CORT binds to CR (KD approximately 0.5 nM) with 5-10-fold higher affinity than to GR (KD approximately 2.5-5 nM).

The ontogeny of the corticoid receptors in the rat hippocampus was examined by in vitro [3H]corticosterone (CORT) binding to soluble molecules in the cytosol, using the selective Type II glucocorticoid agonist, RU 28362, to discriminate between Type I and Type II receptor sites. Type I receptors were undetectable until 8 days after birth. From this age on, the receptor showed adult characteristics for both the binding capacity (Bmax) and affinity (Kd). The Type II receptor concentration increased gradually over the observed period; however, at 3 weeks of age concentrations were still only about 65% those found in adults. The binding affinity of Type II to CORT was high during the first week of life but decreased thereafter towards adult value. These data thus suggest clear distinctions in the developmental patterns of Type I and Type II receptors for corticosteroids in the rat.

We report here on developmental changes in [3H]RU 28362 (glucocorticoid receptor) and [3H]aldosterone (mineralocorticoid receptor) binding capacity in soluble fractions prepared from hippocampal tissue.[3H]RU 28362 binding was low on Day 3 of life (less than 30% of that observed in adults) and increased towards adult values during the second and third weeks of life, a pattern virtually identical to that previously reported for dexamethasone binding. In contrast,[3H]aldosterone binding on Day 3 of life was only slightly lower than that observed in adults and reached adult values by Day 7. Postnatal handling of rat pups, which has been shown to increase dexamethasone binding in hippocampus, resulted in a significant increase in [3H]RU 28362 binding capacity in hippocampus, but had no effect on [3H]aldosterone binding.

Quantitative in vitro autoradiography, cytosol receptor assay in punched brain tissue, and immunocytochemistry have revealed that the glucocorticoid receptor is present in the rat supraoptic nucleus (SON). Based on its binding characteristics the receptor appears to be the type II glucocorticoid receptor. With the use of a monoclonal antibody against purified liver glucocorticoid receptor, immunostaining was found in magnocellular neurosecretory neurons in the SON, but not in magnocellular neurons in the paraventricular nucleus. Immunoreactive cells seem to be concentrated in ventral parts of the SON where vasopressin cells were previously shown to be located. One to 2 weeks after bilateral adrenalectomy, there was a substantial decrease in glucocorticoid receptor immunostaining in magnocellular as well as other types of neurons in various brain regions. Administration of synthetic glucocorticoids (RU 28362 or dexamethasone) induced a robust increase in the intensity of immunostaining in cell nuclei of neurosecretory cells. The presence of glucocorticoid receptors in the SON suggests that glucocorticoids may affect vasopressin synthesis or/and secretion through a direct action on magnocellular neurons.

A series of studies was started to gain insight into the functioning of the canine hypothalamo-pituitary-adrenocortical axis during normo- and hypercortisolemic states. In this first study, we have focused on the binding characteristics of the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) in the brain and pituitary of the adrenalectomized dog. In hippocampal cytosol at 0 C, corticosterone had the highest association rate, followed by cortisol and aldosterone. Cortisol had the most rapid rate of dissociation from MR at 0 C (t1/2 = 45.5 h), followed by aldosterone (70.4 h) and corticosterone (102 h). The selective glucocorticoid RU 28362 associated rapidly with hippocampal GR, attaining maximum binding within 4 h, and dissociated with a t1/2 of 34.8 h. Saturation binding of [3H]cortisol in adrenalectomized dog hippocampal cytosol produced a curvilinear Scatchard plot. After inclusion of RU 28362,[3H]cortisol bound solely to MR [dissociation constant (Kd)= 0.34 nM, Bmax = 72.8 fmol/mg]. GR capacity was determined with [3H]RU 28362 (Kd = 0.39 nM, Bmax = 120 fmol/mg). Competition binding analyses of various steroids for MR and GR revealed markedly different patterns of steroid binding specificity for these receptors. The rank order for displacement of [3H]aldosterone binding of MR was: corticosterone greater than aldosterone = cortisol greater than dexamethasone greater than ZK 91587 greater than RU 26752 greater than spironolactone much greater than RU 38486, and for displacement of [3H]RU 28362 binding of GR: RU 28362 much greater than corticosterone = cortisol greater than dexamethasone greater than aldosterone greater than ZK 91587 greater than RU 26752 = RU 38486 much greater than spironolactone. MR was located in all brain regions examined, with highest levels in the septo-hippocampal complex, whereas GR was rather evenly distributed. Substantial amounts of MR and GR were present in the anterior part of the pituitary as well as in the neurointermediate lobe. Our findings show that the ligand binding specificity of canine MR and GR is remarkably different from that of rodent MR and GR, but is similar to that of recombinant-derived human receptors. Spironolactone and RU 38486 are selective antagonists for MR and GR, respectively. In contrast to other species, the dog has relatively large quantities of MR widely distributed in the brain and pituitary, which makes this species an interesting animal model to study the role of corticosteroid receptor diversity in control of homeostasis.