The neonatal glucocorticoid treatment-produced long-term changes of the pituitary-adrenal function and brain corticosteroid receptors in rats.

Two distinct periods of sensitivity to elevated glucocorticoid hormone levels during postnatal development of the pituitary-adrenal axis were studied. Wistar rats were injected subcutaneously (s.c.) with cortisol (1 mg/kg) on postnatal days 1-5 or 14-18. The steroid treatment during the first postnatal week resulted in a decrease of the morning basal and stress-induced plasma corticosterone levels in 30 day-old male rats, as well as in rats that were injected with cortisol on the third postnatal week. Stress-induced corticosterone levels in 90-day old cortisol-treated rats were determined in blood samples drawn from the tail vein before the restraint stress, immediately after the 20-min long stress, then 60 and 180 min afterwards. Only the rats treated with cortisol during the third week showed a prolonged stress-induced corticosterone secretion, with the highest corticosterone level in 180 min after the restraint stress. The early neonatal cortisol treatment had no effect on (3)H-corticosterone binding in all studied brain areas of the 90-day old rats. The rats treated with cortisol at the 14-17th postnatal days showed a significantly lower (3)H-corticosterone binding in the frontal cortex, hippocampus, and hypothalamus. These findings suggest that the third week of life in rats is more sensitive to elevated levels of corticosterone than the first one. The high level of glucocorticoids at this period has long-term effects on the efficiency of the negative feedback mechanisms provided by hypothalamus-pituitary-adrenal axis.     

Melatonin effects on glucocorticoid receptors in rat brain and pituitary: significance in adrenocortical regulation

1. The effects of chronic melatonin treatment on glucocorticoid binding sites in hippocampus, hypothalamus and pituitary were investigated in rats, subjected to long-term manipulation of circulating corticosterone concentrations. 2. Melatonin treatment decreased the affinity of glucocorticoid receptors. 3. The effect of melatonin was apparent in the presence of normal or enhanced systemic corticosterone levels, but not in long-term adrenalectomized animals.     

Phosphorylation-dephosphorylation of muscarinic acetylcholine receptors: evidence for the in vivo and in vitro release of receptors from rat brain plasma membrane

The effects of phosphorylation on muscarinic acetylcholine receptors (mAChRs) were studied in vitro and in vivo using rat brain plasma membrane and receptors partially purified at least 2500-fold. Purified mAChRs were phosphorylated in vitro by cAMP-dependent protein kinase and dephosphorylated by calcineurin. Phosphorylation of purified mAChRs was enhanced by carbachol and blocked by atropine. The filtrate which passed through glass fiber filters and high speed supernates were assayed for mAChRs by an ammonium sulfate precipitation method. Following incubation of the plasma membrane under phosphorylating conditions and ultracentrifugation at 300,000 g, the mAChRs appeared in the high speed supernate. This release was stimulated by adding carbachol to the incubation medium. In rats treated with carbachol, brain mAChRs redistributed from the heavy into the light membrane fractions. Ultrastructural examination of the light membrane fractions and the 300,000 g supernatant fractions after in vivo and in vitro carbachol treatment calcineurin increased the reincorporation of added partially purified receptors into the plasma membrane. The release and reincorporation of mAChRs strongly imply that there is a translocation and recycling of mAChRs between plasma membrane and cytosol in vivo. The significance and the function of the translocation of mAChRs remain to be investigated.     

On the role of glucocorticoid receptors in brain plasticity

Summary 1. The mapping of glucocorticoid receptors (GR) in the rat central nervous system (CNS) has demonstrated their widespread presence in large numbers of nerve and glial cell populations also outside the classical stress regions.2. The present paper summarizes the evidence that glucocorticoids via GR in the CNS can act as lifelong organizing signals from development to aging. The following examples are given. (a) In the prepubertal and adult offspring, prenatal corticosterone treatment can produce long-lasting changes in striatal dopaminergic communication. (b) In adulthood, the evidence suggests complex regulation by adrenocortical hormones of neurotrophic factors and their receptors in the hippocampal formation. (c) In aging, the strongly GR-immunoreactive pyramidal cell layer of the CA1 hippocampal area appears to be preferentially vulnerable to neurotoxic actions of glucocorticoids, especially in some rat strains.3. Strong evidence suggests that each nerve cell in the CNS is supported by a trophic unit, consisting of other nerve cells and glial cells, blood vessels, and extracellular matrix molecules. Due to multiple actions on nerve and glial cell populations of the different trophic units, the glucocorticoids may exert either an overall trophic or a neurotoxic action. It seems likely that with increasing age, the endangering actions of glucocorticoids on nerve cells prevail over the neurotrophic ones, leading to reduced nerve cell survival in some trophic units.     

Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reserpine-induced central effects: pharmacological evidence for the lack of central effects of reserpine methiodide

Reserpine, an alkaloid from Rauwolfia serpentina, was widely used for its antihypertensive action. However, its use has been reduced because of its sedative and extra pyramidal symptoms. In the present investigation, reserpine methiodide (RMI), a quaternary analogue of reserpine, was synthesized and pharmacologically evaluated in rats and mice for its central (barbiturate hypnosis, spontaneous motor activity, body temperature, and avoidance of conditioned response) and peripheral actions (blood pressure) in comparison with reserpine. The results indicate that reserpine produced a dose-dependent depression of the central nervous system. RMI at doses equal to and double the equimolar doses of reserpine did not produce any behavioural changes compared with control animals. Nevertheless, both reserpine and RMI were found to produce dose-dependent reduction in the blood pressure of anaesthetized rats, although only at higher doses of RMI, indicating that quaternization of reserpine not only attenuated the entry of RMI into the central nervous system, but also reduced its access to the target tissue in the periphery. It is speculated that the hypotensive actions of RMI may also be due to peripheral depletion of catecholamines.     

Rapid glucocorticoid effects on immune cells

Apart from their classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomically mediated effects. Three different mechanisms are currently under discussion as being responsible for these effects: (1) specific interaction with the cytosolic glucocorticoid receptor (cGCR), (2) nonspecific interactions with cellular membranes and (3) specific interactions with membrane-bound glucocorticoid receptors (mGCR). With regard to the first mechanism, there is evidence that although the binding of glucocorticoids to the cGCR-associated multi-protein complex induces the further processes of the classic path, it also leads to a rapid intracellular signalling through other components of the complex (e.g. Src). For the second mechanism, a nonspecific interactive effect with cellular membranes through the intercalation of glucocorticoid molecules is being discussed, which primarily alters cellular functions by influencing cation transport via the plasma membrane and by increasing the proton leak of the mitochondria. With regard to the third, mGCR-mediated mechanism, the first evidence has now been found to suggest a physiological expression of membrane-bound glucocorticoid receptors on human cells, whereas in humans this had previously only been demonstrated on lymphoma cells. The clinical importance and therapeutic relevance of these rapid glucocorticoid effects remains unclear at present, although effects on intracellular signalling, interferences with bioenergetically relevant cell functions and the induction of apoptosis via the mGCR are being discussed. This article gives a detailed presentation of the data available at present concerning rapid glucocorticoid effects on immune cells.     

Ginsenoside RG1 and corticosteroid receptors in rat brain

Old (28 months) male Wistar rats were treated chronically for two weeks with ginsenoside Rg1 or with vehicle delivered via sc implanted Alzet mini-pumps (rate of ginsenoside release 2.4 micrograms/0.5 microliter/h). The number of Type 1 corticosterone-preferring receptor sites (CR) and Type 2 glucocorticoid receptors (GR) was measured in the cytosol of hippocampus tissue of rat brain with an in vitro binding assay. In old rats the Bmax of Type 1 CR and Type 2 GR was reduced by 51.5% and 28.3% respectively. Following the two week treatment with Rg1 the Bmax of Type 1 CR increased by 60% and a receptor concentration was reached which was 21% lower than that observed in the young control animals. Minor differences in affinity of steroid binding to both receptor systems were observed in the groups of rats. The possible binding of ginsenosides to brain corticosteroid receptors in vitro was investigated as well. The inclusion of a 500 fold molar excess of Rg1 in hippocampus cytosol did not displace 3H-corticosterone from its soluble receptor sites. The affinity of Rg1 with these sites in vitro is therefore negligible. In conclusion, the binding capacity of Type 1 CR and Type 2 GR is reduced in the hippocampal brain region of aged rats. Upon chronic infusion of ginsenoside Rg1, only Type 1 CR capacity is restored towards the level observed in young control animals. This finding suggests that in old rats the ginsenoside enhances the CORT signal via Type 1 CR on the function of the hippocampus, which is a limbic brain structure involved in cognition, mood and affect.     

Insulin receptors in rat brain cortex. Kinetic evidence for a receptor subtype in the central nervous system

Binding kinetics of porcine ¹²⁵I-insulin were studied in synaptosomal and microsomal fractions of rat brain cortex. Receptor binding was temperature- and pH-dependent with optimum at 4°C and pH 8.0–8.3. At 15°C, steady state binding was heterogenous, and Scatchard analysis revealed two classes of receptors with K_d of 2 nmol/l and 40 nmol/l in amounts of 50 pmol/g and 200 pmol/g of membrane protein. Dissociation kinetics were biexponential with $\text{T}\text{1}\text{2}$ of about 5 min and 180 min, and in contrast to other cell-types, not influenced by negative cooperativity. No receptor-mediated insulin degradation was detectable at 37°C in the presence of bacitracin. Insulin analogues inhibited ¹²⁵I-insulin binding with potencies relative to porcine insulin (%): human insulin 100, rat insulin (I+II) 71, coypu insulin 47, rat multiplication stimulating activity 8, porcine proinsulin 5, among which the three last values were significantly higher than in rat liver and fat cells. No competition was observed with porcine relaxin and mouse nerve growth factor up to about 1 μmol/l. Receptors were present in all regions of central nervous system with highest concentrations in the cerebral cortex, cerebellum and olfactory bulb, and lowest in the pons, medulla oblongata and spinal cord. In conclusion, insulin receptors in rat brain cortex are functionally different from other tissues regarding the insulin specificity and the absence of negative cooperativity. It is suggested that an insulin receptor subtype in rat brain mediates the growth activity of insulin on nerve cells.     

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.     

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.     

Murine glucocorticoid receptors: new evidence for a discrete receptor influenced by H-2

The glucocorticoid receptor contents in the lungs of females of two congenic strains of mice, B10.A (H-2a) and B10 (H-2b), differing only in the H-2 histocompatibility region of chromosome 17, have been measured by the dextran-charcoal method and by our previously described methods of molecular sieving and ion exchange chromatography [M. Katsumata, C. Gupta, and A. S. Goldman (1985) Arch. Biochem. Biophys. 243, 385-395]. As reported, two receptors, II and IB, are demonstrable by each column chromatographic method, and 5,5-diphenylhydantoin binds to receptor IB but not to receptor II. Receptor IB cannot be detected unless molybdate is added in cytosols prepared with hypotonic buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 10 mM dithiothreitol, pH 7.35) according to S. L. Liu, J. F. Grippo, R. P. Erickson, and W. B. Pratt (1984) J. Steroid Biochem. 21, 633-637], a method which has been reported to give maximal receptor levels. Using hypotonic buffer containing 10 mM molybdate we observed a small but significantly higher content of receptor IB in B10.A mice than that in B10 mice, but no significant difference in receptor II or total receptor content. On the other hand, cytosols prepared with isotonic buffer (50 mM Tris-HCl, 120 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and 10 mM molybdate, a modification of the buffer used in our previous report) contained significantly higher levels of receptor IB and of total binding in pulmonary cytosols of B10.A as compared to those of B10. There was no difference in receptor II content. Molybdate stabilizes receptor IB in both buffers. These results explain the apparent contradiction between our results and those of Liu et al. by showing that the hypotonic buffer used by them allows for determination of maximal levels of receptor II, but permits selective destruction of receptor IB. However, the use of isotonic buffer gives maximal values of both receptors II and IB. With isotonic buffer, it is demonstrated that only the level of receptor IB is influenced by H-2-linked genes.     

Physicochemical properties of type I corticosteroid receptors from rat brain

[3H]Aldosterone binds with high affinity to Type I corticosteroid receptors in cytosols from adrenalectomized rat forebrains. Physicochemical parameters of these receptors were determined in the presence of molybdate, which stabilized receptors and maintained them in a presumably untransformed state. The Stokes' radius of the molybdate-stabilized receptor was 8.1 nm, as determined by gel filtration on Sephacryl S-300. Its sedimentation coefficient was 9.1S in linear sucrose density gradients. The receptor is asymmetric, with an axial ratio of 8-10 and an apparent mol. wt of 303,000 dalton. The [3H]aldosterone-receptor complex is anionic and elutes from DEAE-Trisacryl in a single peak with a maximum at 160 mM KCl. Exposure to heat or salt in the absence of molybdate, conditions which transform other steroid receptors to smaller DNA-binding forms, causes marked instability of the [3H]aldosterone-receptor complex. The [3H]aldosterone-binding protein of rat forebrain, which displays the binding characteristics of a renal Type I (mineralocorticoid) receptor, is similar in size, shape and charge to the molybdate-stabilized oligomeric forms of other steroid hormone receptors.     

Regulation of glucocorticoid receptors in human mononuclear cells: effects of glucocorticoid treatment, Cushing's disease and ketoconazole

Glucocorticoid receptors (GcR) were determined by a whole cell assay in human mononulear leukocytes (hMNL) from control subjects, patients receiving glucocorticoid therapy for systemic diseases and Cushing's disease patients with or without ketoconazole therapy. Prolonged corticosteroid treatment resulted in down-regulation of GcR, while the mean level of GcR in Cushing's disease was normal. In this group, however, receptor levels and morning plasma cortisol values showed a negative correlation, indicating a subtle down-regulatory effect. Furthermore, GcR were unaltered after these patients received ketoconazole, in spite of a marked reduction in morning plasma cortisol and urinary free cortisol. We also observed that ketoconazole was a weak competitor of GcR in intact cells, although it significantly inhibited [3H] dexamethasone binding in cytosolic preparations from rat tissues. The results suggested that GcR in hMNL are down-regulated by synthetic steroids given in vivo, but they showed very mild down-regulation in hypercortisolemic patients suffering from Cushing's disease. Finally, we did not observed either up-regulation or antagonism of GcR by ketoconazole treatment, at the time that cortisol levels of patients with Cushing's disease were reduced. This indicates that the beneficial effects of ketoconazole in Cushing's disease are due to adrenal cortisol suppression and not to interaction with GcR of target cells, and that the process of GcR regulation in hMNL is a complex phenomenon awaiting further elucidation.     

Synthesis of novel arylpyrazolo corticosteroids as potential ligands for imaging brain glucocorticoid receptors

Corticosteroids regulate a variety of essential physiological functions, such as mineral balance and stress. The great interest in these steroids, especially the glucocorticoids, stems from roles they are thought to play in neuropsychiatric disorders, such as severe depression and anxiety.The development of glucocorticoid receptor (GR) ligands which are appropriately labeled with short-lived positron-emitting radioisotopes would allow the non-invasive in-vivo imaging and mapping of brain GRs by means of positron emission tomography (PET). In this context we have synthesized a series of novel arylpyrazolo steroids exhibiting different substitution patterns at the D-ring of the steroid skeleton, as ligands for brain GRs. Special attention was given to 4-fluorophenyl pyrazolo steroids, which are known to display high binding affinity toward the GR. The compounds were evaluated in a competitive radiometric receptor binding assay to determine their relative binding affinities (RBA) to the GR. Some compounds show good binding affinities of up to 56% in comparison to dexamethasone (100%). In initial experiments, selected candidates were labeled with the positron emitter fluorine-18 and in one case with the gamma-emitter iodine-131.     

Participation of the dopaminergic brain system in effects of glucocorticoid hormones

Following the conditioning with dexamethasone, a dose-dependent place preference in non-preferred compartment was observed on the second test day in male Wistar rats. Amphetamine in subthreshold dose exerted no effect if administered alone and induced a place preference in an unbiased paradigm after pre-treatment with dexamethasone. Administration of D2-dopamine receptors' antagonist sulpiride 30 min prior to dexamethasone conditioning completely blocked the acquisition of the place preference. The D1-dopamine receptors' antagonist SCH23390 exerted no effect on the place conditioning. The findings suggest that the D2-dopamine receptors take part in conditioned place preference with dexamethasone.