Paecilomyces tenuipes extract prevents depression-like behaviors in chronic unpredictable mild stress-induced rat model via modulation of neurotransmitters.

The medicinal fungus Paecilomyces tenuipes exhibits a variety of pharmacological effects, including antidepressive effects. The chronic unpredictable mild stress (CUMS)­induced rat model has served an important role in studies involving antidepressants screening. The aim of the present study was to evaluate the antidepressant­like activity of P. tenuipes N45 aqueous extract (PTNE) in a CUMS­induced rat model of behavioral despair depression. Following 4 weeks of PTNE treatment, behavioral tests were conducted to investigate the antidepressant­like activities, and the levels of neurotransmitters and hormones in blood and hypothalamus were measured. The results demonstrated that PTNE treatment significantly increased movement in the forced running test, whereas the immobility time was reduced in the hotplate test and the forced swim test in depression­model rats. PTNE treatment was able to normalize the levels of hormones and neurotransmitters in serum and hypothalamus of CUMS rats. The data demonstrated that PTNE treatment may be a potential pharmaceutical agent in treatment­resistant depression, and the effects of PTNE may be partly mediated through normalizing the levels of neurotransmitters.

The role of mediastinal adipose tissue 11ß-hydroxysteroid d ehydrogenase type 1 and glucocorticoid expression in the development of coronary atherosclerosis in obese patients with ischemic heart disease.

BACKGROUND: Visceral fat deposition and its associated atherogenic complications are mediated by glucocorticoids. Cardiac visceral fat comprises mediastinal adipose tissue (MAT) and epicardial adipose tissue (EAT), and MAT is a potential biomarker of risk for obese patients. AIM: Our objective was to evaluate the role of EAT and MAT 11beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1) and glucocorticoid receptor (GCR) expression in comparison with subcutaneous adipose tissue (SAT) in the development of coronary atherosclerosis in obese patients with coronary artery disease (CAD), and to assess their correlations with CD68 and fatty acids from these tissues. METHODS AND RESULTS: Expression of 11ß-HSD-1 and GCR was measured by qRT-PCR in EAT, MAT and SAT of thirty-one obese patients undergoing coronary artery bypass grafting due to CAD (obese CAD group) and sixteen obese patients without CAD undergoing heart valve surgery (controls). 11ß-HSD-1 and GCR expression in MAT were found to be significantly increased in the obese CAD group compared with controls (p < 0.05). In the obese CAD group, 11ß-HSD-1 and GCR mRNA levels were strongly correlated in MAT. Stearidonic acid was significantly increased in EAT and MAT of the obese CAD group and arachidonic acid was significantly expressed in MAT of the obese male CAD group (p < 0.05). CONCLUSIONS: We report for the first time the increased expression of 11ß-HSD-1 and GCR in MAT compared with EAT and SAT, and also describe the interrelated effects of stearidonic acid, HOMA-IR, plasma cortisol and GCR mRNA levels, explaining 40.2% of the variance in 11ß-HSD-1 mRNA levels in MAT of obese CAD patients. These findings support the hypothesis that MAT contributes locally to the development of coronary atherosclerosis via glucocorticoid action.

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS.

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.

Changes in basal hypothalamo-pituitary-adrenal activity during exercise training are centrally mediated.

The effects of exercise training on hypothalamo-pituitary-adrenal (HPA) function are unclear. We investigated whether pituitary-adrenal adaptation during exercise training is mediated by changes in neuropeptide and corticosteroid receptor gene expression in the brain and pituitary. Sprague-Dawley rats were subject to either daily swimming (DS) or sham exercise (SE) for 45 min/day, 5 days/week, for 2 (2W), 4 (4W), or 6 wk (6W) (n = 7-10/group). Corticosterone (Cort) and catecholamine responses during swimming were robust at 6W compared with 2W and 4W, indicating that HPA response to exercise during training is not attenuated when absolute intensity is progressively increased. In DS, basal (morning) plasma ACTH and Cort levels increased from 2W to 4W but plateaued at 6W, whereas in SE, they increased from 4W to 6W, with 6W values higher than in DS. In DS, there was a transient decrease in glucocorticoid receptor (GR) mRNA in the paraventricular nucleus (PVN) and pituitary and a transient increase in corticotrophin-releasing hormone (CRH) mRNA. In contrast, hippocampal mineralocorticoid receptor mRNA and PVN GR mRNA decreased from 4W to 6W in SE, with 6W values lower than in DS. These findings suggest that exercise training prevents an elevation in basal pituitary-adrenal activity potentially via transient alterations in the gene transcription of PVN and pituitary GR as well as CRH to suppress central drive to the HPA axis. In contrast, the increase in basal pituitary-adrenal activity with repeated sham exercise appears to be associated with decreases in hippocampal MR and PVN GR mRNA expression.

Analysis of the stress response in rats trained in the water-maze: differential expression of corticotropin-releasing hormone, CRH-R1, glucocorticoid receptors and brain-derived neurotrophic factor in limbic regions.

Glucocorticoids and corticotropin-releasing hormone (CRH) are key regulators of stress responses. Different types of stress activate the CRH system; in hypothalamus, CRH expression and release are increased by physical or psychological stressors while in amygdala, preferentially by psychological stress. Learning and memory processes are modulated by glucocorticoids and stress at different levels. To characterize the kind of stress provoked by a hippocampal-dependent task such as spatial learning, we compared the expression profile of glucocorticoid receptor (GR), pro-CRH and CRH-R1 mRNAs (analyzed by RT-PCR), in amygdala, hippocampus and hypothalamus and quantified serum corticosterone levels by radioimmunoassay at different stages of training. mRNA levels of brain-derived neurotrophic factor (BDNF) were also quantified due to its prominent role in learning and memory processes. Male Wistar rats trained for 1, 3 or 5 days in the Morris water-maze (10 trials/day) were sacrificed 5-60 min the after last trial. A strong stress response occurred at day one in both yoked and trained animals (increased corticosterone and hypothalamic pro-CRH and CRH-R1 mRNA levels); changes gradually diminished as the test progressed. In amygdala, pro-CRH mRNA levels decreased while those of BDNF augmented when stress was highest, in yoked and trained animals. Hippocampi, of both yoked and trained groups, had decreased levels of GR mRNA on days 1 and 3, normalizing by day 5, while those of pro-CRH and CRH-R1 increased after the 3rd day. Increased gene expression, specifically due to spatial learning, occurred only for hippocampal BDNF since day 3. These results show that the Morris water-maze paradigm induces a strong stress response that is gradually attenuated. Inhibition of CRH expression in amygdala suggests that the stress inflicted is of physical but not of psychological nature and could lead to reduced fear or anxiety.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Corticosterone infused intracerebroventricularly inhibits energy storage and stimulates the hypothalamo-pituitary axis in adrenalectomized rats drinking sucrose.

When allowed to drink sucrose, bilaterally adrenalectomized (ADX) rats exhibit normal weight gain, food intake, sympathetic neural activity, and ACTH compared with sham-ADX rats. Furthermore, ADX rats drinking sucrose have normal corticotropin-releasing factor (CRF) mRNA throughout brain. In ADX rats without sucrose, all of these variables are abnormal. Systemic corticosterone (B) replacement also restores these variables in ADX rats to normal. To test whether B acts centrally, we infused B or saline intracerebroventricularly into ADX rats under basal conditions and after repeated restraint. Rats were exposed to no stress or 3 h/d restraint for 3 d. Body weights and food and fluid intakes were measured. Brains were analyzed using immunocytochemistry against glucocorticoid receptors (GR) and CRF. Intracerebroventricular B blocked the positive effects of sucrose on metabolism, increased basal ACTH concentrations, and augmented ACTH responses to restraint on d 3. B-infused rats exhibited nuclear GR staining in perirhinal cortex, hippocampus, and hypothalamic paraventricular nuclei, showing that infused B spreads effectively. CRF staining in the paraventricular nucleus of the hypothalamus was higher in B- than in saline-infused rats. We conclude that under basal conditions B acts systemically, but not in the brain, to restore metabolism and neuropeptides after adrenalectomy. By contrast, tonic GR occupancy in brain initiates metabolic and ACTH responses characteristic of stress.

Influence of estradiol on NADPH diaphorase/neuronal nitric oxide synthase activity and colocalization with progesterone or type II glucocorticoid receptors in ovine hypothalamus.

Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.

Postnatal endotoxin exposure results in increased insulin sensitivity and altered activity of neuroendocrine axes in adult female rats.

OBJECTIVES: Severe postnatal infection leads to a systemic inflammatory response with release of cytokines and glucocorticoids, representing a stressful event for the newborn child. The purpose of this study was to mimic this situation and to study the effects of early postnatal endotoxin exposure of female rat pups on metabolic, endocrine and anthropometric variables in adulthood. DESIGN: Female pups were given subcutaneous injections of lipopolysaccharides (LPS; Salmonella enteriditis, 0.05 mg/kg) or vehicle 3 and 5 days after birth. RESULTS: Six hours after injection, LPS-treated rats had higher corticosterone levels than controls. As adults, LPS-exposed female rats showed increased insulin sensitivity (P<0.05), measured with the hyperinsulinemic euglycemic clamp (5 mU/kg per min). They exhibited a higher locomotor activity (P<0.05) and increased skeletal muscle mass in comparison with controls (P<0.05). Basal ACTH and corticosterone levels in LPS-treated rats were elevated (P<0.05), as were corticosterone levels after exposure to a novel environment stress (P<0.05). The adrenals were morphologically changed and enlarged (P<0.05) in LPS-exposed rats at 11 weeks of age, and a higher density of hypothalamic but not hippocampal glucocorticoid receptor protein was found in the LPS-treated rats (P<0.05). Furthermore, circulating progesterone levels were lower (P<0.05) and testosterone tended to be higher. CONCLUSION: The results indicate that postnatal exposure to LPS leads to increased insulin sensitivity in the adult female rat. In addition, LPS-treated rats showed changes in the regulation of hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes. This study suggests that postnatal exposure to an endotoxin such as LPS can induce specific programming of neuroendocrine regulation, with long-term consequences in adult life.

Regulation of hippocampal glucocorticoid receptor gene transcription and protein expression in vivo.

Glucocorticoid receptors (GRs) are glucocorticoid-activated transcription factors that modulate expression of a variety of neuronal genes. Appropriate control of GR expression is therefore critical for maintenance of cellular and organismic homeostasis. The present study assessed glucocorticoid regulation of the GR at the gene, mRNA, and protein level. Removal of circulating glucocorticoids (adrenalectomy) increased GR mRNA expression in CA1 and dentate gyrus (DG). Corticosterone (CORT) replacement normalized GR mRNA expression, whereas high doses slightly decreased GR mRNA in CA1. Parallel increases were observed using a probe complementary to the distal 3' untranslated region, indicating that mRNA changes were not attributable to selection of alternative polyadenylation site. Expression of a GR intronic sequence was also increased by adrenalectomy, consistent with increased gene transcription. Analysis of regional GR protein expression by immunoautoradiography did not reveal changes in GR protein in pyramidal cell layers; however, increased GR signal was seen in the stratum radiatum, indicating redistribution of GR to the cytosol. Western blot analysis confirmed adrenalectomy-induced increases in hippocampal GR levels. Administration of the mineralocorticoid receptor (MR) antagonist spironolactone increased both GR mRNA and protein in CA1 and DG, consistent with MR-mediated inhibition of GR transcription. However, high-dose CORT treatment did not decrease GR mRNA or protein levels. Chronic stress exposure did not downregulate GR mRNA or protein in hippocampus. The results suggest that the hippocampal GR is subject to heterologous regulation by the MR. In contrast, GR autoregulation is only evident during prolonged exposure to high-circulating glucocorticoid levels.

Continuous blockade of brain glucocorticoid receptors facilitates spatial learning and memory in rats.

Previously, a corticosterone surge associated with a learning task was shown to facilitate cognitive processes through brain glucocorticoid receptors (GR) while chronic overexposure to this stress hormone impaired cognition. In the present study we tested the hypothesis that opposing effects on learning and memory might also occur after either phasic or continuous blockade of brain GR by intracerebroventricular (i.c.v.) administration of the GR antagonist RU38486 (aGR). We used a Morris water maze procedure to assess spatial learning and memory abilities in male Wistar rats. The effect of phasic brain GR blockade was studied following daily pretraining administration of 10 and 100 ng/microL aGR i.c.v. on 3 consecutive days. This repetitive aGR treatment impaired spatial learning and memory dose-dependently in comparison with vehicle controls. For continuous brain GR blockade, animals received an i.c.v., infusion of aGR (10 and 100 ng/0.5 microL per h or vehicle) over 10 days. Infusion of 100 ng aGR per hour resulted in a long-lasting facilitation of spatial performance. The 10 ng aGR infusion also caused initially a facilitating effect, which was, however, transient and performance became impaired during retest. Possible anxiolytic properties of the drugs were excluded in view of the animals' behaviour in the elevated plus maze. Both doses of aGR infusion reduced the number of mineralocorticoid receptors in the hippocampus, but only the high dose of aGR resulted in a significant reduction of available GR sites. In conclusion, continuous administration of GR antagonist improves cognitive function, while phasic blockade of brain GR function causes a cognitive deficit.

Developmental changes and molecular weight of immunoreactive glucocorticoid receptor protein in the ovine fetal hypothalamus and pituitary.

Parturition in the sheep is initiated by increased activity of the hypothalamus-pituitary-adrenal (HPA) axis in the fetus. Late in gestation, ACTH secretion increases because of increased stimulation to the HPA axis and a decreased cortisol negative feedback inhibition of ACTH secretion. We hypothesized that this alteration in cortisol negative feedback efficacy might result from a decrease in glucocorticoid receptor (GR) protein in the hypothalamus or pituitary. Fetal hypothalami and pituitaries were collected from healthy animals (74 days gestation to 4 weeks postnatally) and analyzed by Western blot for immunoreactive GR (iGR) protein. We detected two forms of iGR: a form consistent in molecular weight with the GR, and a form which is approximately half the size of the full-length receptor. We were not able to detect a developmental change in the intensity of immuno-staining for the full-length receptor protein in these tissues. However, the proportion of half-length relative to full-length GR increased significantly with developmental age. Exogenous estrogen, androgen, or tamoxifen treatment did not significantly affect the expression of GR protein. In conclusion, we detected a 97 kD iGR protein corresponding to the full-length receptor and a 45 kD iGR protein, or half-length receptor. The proportion of this half-length receptor protein relative to the full-length receptor increased significantly with development.

Glucocorticoids inhibit D1B, but not D2, receptor-mediated effects on hypothalamic atrial natriuretic factor neurons.

Recent evidence suggests that ANF neurons of the hypothalamus are dopamine sensitive, and the catecholamine may exert a direct stimulatory or inhibitory effect on the neurons mediated through D1 or D2 receptors, respectively, in a manner related to the differential dopamine binding sensitivity of the two receptor subtypes. Employing well characterized ANF RIA and colorimetric Northern blot analysis with synthetic oligonucleotide probes complementary to pro-ANF messenger RNA (mRNA), we report here the effect of dexamethasone (DM), a potent synthetic glucocorticoid, on DA-stimulated ANF neurons in long term primary cultures of neonatal rat hypothalamic cells. Although DM alone did not affect basal secretion of immunoreactive ANF, it approximately halved immunoreactive ANF secretion induced by D1 agonist, SKF38393 (P < 0.01). The effect of DM was both time dependent and dose related, with an EC50 of 0.1 nM; it was blocked by 100 nM RU38486 (P < 0.05), a glucocorticoid receptor antagonist, but not by 100 nM RU28318, a mineralocorticoid receptor antagonist. In addition, the effect of DM was mimicked by corticosterone (EC50, 10 nM), but not deoxycorticosterone. The increased expression of pro-ANF mRNA signal induced by the D1 agonist in culture was suppressed by DM in a similar manner. In contrast, DM did not modulate ANF production and secretion induced by D2 agonist, quinpirole. Furthermore, reverse transcription-polymerase chain reaction demonstrated that D1B, but not D2, receptor mRNA expression was selectively suppressed by glucocorticoids. Thus, we conclude that in monolayer cultures of rat hypothalamic neurons, glucocorticoids differentially modulate dopamine receptor-induced responsiveness of ANF neurons by down-regulating D1B, but not D2, receptor-mediated changes. Hence, in severe stress, high levels of circulating glucocorticoids may negate the D1B-induced stimulatory response but allow dopamine to suppress the function of hypothalamic ANF neurons through D2 receptor activation.

Immune and endocrine aspects of social and territorial behavior in male rabbits.

Although there have been some studies of the relation between behavior and mitogen-induced lymphocyte proliferation and immunoglobulin synthesis, few data are available about the effect of behavior on specific lymphokine production. In this study, we describe the effect of social and territorial behaviors on interferon-gamma (IFN-gamma) production by concanavalin A-stimulated peripheral blood mononuclear cells (PBMCs) in pairs of socially naive male rabbits living in a seminatural open-air environment. We also assayed PBMC glucocorticoid receptors (GcRs) and plasma corticosterone (C). Three groups of behaviors were identified: agonistic (Mount and Follow), affiliative (Groom) and territorial (Mark and Dig). Mount was correlated with Follow, while Mark was correlated with Dig. Groom was correlated with all the other behaviors. Groom, Mark, Mount and Follow were all positively correlated with PBMC GcRs. Groom and PBMC GcRs were each negatively correlated with plasma C. The two rabbits in each pair could be distinguished in terms of territorial behavior, since one animal always had a higher score. The animals with the higher level of territorial behavior within the pairs exhibited a significant increase in IFN-gamma production at the end of the experimental period. They also showed a positive correlation between the percentage variations of IFN-gamma production and PBMC GcRs. It is suggested that social factors, especially territorial behavior, affect adrenocortical activity and IFN-gamma production.

Bombesin receptor gene expression in rat pancreatic acinar AR42J cells: transcriptional regulation by glucocorticoids.

BACKGROUND/AIMS: This study investigated the correlation between glucocorticoid-regulated gene expression of the bombesin receptor (BR) and cellular sensitivity to bombesin stimulation in the rat pancreatic acinar cell line AR42J. METHODS: BR gene expression was assessed using a cloned complementary DNA probe and radioligand binding assays. Intracellular Ca2+ mobilization was assessed by dual wavelength spectrophotometry using fura-2 in single cells. RESULTS: Dexamethasone resulted in a rapid dose- and time-dependent decrease of BR messenger RNA levels with maximal inhibition to 25% +/- 2% of controls (n = 4) after 6 hours of hormone treatment. BR messenger RNA half-life was approximately 120 minutes and was not affected by dexamethasone pretreatment; nuclear run-on analysis showed a decreased transcription rate of the BR to approximately 25% of control after hormonal treatment. Radioligand binding studies showed a time-dependent decrease of specific bombesin binding to 25% +/- 8% of control after 48 hours of hormone treatment. Down-regulation of BR gene expression by dexamethasone resulted in a time- and dose-dependent decrease of intracellular Ca2+ mobilization after bombesin stimulation compared with untreated controls. CONCLUSIONS: Glucocorticoids decrease BR gene transcription. The subsequent decrease in cellular BR number renders AR42J cells less sensitive for bombesin-stimulated intracellular Ca2+ mobilization.

Glucocorticoid receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GT1 hypothalamic cell lines.

The synthesis and release of GnRH within a specific subset of neurons in the hypothalamus, which serves as the primary drive to the hypothalamic-pituitary-gonadal (HPG) axis, is subject to various levels of control. Although a number of direct synaptic connections to GnRH-containing neurons have been identified, which presumably provide some regulatory inputs, the mechanisms responsible for hormonal regulation of GnRH synthesis and release mediated by either cell surface or intracellular receptors remain controversial. The recent demonstration that a subset of GnRH-containing neurons in the rat hypothalamus possesses immunoreactive glucocorticoid receptors (GR) implies that this class of steroid hormones could exert a direct effect to regulate the functioning of these neurons and perhaps the HPG axis. We used the GT1-3 and GT1-7 cell lines of immortalized GnRH-secreting hypothalamic neurons as a model to study the direct effects of glucocorticoids on GnRH gene expression. We demonstrated that these cell lines possess GR that bind the synthetic glucocorticoid, dexamethasone, in vitro with high affinity (Kd = 2-3 nM). These receptors are functional, as indicated by their ability to activate transcription from exogenously introduced heterologous glucocorticoid-responsive promoters. Furthermore, dexamethasone represses both the endogenous mouse GnRH gene, decreasing steady state levels of GnRH mRNA, and the transcriptional activity of transfected rat GnRH promoter-reporter gene vectors. Glucocorticoid repression of rat GnRH promoter activity appears to be mediated by sequences contained within the promoter proximal 459 basepairs and not be influenced by the relative basal activity of the GnRH promoter. Thus, our results provide the first direct demonstration of glucocorticoid repression of transcription in a hypothalamic cell line and suggest that GR acting directly within GnRH neurons could be at least partly responsible for negative regulation of the HPG axis by glucocorticoids.

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea.

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin-labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti-GR-receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.

Effect of saiboku-to (TJ-96) on bronchial asthma. Induction of glucocorticoid receptor, beta-adrenaline receptor, IgE-Fc epsilon receptor expression and its effect on experimental immediate and late asthmatic reaction.

A remarkable "steroid sparing" effect of Saiboku-to was noted within 6 to 12 months of treatment in steroid-dependent asthmatic patients. Saiboku-to spared the downregulation of glucocorticoid receptor of human lymphocytes, plasma ACTH, and cortisol levels. It also spared downregulation of beta 2 receptor by beta 2 agonists and suppressed mACh receptor at the same time. Saiboku-to increased tyrosine aminotransferase (TAT) production, which was inhibited by actinomycin-D, thus having steroid-like activity. In mite-allergic asthma, Saiboku-to inhibited the induction of expression of IgE-Fc epsilon R/CD23 in the lymphocytes by mite allergen. It also inhibited IgE production by mite allergens. In experimental asthma in guinea pigs the use of Saiboku-to resulted in a decrease in the number of eosinophils in the bronchoalveolar lavage fluid during late asthmatic response. These findings suggest that Saiboku-to may be effective in inhibiting both the expression of IgE-Fc epsilon R2 and the induction of expression of IgE-Fc epsilon R1. Saiboku-to also has a steroid-like action and polyhedral anti-asthmatic activities.