Brain Glucocorticoid Receptor Research Articles

Expeditious synthesis of steroids containing a 2-methylsulfanyl-acetyl side chain as potential glucocorticoid receptor imaging agents

In our effort to develop imaging agents for brain glucocorticoid receptors, we have prepared several novel glucocorticoids possessing a 2-methylsulfanyl-acetyl side chain. The synthesis was accomplished via a Mitsunobu reaction with thiobenzoic acid starting from cortisol, prednisolone, dexamethasone and triamcinolone acetonide to give the corresponding S-thiobenzoates in 75–82% yield. Subsequent saponification and reaction with methyl iodide afforded C-21 methylthioethers in 68–82% yield. All compounds were tested in an in vitro glucocorticoid receptor-binding assay. Triamcinolone acetonide-based compound 12 showed promising binding affinity of 144% relative to dexamethasone (100%). Compound 12 was selected for radiolabeling with the short-lived positron emitter carbon-11. The radiolabeling was carried out starting from S-thiobenzoate 8 and in situ formation of the corresponding sodium thiolate, which was further reacted with [ 11C]methyl iodide. The obtained radiochemical yield was 20–30%. The specific activity was determined to be 20–40 GBq/μmol at the end-of-synthesis, and the radiochemical purity exceeded 98%.

Overexpressing the glucocorticoid receptor in forebrain causes an aging-like neuroendocrine phenotype and mild cognitive dysfunction.

Repeated stress enhances vulnerability to neural dysfunction that is cumulative over the course of the lifespan. This dysfunction contributes to cognitive deficits observed during aging. In addition, aging is associated with dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis, leading to a delayed termination of the stress response. This delay, in turn, increases exposure to glucocorticoids and exacerbates the likelihood of neural damage. Here we asked whether similar effects could emerge at an early age as a result of genetic variations in the level or function of the brain glucocorticoid receptor (GR). We investigated the effect of forebrain-specific overexpression of GR on LHPA axis activity. Transgenic mice with GR overexpression in forebrain (GRov) display normal basal circulating adrenocorticotropic hormone and corticosterone levels. However, young GRov mice exhibit a number of LHPA alterations, including a blunted initial response to acute restraint stress followed by a delayed turn-off of the stress response. This deficit in negative feedback is paradoxical in the face of elevated GR levels, resembles the stress response in aged animals, and continues to worsen as GRov mice age. The neuroendocrine dysregulation in young GRov mice is coupled with a mild cognitive deficit, also consistent with the accelerated aging hypothesis. The molecular basis of this phenotype was examined using microarray analysis of the hippocampus, which revealed a broad downregulation of glutamate receptor signaling in GRov mice. Thus, even in the absence of chronic stress, elevation of GR gene expression can lead to an increased allostatic load and result in an "aging-like" phenotype in young animals.

Salicylate impacts the physiological responses to an acute handling disturbance in rainbow trout

While salicylates (non-steroidal anti-inflammatory drugs) have been detected in the aquatic environment, few studies have focused on the mechanism of action of these pharmaceuticals on aquatic organisms. We reported previously that salicylate disrupted the acute trophic hormone-stimulated corticosteroidogenesis in rainbow trout ( Oncorhynchus mykiss) interrenal tissue in vitro. Here, we tested the hypothesis that this drug will inhibit the adaptive plasma cortisol response and the associated metabolic response to an acute stressor in trout. Fish were fed salicylate-laced feed (100 mg/kg body weight) for 3 days, subjected to an acute (5 min) handling disturbance and sampled 1, 4 and 24 h after the stressor exposure. Salicylate treatment attenuated the stressor-induced plasma cortisol but not glucose or lactate elevations. The disruption of cortisol response corresponded with a significant reduction in transcript levels of the steroidogenic acute regulatory protein (StAR), but not peripheral-type benzodiazepine receptor, cytochrome P450 side-chain cleavage or 11β-hydroxylase. Salicylate did not modify the stressor-induced elevation of brain glucocorticoid receptor (GR) protein expression, while liver GR protein content was reduced. Salicylate impact on liver metabolic capacity involved depressed liver glycogen content, whereas no significant changes in liver hexokinase, glucokinase, lactate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase, aspartate aminotransferase and alanine aminotransferase activities were observed. Taken together, salicylate impairs the stressor-mediated plasma cortisol response and the associated liver metabolic capacity in trout. The mode of action of salicylate involves disruption of StAR and liver GR, two key proteins critical for cortisol production and target tissue responsiveness to this steroid, respectively.

Synthesis and radiopharmacological characterization of [11C]AL-438 as a nonsteroidal ligand for imaging brain glucocorticoid receptors

The radiosynthesis and the radiopharmacological characterization of [ 11 C]AL-438 as a nonsteroidal ligand for the glucocorticoid receptor (GR) is described. Radiolabeling of the corresponding desmethyl precursor 10 with [ 11C]MeI gave [ 11 C]AL-438 in decay-corrected radiochemical yields of 30 ± 4% (based upon [ 11C]CO 2) within 35 min at a specific radioactivity of 10–15 GBq/μmol at the end-of-synthesis. The radiopharmacological evaluation of [ 11 C]AL-438 involved biodistribution and small animal PET imaging in rats, and autoradiography studies using rat brain sections. Biodistribution studies were performed in male Wistar rats and demonstrated high radioactivity uptake in pituitary and brain. However, the inability of high dose corticosterone to block binding would suggest that the radioactivity accumulation in the brain was not receptor-mediated.

Cellular co-localization of protein phosphatase 5 and glucocorticoid receptors in rat brain

Glucocorticoid receptors are widely expressed in brain, where they are thought to play a role in controlling neurogenesis and to mediate many of the central nervous system effects of stress. In non-neuronal cells, protein phosphatase 5 (PP5) has been found in complexes with heat shock protein 90 and glucocorticoid receptors and may be a negative modulator of glucocorticoid receptor function. In the present study, we used co-immunofluorescence analysis to examine whether PP5 and glucocorticoid receptors are co-expressed at the cellular level in rat brain. In several regions containing major populations of glucocorticoid receptor expressing neurons, PP5 and glucocorticoid receptors were co-localized at the cellular level. These include pyramidal cells of the hippocampal CA1 and CA2 regions and dentate gyrus granule cells, cerebellar Purkinje neurons, cortical pyramidal neurons, neurons of the central nucleus of the amygdala and parvocellular neurons of the hypothalamic paraventricular nucleus. There are also neuronal populations that are stained strongly for glucocorticoid receptors, such as cerebellar granule cells, where PP5 is either absent or below detection limits. Likewise, numerous neuronal populations contain PP5, but not glucocorticoid receptors. Whereas glucocorticoid receptors are expressed in both neurons and glial cells throughout the brain, PP5 appears to be primarily expressed in neurons. These studies suggest that glucocorticoid receptors may be differentially regulated by phosphatase action in different populations of central nervous system cells. Co-localization of PP5 and glucocorticoid receptors in brain regions involved in feedback control of the hypothalamus–pituitary–adrenal axis suggests that PP5 may be an important modulator of glucocorticoid receptor responses in this pathway.

Salicylate Disrupts Interrenal Steroidogenesis and Brain Glucocorticoid Receptor Expression in Rainbow Trout

Varying levels of pharmaceuticals, including salicylate, ibuprofen, and acetaminophen, have been reported in the aquatic environment, but few studies have actually addressed the impact of these drugs on aquatic organisms. We tested the hypothesis that these pharmaceuticals are endocrine disruptors in fish by examining their impact on interrenal corticosteroidogenesis in rainbow trout. Indeed, acute adrenocorticotrophic hormone (ACTH)-mediated cortisol production in trout interrenal cells in vitro was significantly depressed (20-40%) by these pharmaceutical drugs. Furthermore, we investigated whether this interrenal dysfunction involved inhibition of the steroidogenic capacity in rainbow trout. To this end, we fed trout salicylate-laced feed (100 mg/kg body weight) for 3 days and assessed the transcript levels of key proteins involved in corticosteroidogenesis, including steroidogenic acute regulatory protein (StAR), peripheral-type benzodiazepine receptor (PBR), cytochrome P450 cholesterol side chain cleavage (P450scc), and 11beta-hydroxylase. Salicylate treatment did not affect the resting plasma cortisol or glucose levels, whereas the acute ACTH-stimulated cortisol production was significantly depressed in the interrenal tissue. This disruption of steroidogenesis by salicylate corresponded to a significant drop in the gene expression of StAR and PBR, but not P450scc or 11beta-hydroxylase, compared to the sham-treated fish. Also, brain glucocorticoid receptor (GR) protein content and not GR mRNA level was significantly reduced by salicylate. Taken together, salicylate is a corticosteroid disruptor in trout and the targets include the key rate-limiting step in interrenal steroidogenesis and brain glucocorticoid signaling.

Effects of ginsenosides extracted from ginseng stem and leaves on glucocorticoid receptor in different viscera in heat-damaged rats

To evaluate the effects of ginsenosides (GSS) extracted from ginseng stem and leaves on glucocorticoid receptor (GR) in different viscera in heat-damaged rats, and to find out its action mechanism. Thirty-two male SD rats were divided into control group and experimental group, and fed 2 mg/d GSS and equal-quantity of distilled water respectively for 7 days. Eight rats of each group were exposed to (42+/-1) degrees C for one hour. The binding activities of GR in brain, thymus, lung and liver cytosols in rats were detected by radioligand binding assay. The expression levels of GR mRNA in brain and liver cytosols were determined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Plasma adrenocorticotropin (ACTH) and corticosterone (CS) concentrations were determined by radioimmunoassay. The binding activities of GR in brain, lung and liver cytosols, and the expression levels of GR mRNA in brain and liver cytosols were all higher in the GSS-treated and heat-damaged rats than those in the untreated heat-damaged rats (P<0.05 or P<0.01). There were no significant differences in plasma concentrations of ACTH and CS between the GSS-treated heat-damaged rats and the untreated heat-damaged rats. GSS can lessen the descending degree of the binding activity of GR in brain, thymus, lung and liver cytosols, and such efficacy of GSS may be related to improvement of the expression of GR mRNA.

Brain Glucocorticoid Receptor and Heat Shock Protein 70 Levels in Rats Exposed to Acute, Chronic or Combined Stress

The pattern and intensity of glucocorticoid receptor (GR) and heat shock 70 protein (Hsp 70) changes in the hippocampus and brain cortex of adult Wistar rat males exposed to acute (immobilization, cold) and chronic (social isolation, crowding, daily swimming) stress or their combinations were followed by Western immunoblotting. Plasma ACTH and CORT were measured by chemiluminescent method and RIA. A significant decrease in cytosol GR and Hsp 70 was observed after acute stress, while chronic stresses led to negligible changes in both these proteins and caused a reduced responsiveness to a novel acute stress. This was valid irrespective of the type of chronic or acute stress combinations for both hippocampal and cortical GR and Hsp 70. The results support the hypothesis that chronic stress-induced deregulation of the LHPA axis may be caused, at least in part, by partial disruption of intracelullar negative feedback control in the higher centers of the brain.

Central and peripheral effects of repeated noise stress on hypothalamic–pituitary–adrenocortical axis in pigs

The present study was performed to examine the effects of a repeated noise exposure (4 weeks) on basal and noise-induced plasma adrenocorticotrophin (ACTH), cortisol and corticosteroid binding globulin (CBG) levels, brain glucocorticoid receptors (GR) and corticotropin-releasing hormone (CRH) concentrations as well as on the morphology of adrenal glands in German Landrace pigs. Chronically cannulated pigs were exposed to either a daily stimulation with broad-band noise (2 h, 90 dB) (N1), or to the same stimulus three times a week (N2), or to an equal handling procedure without noise stimulation. Noise exposure caused an increase of basal CBG concentrations in N1 animals in the first week. During noise session, an increase of plasma ACTH and cortisol levels in N1 animals was measured in week 1 followed by a subsequent decrease until week 4. The ACTH and cortisol response of the N2 animals increased after week 1 and was significantly elevated in week 4. Furthermore, noise stress caused an increased hippocampal GR binding of N2 pigs, but had no effects on GR binding in the amygdala and on hypothalamic CRH levels. There were also significant structural modifications in the adrenal gland of N1 pigs resulting in differentiated effects on the adrenal cortex and medulla. The present results show that repeated noise exposure of pigs causes considerable alterations at different levels of the HPA system in association with the frequency of noise stimulation. This may have substantial impact on the general vulnerability of the animal with respect to productivity, health and welfare.

Ah Receptor-Mediated Impairment of Interrenal Steroidogenesis Involves StAR Protein and P450scc Gene Attenuation in Rainbow Trout

The objective of the study was to investigate the impact of aryl hydrocarbon receptor (AhR) activation on interrenal steroidogenesis in rainbow trout. To this end, fish were fed AhR agonist (beta-naphthoflavone (BNF): 10 mg/kg body mass/day) and antagonist (alpha-naphthoflavone (ANF): 10 mg/kg body mass/day) either singly or in combination (ABNF) for 5 days to elucidate the mechanisms involved in AhR-mediated depression of cortisol production. Liver AhR protein expression was significantly elevated only with ABNF, but not with BNF and ANF compared to the control group. However, all three treatments (BNF, ANF, and ABNF) significantly elevated cytochrome P450 1A1 (CYP1A1) gene and protein expression in the kidney and liver, respectively. Also, these three treatment groups had significantly depressed ACTH-stimulated cortisol production in vitro compared to the control group. This attenuation of interrenal steroidogenesis corresponded with a lower mRNA abundance of steroidogenic acute regulatory (StAR) protein and cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), but not 11beta-hydroxylase. Furthermore, in vitro incubation of head kidney pieces with 7-3H-pregnenolone failed to show any treatment effects on pathways downstream of P450scc, except for a significantly higher conversion to progesterone in the BNF and ANF groups. Plasma cortisol and glucose levels showed no significant change between the treated groups and control, but liver and brain glucocorticoid receptor (GR) protein expression was higher in the BNF group, and ANF abolished this response. Taken together, both BNF and ANF impaired cortisol production, and the mechanism may involve attenuation of StAR and P450scc, the rate limiting steps in steroidogenesis. Overall, endocrine disruption by xenobiotics acting via AhR includes impaired cortisol biosynthesis and abnormal cortisol target tissue GR responses in rainbow trout.

Glucocorticoid Receptor Function In Vitro in Patients with Major Depression

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamic–pituitary–adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Interestingly, a number of studies have also demonstrated that GR function is reduced in vitro, in peripheral tissues of depressed patients, as shown by a decreased sensitivity to the effects of glucocorticoids on immune and metabolic functions. This paper reviews the in vitro studies that have examined GR function in patients with major depression, and the possible molecular mechanisms involved in GR resistance. Since several studies have demonstrated similar regulation of GR in the brain and in peripheral tissues in humans, and given limited access to brain GR in clinical populations, this review claims that in vitro studies are of particular relevance to understand the molecular mechanisms underlying GR abnormalities in patients with major depression and its regulation by antidepressant treatment.

Effect of chronic lithium treatment on glucocorticoid and 5-HT1A receptor messenger RNA in hippocampal and dorsal raphe nucleus regions of the rat brain

The therapeutic mechanism of action of lithium in the treatment of bipolar disorder is not well understood. Dysfunction of both 5-HT1A receptor mediated neurotransmission and the glucocorticoid receptor is associated with mood disorders, and preclinical studies suggest that lithium treatment can modulate these receptor subtypes. In this study, we investigated the effect of chronic lithium treatment on 5-HT1A receptors and glucocorticoid receptors in the rat brain. Male Sprague-Dawley rats were treated with lithium (3 mmol/kg/day) or saline for 28 days via subcutaneous implanted mini-osmotic pumps. After 28 days of treatment, the expression of mRNA for 5-HT1A receptors and glucocorticoid receptors in the rat hippocampus and dorsal raphe nucleus was determined by in situ hybridization histochemistry. Chronic administration of lithium decreased mRNA coding for post-synaptic 5-HT1A receptors in hippocampal subregions but not for somatodentritic 5-HT1A receptors in the dorsal raphe nucleus. Chronic administration of lithium did not affect mRNA coding for glucocorticoid receptors in hippocampal subregions or in the dorsal raphe nucleus. Mean plasma lithium levels in the lithium-treated group were 0.50 ± 0.03 mmol/l; all animals appeared healthy and maintained a normal increase in body weight. Given recent reports implicating hypersensitive post-synaptic 5-HT1A receptors in bipolar manic patients, the present study suggests that down-regulation of this receptor population may be important in the therapeutic mechanism of action of lithium.

Effect of chronic lithium treatment on glucocorticoid and 5-HT1A receptor messengerRNA in hippocampal and dorsal raphe nucleus regions of the rat brain

The therapeutic mechanism of action of lithium in the treatment of bipolar disorder is not well understood. Dysfunction of both 5-HT(1A) receptor mediated neurotransmission and the glucocorticoid receptor is associated with mood disorders, and preclinical studies suggest that lithium treatment can modulate these receptor subtypes. In this study, we investigated the effect of chronic lithium treatment on 5-HT(1A) receptors and glucocorticoid receptors in the rat brain. Male Sprague-Dawley rats were treated with lithium (3 mmol/kg/day) or saline for 28 days via subcutaneous implanted mini-osmotic pumps. After 28 days of treatment, the expression of mRNA for 5-HT(1A) receptors and glucocorticoid receptors in the rat hippocampus and dorsal raphe nucleus was determined by in situ hybridization histochemistry. Chronic administration of lithium decreased mRNA coding for post-synaptic 5-HT(1A) receptors in hippocampal subregions but not for somatodentritic 5-HT(1A) receptors in the dorsal raphe nucleus. Chronic administration of lithium did not affect mRNA coding for glucocorticoid receptors in hippocampal subregions or in the dorsal raphe nucleus. Mean plasma lithium levels in the lithium-treated group were 0.50 +/- 0.03 mmol/l; all animals appeared healthy and maintained a normal increase in body weight. Given recent reports implicating hypersensitive post-synaptic 5-HT(1A) receptors in bipolar manic patients, the present study suggests that down-regulation of this receptor population may be important in the therapeutic mechanism of action of lithium.

12th Workshop of the Central European Division e.V. of the International Isotope Society

AbstractV Snieckus [Queen's University, Canada]—The Directed Ortho Metalation—Cross Coupling Symbiosis in Aromatic Synthesis.N Geach, GP Randall, CJ Winfield [Scynexis, UK]—Isotopically Labelled 4‐Formylimidazole.A Studer [Marburg University, Germany]—New Concepts in Tin‐Free Radical Chemistry.AC Spivey, C Noban [Imperial College London, UK]. T Jones [Sheffield University, UK], GJ Ellames, A Kohler [Sanofi‐Synthélabo, UK], H Wadsworth [Amersham Biosciences, UK]—Opportunities for Isotope Labelling via Solid Phase Synthesis with Organogermanium Linkers.J Rieke‐Zapp [Aventis Pharma, Germany]—Optimization of Reaction Conditions on mg‐Scale by Use of Screening Devices.S Stone‐Elander [Karolinska Institute, Sweden]—Rerouting Radiochemistry with Microwaves.J Goehl, B Perry, D Leuck, J Windels, M Marx [EaglePicher Pharmaceutical, USA]—GMP and Radiolabelled Syntheses of Capsaicin.J Krüger, B Manmontri, G Fels [Paderborn University, Germany]—H/D‐Exchange in Aromatic Systems Using Transition Metal Catalysis.F Wüst, T Kniess, R Bergmann [Research Centre Rossendorf, Germany]—Synthesis of a 11C‐Labelled Nonsteroidal Glucocorticoid Receptor Ligand as Potential Radiotracer for Imaging Brain Glucocorticoid Receptors with Positron Emission Tomography (PET).P Mäding, F Füchtner [Research Centre Rossendorf, Germany], CS Hilger [Schering AG, Germany]. M Halks‐Miller, R Horuk [Berlix Biosciences, USA]—18F‐Labelling of a Potent Nonpeptide CCR1 Antagonist for the Diagnosis of the Alzheimer's Disease.L Matei, C Postolache, V Fugaru, E Condac, A Dinischiotu, M Costache [Bukarest University, Romania]—Use of [1,2‐3H] Testosterone in 5α‐ Reductase Enzymatic Activity Dosing in Dermal Fibroblast Cultures from Polycystic Ovarian Patients.L Matei, C Postolache, V Fugaru [Bukarest University, Romania]—Synthesis and Stability Studies of Acyclovir Labelled with Tritium.J Atzrodt, V Derdau [Aventis Pharma, Germany]—Studies on Directed Ortho‐Metalation (DoM) as Convenient Tool for the Synthesis of Labeled Compounds.

PCB disruption of the hypothalamus-pituitary-interrenal axis involves brain glucocorticoid receptor downregulation in anadromous Arctic charr.

We examined whether brain glucocorticoid receptor (GR) modulation by polychlorinated biphenyls (PCBs) was involved in the abnormal cortisol response to stress seen in anadromous Arctic charr (Salvelinus alpinus). Fish treated with Aroclor 1254 (0, 1, 10, and 100 mg/kg body mass) were maintained for 5 mo without feeding in the winter to mimic their seasonal fasting cycle, whereas a fed group with 0 and 100 mg/kg Aroclor was maintained for comparison. Fasting elevated plasma cortisol levels and brain GR content but depressed heat shock protein 90 (hsp90) and interrenal cortisol production capacity. Exposure of fasted fish to Aroclor 1254 resulted in a dose-dependent increase in brain total PCB content. This accumulation in fish with high PCB dose was threefold higher in fasted fish compared with fed fish. PCBs depressed plasma cortisol levels but did not affect in vitro interrenal cortisol production capacity in fasted charr. At high PCB dose, the brain GR content was significantly lower in the fasted fish and this corresponded with a lower brain hsp70 and hsp90 content. The elevation of plasma cortisol levels and upregulation of brain GR content may be an important adaptation to extended fasting in anadromous Arctic charr, and this response was disrupted by PCBs. Taken together, the hypothalamus-pituitary-interrenal axis is a target for PCB impact during winter emaciation in anadromous Arctic charr.

Reduced brain CRH and GR mRNA expression precedes obesity in juvenile rats bred for diet-induced obesity

To assess the role of endogenous peptides involved in stress responsivity in the development of diet-induced obesity (DIO), selectively bred DIO and diet-resistant (DR) male were weaned onto a low fat (4.5%) chow diet at 3 weeks of age and then fed either chow or a 31% fat by energy content (high energy (HE)) diet for 9 days beginning at 4 weeks of age. Regardless of diet, DIO rats gained more weight than DR rats but did not show the selective DIO weight gain trait characteristic of older DIO rats fed HE diet. At this early age, both DR and DIO rats on HE diet ate more and had higher leptin levels but gained less body weight and had lower feed efficiency (body weight gain (g)/food intake (kcal)) than their chow-fed controls. HE diet also prevented the decline in 24 h urine corticosterone levels from the third to fifth week observed in chow-fed rats. Terminally, DIO rats had lower hippocampal glucocorticoid receptor (GR) and amygdalar central nucleus corticotrophin-releasing hormone (CRH) mRNA than DR rats, regardless of their diets. Taken together with prior studies in these rats, there appears to be a critical period between 3 and 5 weeks of age when DIO and DR rats are not phenotypically different and hypothalamo–pituitary–adrenal (HPA) function is rapidly changing. The reduced expression of brain GR and CRH expression at the end of this period might contribute to the propensity of DIO rats to become obese selectively on HE diet after 5 weeks of age.

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 γ-emitter iodine-131.

Effect of chronic administration of selective glucocorticoid receptor antagonists on the rat hypothalamic-pituitary-adrenocortical axis.

The effects of the selective glucocorticoid receptor (GR) antagonists ORG 34850, ORG 34116, and ORG 34517 on the rat hypothalamic-pituitary-adrenocortical (HPA) system were investigated. To assess the potency of the compounds to occupy GR in the brain and pituitary, we applied a single acute subcutaneous (s.c.) injection (10 mg/kg). ORG 34517 was most potent to occupy GR in the anterior pituitary and distinct brain areas, whereas all compounds were unable to occupy mineralocorticoid receptor (MR). Chronic administration of ORG 34850, ORG 34116, and ORG 34517 (20 mg/kg/day) for 1, 3, and 5 weeks resulted only in minor changes in brain GR levels. However, profound increases of hippocampal MR were observed virtually at all time points. Treatment with ORG 34850 and ORG 34116 elicited episodic increases in HPA axis activity, whereas ORG 34517 did not cause any changes in HPA activity. Thus, the GR antagonists exert distinct effects on the HPA axis, which may be pertinent for the proposed antidepressant activity of these compounds.

The glucose paradox of cerebral ischemia – a possible resolution

The glucose paradox of cerebral ischemia, the aggravation of delayed neuronal damage by preischemic hyperglycemia, is a well‐documented phenomenon. Recently, we demonstrated that corticosterone (CT) plasma levels were significantly elevated within 30 min after glucose administration (i.p.) only to return to baseline levels by 120 min. In this study we examined the postulate that preischemic glucose loading aggravates the postischemic outcome via the induction of a short‐lived, massive elevation in CT plasma levels. Hence, we tested the ability of the CT synthesis inhibitor metyrapone (MT) and the glucocorticoid receptor antagonist, mifepristone (RU486), to attenuate the aggravated delayed neuronal damage in a rat model of cardiac‐arrest‐induced transient global cerebral ischemia (TGI). Seven different groups of 24‐h‐fasted rats were used and were administered several treatments prior to TGI that were aimed at either inhibiting CT synthesis (MT, 100 mg/kg, i.p.) or at blocking the brain glucocorticoid receptors (RU486, 40 mg/kg, i.p.). Plasma glucose and CT levels were measured 2 min pre‐TGI. Neuronal damage was assessed 7 days post‐TGI in hippocampal slices using electrophysiological means. Plasma glucose levels were not reduced in MT‐ or RU486‐treated rats. Both MT and RU 486 prevented preischemic hyperglycemia‐aggravated ischemic damage, the former presumably via inhibition of CT synthesis, the latter by glucocorticoid receptor antagonism. The present data strongly suggest that preischemic hyperglycemia‐aggravated neuronal damage, i.e. the glucose paradox, results from glucose‐induced CT release, rather than intensification of lactic acidosis in the ischemic brain.

Modulation of cellular expression of glucocorticoid receptor and glucocorticoid response element-DNA binding in rat brain during alcohol drinking and withdrawal.

To define the molecular mechanisms of abnormal hypothalamic pituitary adrenal (HPA) axis during ethanol dependence, we investigated the effect of chronic ethanol treatment (15 days) and its withdrawal (24 h) on the expression of glucocorticoid receptors (GRs) and glucocorticoid response element (GRE)-DNA binding in the rat brain. The effects of chronic mianserin [serotonin (5-HT)(2A/2C) antagonist] treatment on these parameters in various brain structures of control diet-fed and ethanol-fed rats were also investigated. It was found that ethanol treatment and withdrawal significantly decreased the GR protein levels in cortical (cingulate gyrus, frontal, parietal, and piriform cortex) and amygdaloid (central, medial, and basolateral) structures and paraventricular nucleus (PVN) of hypothalamus of rats. It was also observed that ethanol treatment produced significant reductions in GR protein levels in various hippocampal structures (CA1, CA2, CA3, and dentate gyrus), but these changes were normalized during ethanol withdrawal. Ethanol treatment also significantly decreased GRE-DNA binding in rat cortex and hippocampus, which remained decreased in the cortex but reverted to normal in hippocampus during ethanol withdrawal. Chronic mianserin (alone) treatment had no effect on cortical GRE-DNA binding and GR protein levels in cortical, amygdaloid, or PVN structures but significantly decreased the GR protein expression in various hippocampal structures and GRE-DNA binding in whole hippocampus. However, when administered concurrently with ethanol treatment, mianserin significantly antagonized the reductions in cortical GRE-DNA binding and in GR protein expression in cortical, PVN, and central, but not medial and basolateral, amygdaloid structures during ethanol withdrawal. On the other hand, mianserin treatment along with ethanol administration significantly decreased the hippocampal GRE-DNA binding and GR protein expression in various hippocampal structures during ethanol withdrawal. Furthermore, ethanol treatment and its withdrawal or mianserin treatment had no effect on the neuron-specific nuclear protein levels in the various brain structures. Taken together, these results indicate that interaction of 5-HT(2A/2C) receptors with GRs in cortical, central amygdaloid, and PVN structures may play a role in neural mechanisms of alcohol dependence. It is possible that decreased GR expression in PVN may be responsible for the abnormal HPA axis during ethanol exposure and withdrawal.