Cytosolic Glucocorticoid Receptor Research Articles

The effect of lead intoxication on endocrine functions

Studies on the effects of lead on the endocrine system are mainly based on occupationally lead-exposed workers and experimental animal models. Although evidence is conflicting, it has been reported that accumulation of lead affects the majority of the endocrine glands. In particular, it appears to have an effect on the hypothalamic-pituitary axis causing blunted TSH, GH, and FSH/LH responses to TRH, GHRH, and GnRH stimulation, respectively. Suppressed GH release has been reported, probably caused by reduced synthesis of GHRH, inhibition of GHRH release or reduced somatotrope responsiveness. Higher levels of PRL in lead intoxication have been reported. In short-term lead-exposed individuals, high LH and FSH levels are usually associated to normal testosterone concentrations, whereas in long-term exposed individuals' low testosterone levels do not induce high LH and FSH concentrations. These findings suggest that lead initially causes some subclinical testicular damage, followed by hypothalamic or pituitary disturbance when longer periods of exposure take place. Similarly, lead accumulates in granulosa cells of the ovary, causing delays in growth and pubertal development and reduced fertility in females. In the parenchyma of adrenals histological and cytological changes are demonstrated, causing changes in plasma basal and stress-mediated corticosterone concentrations and reduced cytosolic and nuclear glucocorticoid receptor binding. Thyroid hormone kinetics are also affected. Central defect of the thyroid axis or an alteration in T4 metabolism or binding to proteins may be involved in derangements in thyroid hormone action. Lead toxicity involves alterations on calcitropic hormones' homeostasis, which increase the risk of skeletal disorders.

Genomic and nongenomic effects of glucocorticoids

The strong anti-inflammatory and immunosuppressive effects of glucocorticoids are mediated primarily by the cytosolic glucocorticoid receptors. These receptors are members of the steroid hormone receptor family, a superfamily of ligand-inducible transcription factors, and exert genomic effects that can result in increased expression of regulatory-including anti-inflammatory-proteins (transactivation), or decreased production of proinflammatory proteins (transrepression). Transactivation is thought to be responsible for numerous adverse effects of glucocorticoids; transrepression is thought to be responsible for many of the clinically desirable anti-inflammatory and immunosuppressive effects of glucocorticoids. Optimized glucocorticoids, such as selective glucocorticoid receptor agonists, are being developed to try to minimize the adverse effects many patients experience. Glucocorticoids also exert their effects via rapid, nongenomic mechanisms that can be classified as involving nonspecific interactions of glucocorticoids with cellular membranes, nongenomic effects that are mediated by cytosolic glucocorticoid receptors, or specific interactions with membrane-bound glucocorticoid receptors. Increased understanding of these mechanisms of glucocorticoid action could enable the development of novel drugs with which to treat patients with inflammatory and autoimmune disease.

Molecular Mechanisms of Corticosteroid Resistance

Most patients with asthma are successfully treated with conventional therapy. Nevertheless, there is a small proportion of asthmatic patients, including present cigarette smokers and former cigarette smokers, who fail to respond well to therapy with high-dose glucocorticoids (GCs) or with supplementary therapy. In addition, high doses of steroids have a minimal effect on the inexorable decline in lung function in COPD patients and only a small effect on reducing exacerbations. GC insensitivity, therefore, presents a profound management problem in these patients. GCs act by binding to a cytosolic GC receptor (GR), which is subsequently activated and is able to translocate to the nucleus. Once in the nucleus, the GR either binds to DNA and switches on the expression of antiinflammatory genes or acts indirectly to repress the activity of a number of distinct signaling pathways such as nuclear factor (NF)-kappaB and activator protein (AP)-1. This latter step requires the recruitment of corepressor molecules. Importantly, this latter interaction is mutually repressive in that high levels of NF-kappaB and AP-1 attenuate GR function. A failure to respond may therefore result from reduced GC binding to GR, reduced GR expression, enhanced activation of inflammatory pathways, or lack of corepressor activity. These events can be modulated by oxidative stress, T-helper type 2 cytokines, or high levels of inflammatory mediators, all of which may lead to a reduced clinical outcome. Understanding the molecular mechanisms of GR action, and inaction, may lead to the development of new antiinflammatory drugs or may reverse the relative steroid insensitivity that is characteristic of patients with these diseases.

Radiation-Induced Hyposuppression of the Hypothalamic-Pituitary-Adrenal Axis is Associated with Alterations of Hippocampal Corticosteroid Receptor Expression

Therapeutic brain irradiation in children can cause a progressive decline in cognitive functions through a diminished capability to learn and memorize. Because of the known involvement of the hippocampus in memory consolidation, this study was aimed at examining the late effects of gamma radiation on hypothalamic-pituitary-adrenal (HPA) axis activity and hippocampal corticosteroid receptor expression in an animal model of cranial radiotherapy. In the late-response phase, the basal and stress-induced corticosterone levels were not affected by radiation, but the suppression of glucocorticoid negative feedback by dexamethasone was attenuated in irradiated rats. Western blot analyses showed that exposure to radiation led to a decrease of cytosolic glucocorticoid receptor (GR) levels and a concomitant elevation of mineralocorticoid receptor (MR). The results obtained were complemented by those of RT-PCR, since the ratio of GR/MR mRNA was also decreased after radiation exposure. Dexamethasone appeared to be much less effective in shifting GR to the nuclear compartment in irradiated rats than in sham-irradiated animals. However, the expression of chaperones that aid GR intracellular trafficking, Hsp90 and Hsp70, remained unaffected. In conclusion, our data suggest that the hallmark of the late response to gamma radiation is a hyposuppressive state of the HPA axis that is associated with a decrease in both the GR/MR ratio and the nuclear accumulation of dexamethasone-activated GR in the hippocampus.

Liver glucocorticoid receptor and heat shock protein 70 levels in rats exposed to different stress models

The aim of the present study was to define the stress-induced pattern of cytosolic glucocorticoid receptor (GR) and Hsp70 protein in the liver of male Wistar rats exposed to different stress models: acute (2 h/day) immobilization or cold (4 degrees C); chronic (21 days) isolation, crowding, swimming or isolation plus swimming and combined (chronic plus acute stress). Changes in plasma levels of corticosterone were studied by radioimmunoassay (RIA). The results obtained by Western immunoblotting showed that both acute stressors led to a significant decrease in cytosolic GR and Hsp70 levels. Compared to acute stress effects, only a weak decrease in the levels of GR and Hsp70 was demonstrated in chronic stress models. Chronically stressed rats, which were subsequently exposed to novel acute stressors (immobilization or cold), showed a lower extent of GR down-regulation when compared to acute stress. The exception was swimming, which partially restores this down-regulation. The observed changes in the levels of these major stress-related cellular proteins in liver cytosol lead to the conclusion that chronic stressors compromise intracellular GR down-regulation in the liver.

Mitochondrial effects of dexamethasone imply both membrane and cytosolic-initiated pathways in HepG2 cells

Glucocorticoid treatment is often linked to increased whole-body energy expenditure and hypermetabolism. Glucocorticoids affect mitochondrial energy production, notably in the liver, where they lead to mitochondrial uncoupling reducing the efficacy of oxidative phosphorylation. However, the signaling pathways involved in these phenomena are poorly understood. Here we treated HepG2 cells with dexamethasone for different times and, by using different combinations of inhibitors, we showed that dexamethasone treatment leads to recruitment of two main signaling pathways. The first one involves a G-protein coupled membrane glucocorticoid binding site and rapidly decreases complexes I and II activities while complex III activity is upregulated in a p38MAPK dependent mechanism. The second one implies the classical cytosolic glucocorticoid receptor and triggers long-term transcriptional increases of respiration rates and of complex IV activity and quantity. We concluded that mitochondria are the target of multiple dexamethasone-induced regulatory pathways that are set up gradually after the beginning of hormone exposure and that durably influence mitochondrial oxidative phosphorylation.

Modeling of Corticosteroid Effects on Hepatic Low-Density Lipoprotein Receptors and Plasma Lipid Dynamics in Rats

This study examines methylprednisolone (MPL) effects on the dynamics of hepatic low-density lipoprotein receptor (LDLR) mRNA and plasma lipids associated with increased risks for atherosclerosis. Normal male Wistar rats were given 50 mg/kg MPL intramuscularly (IM) and sacrificed at various times. Measurements included plasma MPL and CST, hepatic glucocorticoid receptor (GR) mRNA, cytosolic GR density and hepatic LDLR mRNA, and plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), and triglycerides (TG). MPL showed bi-exponential disposition with two first-order absorption components. Hepatic GR and LDLR mRNA exhibited circadian patterns which were disrupted by MPL. Down-regulation in GR mRNA (40-50%) was followed by a delayed rebound phase. LDLR mRNA exhibited transient down-regulation (60-70%). Cytosolic GR density was significantly suppressed but returned to baseline by 72 h. Plasma TC and LDLC showed increases (55 and 142%) at 12 h. A mechanistic receptor/gene pharmacokinetic/pharmacodynamic model was developed to describe CS effects on hepatic LDLR mRNA and plasma cholesterols. Our PK/PD model was able to satisfactorily capture the MPL effects on hepatic LDLR, its relationship to various plasma cholesterols, and builds the foundation to explore this area in the future.

Modeling receptor/gene-mediated effects of corticosteroids on hepatic tyrosine aminotransferase dynamics in rats: dual regulation by endogenous and exogenous corticosteroids

Receptor/gene-mediated effects of corticosteroids on hepatic tyrosine aminotransferase (TAT) were evaluated in normal rats. A group of normal male Wistar rats were injected with 50 mg/kg methylprednisolone (MPL) intramuscularly at the nadir of their plasma corticosterone (CST) rhythm (early light cycle) and sacrificed at various time points up to 96 h post-treatment. Blood and livers were collected to measure plasma MPL, CST, hepatic glucocorticoid receptor (GR) mRNA, cytosolic GR density, TAT mRNA, and TAT activity. The pharmacokinetics of MPL showed bi-exponential disposition with two first-order absorption components from the injection site and bioavailability was 21%. Plasma CST was reduced after MPL dosing, but resumed its daily circadian pattern within 36 h. Cytosolic receptor density was significantly suppressed (90%) and returned to baseline by 72 h resuming its biphasic pattern. Hepatic GR mRNA follows a circadian pattern which was disrupted by MPL and did not return during the study. MPL caused significant down-regulation (50%) in GR mRNA which was followed by a delayed rebound phase (60-70 h). Hepatic TAT mRNA and activity showed up-regulation as a consequence of MPL, and returned to their circadian baseline within 72 and 24 h of treatment. A mechanistic receptor/gene-mediated pharmacokinetic/pharmacodynamic model was able to satisfactorily describe the complex interplay of exogenous and endogenous corticosteroid effects on hepatic GR mRNA, cytosolic free GR, TAT mRNA, and TAT activity in normal rats.

Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists

Glucocorticoids (GC) are the most common used anti-inflammatory and immunosuppressive drugs in the treatment of rheumatic and other inflammatory diseases. Their therapeutic effects are considered to be mediated by four different mechanisms of action: the classical genomic mechanism of action caused by the cytosolic glucocorticoid receptor (cGCR); secondary non-genomic effects which are also initiated by the cGCR; membrane-bound glucocorticoid receptor (mGCR)-mediated non-genomic effects; non-specific, non-genomic effects caused by interactions with cellular membranes. The classical, genomic mechanism of GC-action can be divided into two processes: “transrepression”, which is responsible for a large number of desirable anti-inflammatory and immunomodulating effects, and “transactivation” which is associated with frequently occurring side effects as well as with some immunosuppressive activities [Ehrchen, J., Steinmuller, L., Barczyk, K., Tenbrock, K., Nacken, W., Eisenacher, M., Nordhues, U., Sorg, C., Sunderkotter, C., Roth, J., 2007. Glucocorticoids induce differentiation of a specifically activated, anti-inflammatory subtype of human monocytes. Blood 109, 1265–1274]. Great efforts have been made to diminish glucocorticoid-induced adverse effects, but the improvement of conventional glucocorticoids has almost reached its limits. As a consequence, new variations of the conventional “good old drugs” are being tested and nitro-steroids and long circulating liposomal glucocorticoids indeed show promising results. Nevertheless, crux of the matter should be the design of qualitatively new drugs, such as selective glucocorticoid receptor agonists (SEGRAs). These innovative steroidal or non-steroidal molecules induce transrepression, while transactivation processes are less affected. First reports on two different GCR ligands, A276575 and ZK216348, show promising results. Here, we review the above-mentioned mechanisms of glucocorticoid action and give particular attention to the development of optimized glucocorticoids and SEGRAs.

Assessing the dynamics of nuclear glucocorticoid-receptor complex: adding flexibility to gene expression modeling.

A retrospective analysis was performed to modify our fourth-generation pharmacodynamic model for glucocorticoid receptor (GR) dynamics with incorporation of more physiological features. This modified model was developed by integrating previously reported free cytosolic GR and GR mRNA data following single (10, 50 mg/kg) and dual (50 mg/kg at 0 and 24 hr) intravenous doses of methylprednisolone (MPL) in adrenalectomized (ADX) male Wistar rats with several in vitro studies describing real-time kinetics of the transfer of rat steroid-receptor complex from the cell cytosol to the nucleus. Additionally, free hepatic cytosolic GR and its mRNA data from a chronic infusion dosing study of MPL (0.1 and 0.3 mg/kg/hr) in male ADX Wistar rats were used to verify the predictability of the model. Incorporation of information regarding in vitro receptor kinetics allowed us to describe the receptor-mediated pharmacogenomic effects of MPL for a larger variety of genes in rat liver from microarray studies. These included early responsive gene like CCAAT/enhancer binding protein-beta (CEBP-beta), a transcription factor, as well as the later responsive gene for tyrosine aminotransferase (TAT), a classical biomarker of glucocorticoid (GC) genomic effects. This more mechanistic model of GR dynamics can be applied to characterize profiles for a greater number of genes in liver.

The Effect of Repeated Physical Exercise on Hippocampus and Brain Cortex in Stressed Rats

Sensitivity of target cells to glucocorticoids is regulated by the expression of intracellular glucocorticoid receptor (GR), which mediates the effects of glucocorticoids. The level of GR and of its nuclear transporter protein 70 (Hsp70) were followed in hippocampus and brain cortex of adult Wistar rat males exposed to acute (immobilization, cold) and chronic (social isolation, isolation, and 15 min daily swimming) stress or their combinations. Changes in plasma levels of adenocorticotropic hormone and corticosterone were also studied. A significant decrease in cytosol GR and Hsp70 was observed after acute stress. Opposite to that, chronic stress led to negligible changes in both cytosol GR and Hsp70 levels. Isolation, as chronic psychosocial stressor, caused reduced responsiveness to novel acute stressors, judged by the cytosol GR and Hsp70 levels. This was not observed if chronic isolation was combined with 15 min daily swimming prior to acute exposure to immobilization. The data suggest that repeated physical exercise may, at least in some cases, diminish detrimental effects of chronic social isolation on limbic-hypothalamic-pituitary-adrenocortical axis, as judged by the levels of GR and Hsp70 in the Wistar rat brain.

Saikokaryukotsuboreito, a herbal medicine, prevents chronic stress-induced dysfunction of glucocorticoid negative feedback system in rat brain

Disruption of the hypothalamo–pituitary–adrenal (HPA) axis characterized by dysfunction of the glucocorticoid negative feedback system is frequently observed in human depressives and is thought to involve a reduction in glucocorticoid receptor (GR) function in the feedback sites including the brain. Recently, we found that chronic stress in rats induces similar HPA disruption that is caused by abolishment of feedback ability in the prefrontal cortex (PFC) and hippocampus, which involves decreased cytosolic GRs or increased nuclear GRs, respectively. Also, we found that saikokaryukotsuboreito (SRBT), a herbal medicine, prevents the chronic stress-induced HPA disruption. We therefore examined here the effects of this drug on the chronic stress-induced changes in GRs in the PFC and hippocampus. Chronic stress was induced in rats by water immersion and restraint (2 h/day) for 4 weeks. SRBT significantly prevented decreased cytosolic GRs in the PFC and increased nuclear GRs in the hippocampus in the chronically stressed rats. Moreover, SRBT significantly prevented the abolishment of feedback ability in both regions. These results suggest that the beneficial effects of SRBT on the GR level are involved in its ameliorating actions on the HPA disruption. This finding provides information important for the prevention and treatment of depression.

Region‐specific regulation of glucocorticoid receptor/HSP90 expression and interaction in brain

The hippocampal glucocorticoid receptor (GR) is involved in negative feedback regulation of the hypothalamo-pituitary-adrenal axis and is believed to transduce the deleterious effects of glucocorticoids in depression and age-related memory loss. Regulation and intracellular trafficking of the GR are critical determinants of GR action in both health and disease. Here, we show dynamic regulation of GR and its interaction with its principal intracellular chaperone, heat-shock protein (HSP) 90, across the circadian cycle. Our initial experiments indicate that cytosolic hippocampal GR protein is elevated in the evening (PM), whereas nuclear GR and cytosolic HSP90, HSP70 and heat-shock cognate 70 (HSC70), are unchanged. In contrast, there are no changes in examined proteins in the hypothalamus. Immunoprecipitation experiments reveal increased GR-HSP90 associations in the hippocampus in the PM, whereas binding in the hypothalamus is decreased in the PM. Given that GR requires HSP90 for ligand binding, the data suggest that circadian GR signaling capacity is regulated in a region-specific pattern.

Proteomic identification of glucocorticoid receptor interacting proteins

The glucocorticoid receptor (GR) acts as a ligand dependent transcription factor but can also cross talk with other signaling pathways via protein-protein interactions. In this paper we describe methods to study novel cytosolic GR interacting proteins, using mAb based immunoaffinity chromatography of GR from rat liver cytosol. Co-purifying proteins were identified by 2-DE in combination with MALDI-TOF-MS. Non-liganded/non-activated and in vitro liganded/activated GR, respectively, co-purifies with specific sets of proteins. Of these 34 were conclusively identified, seven have previously been reported to be part of the GR-complex, revealing 27 new possible interacting candidates for the GR-complex. Of the novel GR interacting proteins the major vault protein, TATA binding interacting protein 49a and glycoprotein PP63 were of special interest. Furthermore, using 2-D DIGE we show that the set of proteins interacting with non-liganded GR is distinctly different in protein amount compared to the proteins found with liganded/activated GR. This suggests the presence of different GR complexes in the cell, which was further substantiated by the finding of several separate GR native protein complexes, "GR-receptosomes", using blue native gel electrophoresis. Our findings suggest the existence of several new mechanisms for GR signaling and regulation.

Cytosolic glucocorticoid receptor in the testis of Bufo arenarum: Seasonal changes in its binding parameters

Glucocorticoids (GC) are the hormonal mediators of stress. In mammals, high levels of GC have negative effects on reproductive physiology. For instance, GC can inhibit testicular testosterone synthesis by acting via glucocorticoid receptors (GR), the extent of the inhibition being dependent on GC levels. However, the effect of GC on testicular function and even the presence of GR in amphibians are still unclear. The purpose of this work was to characterise testicular cytosolic GR in Bufo arenarum, determining the seasonal changes in its binding parameters as well as the intratesticular localisation. The binding assays were performed in testis cytosol with [ 3H]dexamethasone (DEX) and [ 3H]corticosterone (CORT). Binding kinetics of DEX and CORT fitted to a one-site model. Results were expressed as means ± standard error. Apparent number of binding sites (Bapp) was similar for both steroids (Bapp DEX = 352.53 ± 72.08 fmol/mg protein; Bapp CORT = 454.24 ± 134.97 fmol/mg protein) suggesting that both hormones bind to the same site. Competition studies with different steroids showed that the order of displacement of [ 3H]DEX and [ 3H]CORT specific binding is: DEX ∼ RU486 ∼ deoxycorticosterone (DOC) > CORT > aldosterone > RU28362 > progesterone >>> 11-dehydroCORT. The affinity of GR for DEX ( K d = 11.2 ± 1.5 nM) remained constant throughout the year while circulating CORT clearly increased during the reproductive season. Therefore, testis sensitivity to GC action would depend mainly on inactivating mechanisms (11β-hydroxysteroid dehydrogenase type 2) and CORT plasma levels. Since total and free CORT are higher in the reproductive than in the non-reproductive period, the magnitude of GC actions could be higher during the breeding season. The intratesticular localisation of the GR was determined after separation of cells by a Percoll density gradient followed by binding assays in each fraction. DEX binds to two different fractions corresponding to Leydig and Sertoli cells. In conclusion, in the testis of B. arenarum GC could regulate the function of both cellular types particularly during breeding when CORT reaches the highest plasma concentration.

Non-genomic glucocorticoid effects to provide the basis for new drug developments

Glucocorticoids act via genomic and non-genomic actions. The genomic glucocorticoid actions are well known and new details on processes of transactivation and transrepression have been reported recently. Here we describe the current knowledge on non-genomic glucocorticoid actions and discuss why these actions are considered to be of therapeutic relevance. It is assumed that rapid non-genomic glucocorticoid effects are mediated by three different mechanisms: (1) physicochemical interactions with cellular membranes (non-specific non-genomic effects); (2) membrane-bound glucocorticoid receptor (mGCR)-mediated non-genomic effects; and (3) cytosolic glucocorticoid receptor (cGCR)-mediated non-genomic effects. With regard to the first mechanism, we discuss here lazaroids and the novel development of drug targeting with liposomes as the carrier system for glucocorticoids. The clinical use of the latter two mechanisms is still speculative, but intriguing ideas are being discussed in this regard.

Alteration of glucocorticoid receptor subcellular distribution by hyperthermic stress

The aim of the present study was to examine intracellular redistribution of the glucocorticoid receptor (GR) in rat liver cells during a 24-h time period after exposure of the animals to 41?C whole body hyperthermic stress. The level of the receptor protein in the cytoplasmic and nuclear compartments was measured by immunoblotting procedures applied to both crude cytosol and immunopurified GR, as well as by immunocytochemical analyses applied to both paraffin-embedded liver sections and unfixed nuclear smears. All the experimental approaches employed in the study provided similar results, demonstrating that the transient stress-related decline of the cytoplasmic GR observed during the first five hours after exposure of the animals to whole-body hyperthermic stress is accompanied by enhanced nuclear accumulation of the receptor. The study can contribute to a better understanding of the influence of stress on the glucocorticoid signal transduction pathway. .

Differential effects of selected natural compounds with anti-inflammatory activity on the glucocorticoid receptor and NF-κB in HeLa cells

Natural compounds have been used in the treatment of various diseases for centuries. Herein, we investigated the effects of structurally diverse alkaloids with anti-inflammatory activity (berberine, sanguinarine, chelerythrine, and colchicine) on two important anti-inflammatory and pro-inflammatory players, i.e. glucocorticoid receptor (GR) and nuclear factor kappa B (NF-κB), respectively. Sanguinarine and chelerythrine elicited nuclear translocation of the p65 subunit of NF-κB. The nuclear import of p65 was strongly augmented by these akaloids in non-stimulated cells as well as in cells challenged with tumor necrosis factor alpha (TNFα). Colchicine and berberine had no effect on p65 nuclear translocation regardless of the presence or absence of TNFα. Colchicine caused rapid degradation of the GR protein, whereas berberine had no effect on GR content or cellular localization. Sanguinarine and chelerythrine induced accumulation of GR in the nucleus with concomitant diminution of cytosolic GR. Analyses on the transcriptional activity of GR and NF-κB monitored by reporter assays using HeLa cells transiently transfected with glucocorticoid response element (pGRE-LUC) and/or NF-κB elements fused to luciferase gene (pNF-κB-luc) showed that none of the compounds tested had the capability to trigger GR and/or NF-κB transcriptional activities, respectively. The ligand binding assay showed that colchicine and berberine are not GR ligands whereas sanguinarine and chelerythrine significantly decreased binding of 3H-labelled dexamethasone to GR. In conclusion, structurally diverse natural antiflogistics displayed differential effects on GR and NF-κB in HeLa cells.

Synthesis and pharmacological evaluations of new steroidal anti-inflammatory antedrugs: 9α-Fluoro-11β,17α,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16α-carboxylate (FP16CM) and its derivatives

In continuing efforts to develop potent anti-inflammatory steroids without systemic adverse effects, methyl 9α-fluoro-11β,17α,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16α-carboxylate (FP16CM) and its 16-alkoxycarbonyl derivatives (FP16CE, FP16CP and FP16CB) were synthesized based on the antedrug concept. The steroids were evaluated for their pharmacological activities and adverse systemic effects. All steroidal antedrugs showed both binding affinity to the glucocorticoid receptor in liver cytosol and inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cell. These compounds also inhibited croton-oil-induced ear edema and showed no systemic effects such as thymus atrophy and suppression of corticosterone level after 5-day treatment. Among those compounds tested, FP16CM showed the highest activities in receptor binding, NO inhibition and ear edema, these activities were comparable to those of prednisolone. Hydrolysis study in plasma showed that FP16CB was hydrolyzed rapidly, with the half-live ( T 1/2) of 3.2 min and the half-lives of other compounds were between 16.9 and 29.4 min. These results support the antedrug concept, of which the decrease in systemic adverse effects is attributed to fast hydrolysis to inactive metabolite in the systemic circulation.

Glucocorticoids stimulate the activity of large-conductance Ca 2+-activated K + channels in pituitary GH 3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH 3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca 2+-activated K + current ( I K(Ca)) with an EC 50 value of 21 ± 5 μM. Dexamethasone-induced increase in I K(Ca) density was suppressed by paxilline (1 μM), yet not by glibenclamide (10 μM), pandinotoxin −Kα (1 μM) or mifepristone (10 μM). Paxilline is a blocker of large-conductance Ca 2+-activated K + (BK Ca) channels, while glibenclamide and pandinotoxin-Kα are blockers of ATP-sensitive and A-type K + channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 μM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BK Ca-channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca 2+-sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 μM) also increased BK Ca-channel activity. Dexamethasone-mediated stimulation of I K(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.