Glucocorticoids exacerbate peroxynitrite mediated potentiation of glucose deprivation-induced death of rat primary astrocytes

Glucocorticoids have been implicated in the exacerbation of several types of neurotoxicity in various neuropathological situations. In this study, we investigated the effect of a glucocorticoid dexamethasone on glucose deprivation induced cell death of immunostimulated rat primary astrocytes, which is dependent on the production of peroxynitrite from the immunostimulated cells [Choi et al. Glia, 31(2001) 155-164; J. Neuroimmunol. 112 (2001) 55-62]. Glucose deprivation in immunostimulated rat primary astrocytes results in the release of lactate dehydrogenase (LDH) after 5 h and co-treatment with dexamethasone (1-1000 nM) dose-dependently increased LDH release. Treatment of the exogenous peroxynitrite generator SIN-1 (20 microM), plus glucose deprivation, also increased LDH release after 6 h and co-treatment with dexamethasone dose-dependently increased LDH release. A glucocorticoid receptor antagonist, RU-486, reversed the potentiation of cell death by dexamethasone. Glucose deprivation in immunostimulated cells decreased the intracellular ATP levels, which preceded LDH release from the cell, and co-treatment with dexamethasone dose-dependently potentiated the depletion of intracellular ATP levels. In addition, dexamethasone further deteriorated SIN-1 plus glucose deprivation-induced decrease in mitochondrial transmembrane potential in rat primary astrocytes, which was reversed by RU-486. The results from the present study suggest that glucocorticoids may be detrimental to astrocytes in situations where activation of glial cells are observed, including ischemia and Alzheimer's disease, by mechanisms involving depletion of intracellular ATP levels and deterioration of mitochondrial transmembrane potentials.     

Characterization of Glucocorticoid Type II Receptors in Neuronal and Glial Cultures from Rat Brain

The purpose of this study was to characterize and compare the properties of glucocorticoid Type II receptors in neuronal and astrocyte glial cultures prepared from rat brain. Type II receptors in cytosol prepared from cultured cells were labeled with [³ H]dexamethasone (DEX) at 0°C. The binding was saturable and specific, with a complete displacement by unlabeled DEX or RU 28362 (a pure glucocorticoid). Scatchard analysis of [³ H]DEX binding suggested a single class of receptors with a slightly lower dissociation constant (K_d) in neuronal (1.13 nM) versus astrocyte glial (1.64 nM) cytosol. The number of binding sites (B_max) in astrocyte glial cultures was four times that in neuronal cultures on a per milligram protein basis (120.3 versus 29.3 fmol/mg protein). The presence of Type II receptors in cultured neurons and astrocyte glia was further confirmed by immunofluorescent staining with a monoclonal antibody against this receptor (BuGR-2). The steroid specificity of Type II receptors was studied by examining the displacement of [³ H]DEX binding to cytosol with unlabeled steroids. For both types of cultures, the potency series for competition was RU 28362> DEX> corticosterone> > aldosterone. Switching cultured cells from serum-supplemented to serum-free medium reduced [³ H]DEX binding at low concentrations (0.5 to 5 nM) of the ligand in both types of culture, thus resulting in a decrease in the apparent affinity. This treatment did not, however, have any significant effect on the total number of binding sites. In summary, these results demonstrate that both neuronal and astrocyte glial cells in culture contain specific glucocorticoid Type II receptors, which resemble those seen in the brain and peripheral tissues.     

Implication of glucocorticoid receptors in the stimulation of human glioma cell proliferation by dexamethasone

Dexamethasone is frequently used in the therapy of brain tumor patients. We investigated the effect of dexamethasone on the proliferation of three short-term and four established human glioma cell lines in vitro, using a microculture tetrazolium assay to determine growth rates. In one short-term culture and in one established cell line dexamethasone consistently stimulated the proliferation in concentration-dependent way. The proliferation was maximally enhanced at a concentration of approximately 0.1 μM. In these two cell lines a relatively high level of glucocorticoid receptors was present, whereas low levels of glucocorticoid receptors were found in the other cell lines. In addition, we demonstrated that the stimulatory effects of dexamethasone on the proliferation of the glioma cell lines can be antagonized by the antiglucocorticoid RU38486. The results demonstrate unequivocally that the glucocorticoid receptor plays a role in the growth stimulating effect of dexamethasone.     

Dexamethasone and forskolin synergistically increase [Met]enkephalin accumulation in mixed brain cell cultures

Possible synergistic effects of the glucocorticoid dexamethasone (DEX, 10⁻⁷ M) and the adenylate cyclase agonist forskolin (FSK, 10⁻⁵ M) on [Met⁵]enkephalin (ME) accumulation were examined in enriched rat glial cultures and in mixed neuronal/glial cultures. In enriched glial cultures, DEX and FSK each stimulated the accumulation of ME 2–3-fold over basal media levels, but there was little additional stimulation when these agonists were combined. In contrast, mixed neuronal/glial cultures showed only weak responses to DEX or FSK alone, but the combination of these agonists produced a pronounced synergistic effect on media ME accumulation (6–10-fold over basal levels). The DEX effect was mediated via a classical glucocorticoid receptor, since DEX was potent (acting over a concentration range of 10⁻¹¹−10⁻⁷ M), mimicked by corticosterone (10⁻⁶ M), and blocked by the glucocorticoid receptor antagonist RU486. There was a pronounced time lag (2 days) for the synergistic effects of DEX+FSK to develop. In situ hybridization and immunocytochemical studies suggested that astrocytes were the major source for the increased ME production in all mixed neuronal/glial cultures examined. Creating a mixed culture by plating fetal neurons onto confluent, enriched P7 glial cultures inhibited accumulation of ME in the media. DEX+FSK, but neither agonist alone, overcame this neuronal inhibition and increased accumulation of media ME to levels identical to levels in stimulated enriched glial cultures. The net effect was a 6-fold increase in ME accumulation in the mixed neuronal/glial cultures relative to a 2.5-fold increase in the enriched glial cultures. Neuronal inhibition of basal glial ME production could explain the similar synergistic effects of DEX+FSK observed in all mixed neuronal/glial cultures examined, and may be important in suppressing ME production by astrocytes in the brain.     

Rat C6 glioma cells contain type I as well as type II corticosteroid receptors

Rat brain cytosol contains Type I corticosteroid receptors. Unlike Type II (glucocorticoid) receptors, Type I receptors have high affinity for the endogenous corticosteroids - aldosterone, deoxycorticosterone, and corticosterone - and much lower affinities for synthetic glucocorticoids. In the present study, we report that Type I corticosteroid receptors are present in C6 glioma cells. Type I receptors were identified in C6 cell cytosol and whole cells by the binding of [3H]aldosterone. The specific glucocorticoid RU 26988 was used to block Type II receptors. Measured in whole C6 cells, Type I receptors had a density of 2.1 +/- 1.1 fmol/10(6) cells and a dissociation constant (Kd) for [3H]aldosterone of 0.41 +/- 0.06 nM. The density of Type I receptors was only 2% of the density of Type II corticosteroid receptors (96 +/- 7 fmol/10(6) cells), measured in whole C6 cells by [3H]triamcinolone binding. The steroid specificity of glial cytosolic Type I receptors (deoxycorticosterone greater than corticosterone greater than aldosterone greater than dexamethasone greater than triamcinolone much greater than RU 26988) was identical to the steroid specificity of Type I receptors in rat brain cytosol. The potency of deoxycorticosterone was somewhat reduced when measured in whole cells. The steroid specificity of the Type I receptor differed markedly from that of the Type II (glucocorticoid) receptor (triamcinolone greater than dexamethasone greater than RU 26988 corticosterone greater than deoxycorticosterone greater than aldosterone). Since Type I receptors in the kidney mediate effects of aldosterone upon renal transport of sodium and potassium, it is proposed that glial Type I corticosteroid receptors may be involved in the regulation of glial ion transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further studies of brain aldosterone binding sites employing new mineralocorticoid and glucocorticoid receptor markers in vitro

We have used synthetic markers of the glucocorticoid (GC) receptor (RU 28362) and of the mineralocorticoid (MC) receptors (RU 26752 and RU 28318) to characterize the specificity of the sites binding aldosterone (ALDO), dexamethasone (DEX) and corticosterone (CORT) in cytosol of hippocampus. The results obtained suggest that ALDO was bound mostly to a MC receptor, as the relative binding affinity (RBA) of the GC receptor marker (and that of the previously studied RU 26988) was negligible for this site, in contrast to the high RBA displayed by RU 26752. DEX was bound for a large part to a GC receptor, as RU 28362 competed for this site, although the MC receptor marker still showed some affinity. An intermediate effect of both marker types was obtained with CORT. RU 28318 was a weak competitor for either the GC or the MC binding site. Thus, RU 28362 and RU 26752 allowed the discrimination of two to three receptors in the hippocampus, similarly to those described in the kidney. Finally, we have demonstrated the usefulness of these synthetic markers in identifying MC binding sites in several brain regions and also in the hippocampus during ontogenetic development.     

Dexamethasone induces cell death which may be blocked by NMDA receptor antagonists but is insensitive to Mg2+ in cerebellar granule neurons

Since dexamethasone may elevate the Ca2+ influx through NMDA receptors, we have investigated mechanisms of dexamethasone toxicity in rat cerebellar granule neurons. Dexamethasone concentrations over 0.1 microM induced cell death that reached about 20% of the death induced by glutamate. Dexamethasone-induced cell death was reduced by more than 80% by the mineralocorticoid antagonist RU 28318 or the NMDA receptor antagonists MK 801 and CGP 39551, whereas RU 28318 rescued only approximately 30% of cells treated with glutamate, indicating that dexamethasone requires NMDA receptors to induce acute neuronal toxicity and that a fraction of the neurons showed this toxicity. Mg2+ reduced the cell death induced by glutamate at potassium concentrations of 1 mM and 5 mM, but not at 25 mM. In contrast, cell death induced by dexamethasone was not significantly reduced by Mg2+ in any of the potassium concentrations. Both glutamate and dexamethasone induced toxicity with translocation of the apoptosis inducer NGFI-B to the mitochondria seen after 30 min-2 h concomitant with activation of apoptosis inducing factor (AIF) and caspase-3. In conclusion, dexamethasone induces a rapid toxicity which is blocked by NMDA receptor antagonists other than Mg2+, and involves mitochondrial apoptosis inducer NGFI-B.     

Functional interference of dexamethasone on some morphine effects: hypothesis for the steroid-opioid interaction

The effect of dexamethasone (DEX) and its interaction with morphine has been studied on transmurally-stimulated guinea-pig ileum preparation, gastrointestinal transit and analgesia. TRANSMURALLY-STIMULATED GUINEA-PIG ILEUM PREPARATION: DEX dose-dependently reduced the contractions of the ileum. Proteic synthesis inhibitors did not modify the inhibition induced by DEX whereas RU-38486, a glucocorticoid antagonist receptor, antagonized completely the inhibitory effect of DEX. GASTROINTESTINAL TRANSIT: DEX was found to antagonize morphine-, atropine- and verapamil-induced constipation. Cycloheximide does not modify the DEX effects. RU-38486 reverses both the inhibitory action of DEX on gastrointestinal transit and its reducing effect on morphine-induced constipation. ANALGESIA: DEX reduced the antinociception induced by mu agonists, morphine, DAMGO and beta endorphin whereas the steroid exerted little or no influence on the antinociception induced by a delta1 agonist, DPDPE and delta2 agonist deltorphin II. DEX potentiated the antinociception induced by the K agonist, U50,488. Cycloheximide, a protein synthesis inhibitor, prevented the antagonism by DEX of responses to the mu opioid agonists. Finally, i.c.v. injection of DEX significantly reduced morphine analgesia in Swiss mice whereas no effects were observed in DBA/2J and C57BL/6 mice. In addition, i.p. injection of DEX significantly reduced morphine analgesia in all three strains. Our data indicate that in the rodent brain there is an important functional interaction between the corticosteroid and the opioid systems at least at the mu. receptor level, while delta and K receptors are modulated in different ways. These results, particularly the effects of drug interaction for i.c.v. administration, strongly confirm a central site for DEX and RU 38486 action as well as the use of different genetic strains may provide a useful approach for studying DEX-morphine analgesia interaction.     

Pharmacological mechanisms mediating phencyclidine-induced apoptosis of striatopallidal neurons: the roles of glutamate, dopamine, acetylcholine and corticosteroids

Phencyclidine (PCP) has recently been shown to induce apoptosis of a subpopulation of striatopallidal neurons which lie in the dorsomedial caudate-putamen. The pharmacological mechanisms underlying this PCP-induced striatal death were investigated in a series of small experiments. Striatal silver-methenamine-stained sections from rats injected acutely with dizocilpine (MK-801; 1.5-5 mg/kg, i.p.) were analysed to determine whether other non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists could induce apoptotic-like changes in striatal cells. The effects of amphetamine (3-12 mg/kg, i.p.) were similarly investigated as PCP can elevate extracellular dopamine levels and dopamine has the potential to be neurotoxic. The potential involvement of dopamine transmission in PCP-induced striatal apoptosis was also tested by determining the effect of co-administering SCH23390 (D1 dopamine receptor antagonist) and quinpirole (D2 dopamine receptor agonist) on PCP (80 mg/kg, s.c.)-induced striatal apoptotic-like cell death. Equivalent experiments were performed using scopolamine (cholinergic antagonist) as this drug blocks PCP-induced damage of the retrosplenial cortex and RU38486 (corticosteroid receptor antagonist) as a similar subpopulation of striatal neurons undergoes apoptosis following dexamethasone administration. Injection of neither MK-801 nor amphetamine induced elevations of apoptotic-like cells in the striatum nor did co-administration of SCH23390 or scopolamine affect the levels of PCP-induced striatal cell death. In contrast, quinpirole elevated the levels of PCP-induced apoptotic-like striatal cell death and RU38486 markedly reduced it. Within the retrosplenial cortex, scopolamine lowered PCP-induced apoptotic-like cell death whereas RU38486 was without effect. These results suggest that PCP-induced striatal apoptosis results from a corticosteroid-dependent mechanism. The results further demonstrate that different pathological mechanisms underlie PCP-induced neuronal damage in the striatum and the retrosplenial cortex.     

Direct targeting of hippocampal neurons for apoptosis by glucocorticoids is reversible by mineralocorticoid receptor activation

An important question arising from previous observations in vivo is whether glucocorticoids can directly influence neuronal survival in the hippocampus. To this end, a primary postnatal hippocampal culture system containing mature neurons and expressing both glucocorticoid (GR) and mineralocorticoid (MR) receptors was developed. Results show that the GR agonist dexamethasone (DEX) targets neurons (microtubule-associated protein 2-positive cells) for death through apoptosis. GR-mediated cell death was counteracted by the MR agonist aldosterone (ALDO). Antagonism of MR with spironolactone ([7alpha-(acetylthio)-3-oxo-17alpha-pregn-4-ene-21 carbolactone] (SPIRO)) causes a dose-dependent increase in neuronal apoptosis in the absence of DEX, indicating that nanomolar levels of corticosterone present in the culture medium, which are sufficient to activate MR, can mask the apoptotic response to DEX. Indeed, both SPIRO and another MR antagonist, oxprenoate potassium ((7alpha,17alpha)-17-hydroxy-3-oxo-7-propylpregn-4-ene-21-carboxylic acid, potassium salt (RU28318)), accentuated DEX-induced apoptosis. These results demonstrate that GRs can act directly to induce hippocampal neuronal death and that demonstration of their full apoptotic potency depends on abolition of survival-promoting actions mediated by MR.     

Glucocorticoids enhance the potency of Schwann cell mitogens

Previous studies have documented that cultured Schwann cells require serum-containing medium to respond maximally to mitogens. We now report that Schwann cells are able to proliferate to a mitogenic response in a serum-free defined medium termed oligodendrocyte defined media (ODM). Glucocorticoids are the essential component of ODM which allow Schwann cell proliferation in the serum-free medium. Charcoal treatment of the fetal calf serum decreases the mitogenic potency of the axolemma-enriched fraction (AEF) by 50%. The addition of 2 μM hydrocortisone to charcoal-treated fetal calf serum restores 75% of the lost mitogenicity. These observations are consistent with the view that glucocorticoids present in fetal calf serum are potent co-mitogens essential for AEF-induced Schwann cell proliferation. The synthetic glucocorticoid, dexamethasone, is a more potent co-mitogen than hydrocortisone, with a maximal effect at concentrations less than 10 nM. In contrast, other steroids including aldosterone, progesterone, testosterone, and 17β-estradiol have no effect on enhancing the mitogenic response of Schwann cells to the AEF. The glucocorticoid antagonists RU 486 and dehydroepiandrosterone (DHEA), but not the antiestrogenic compound tamoxifen, block AEF-induced Schwann cell proliferation. These results suggest that glucocorticoid-induced Schwann cell proliferation is mediated through a glucocorticoid receptor (GR) mechanism. We detected immunoreactivity to the GR in the cytoplasm, but not in the nuclei of Schwann cells grown in ODM lacking dexamethasone. The addition of 100 nM dexamethasone to these cultures resulted in immunoreactivity in the nucleus. This data suggests that glucocorticoids working through the GR are potent co-mitogens for Schwann cell proliferation. © 1994 Wiley-Liss, Inc.     

Real-time imaging of glucocorticoid receptor dynamics in living neurons and glial cells in comparison with non-neural cells.

To investigate the intracellular trafficking of glucocorticoid receptor (GR) in response to various conditions in a single living cell, a green fluorescent protein (GFP) and rat GR chimera construct (GFP-GR) was prepared. We transiently transfected GFP-GR into primary cultured rat hippocampal neurons, cortical glial cells, and non-neural cells, e.g. COS-1 cells and CV-1 cells, and compared the dynamic changes in subcellular localization of GFP-GR in these cells. When GFP-GR was expressed in the cells, GFP-GR efficiently transactivated the mouse mammary tumour virus promoter in response to dexamethasone (DEX). The cytoplasm-to-nuclear translocation of GFP-GR induced with 10(-7) m DEX, a specific agonist of GR, at 37 degrees C was completed within 30 min in all cell types used, and the rate of nuclear translocation was dependent on the ligand dose. The translocation of GFP-GR into the nucleus from the cytoplasm was induced in a ligand-specific manner, similar to that of the native GR. The disruption of microtubules by colchicine or nocodazole showed no significant effect on the DEX-induced GFP-GR translocation from the cytoplasmic region to the nuclear region. The cells were not deteriorated during time-lapse imaging analysis for 1 h at 37 degrees C. The present findings suggest that the subcellular localization of GFP-GR is dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GR among different types of cells in vitro.     

Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord

Apoptosis is an important cause of secondary cell death in spinal cord injury (SCI). SCI induces the expression of the low affinity neurotrophin receptor p75 (p75NTR), that in the absence of the high affinity component, TrkA, can promote cell death by apoptosis. We therefore hypothesized that a reduction of p75NTR expression in SCI may increase tissue sparing and therefore improve recovery of function. As a tool to test our hypothesis we used the synthetic glucocorticoid dexamethasone (DEX) to down-regulate p75NTR expression. A standardized thoracic spinal cord contusion injury was produced in female rats. Laminectomized and SCI rats received various doses of DEX immediately after injury and the treatment was continued daily for 7 days. DEX, given at high doses (20 mg/kg, s.c.) but not at low doses (1 or 8 mg/kg) prevented the increase in p75NTR mRNA and protein in SCI rats, without affecting the expression of TrkA. High doses of DEX also reduced cellular apoptosis both in white and gray matters. This effect correlated with the ability of DEX to accelerate behavioral recovery of function measured by a combined behavioral score. These data suggest that reduction of p75NTR in SCI may be a therapeutic strategy to limit cell and tissue damage and therefore to improve recovery of function in SCI patients.     

Possible involvement of 5alpha-reduced neurosteroids in adrenergic and serotonergic stimulation of GFAP gene expression in rat C6 glioma cells

Influence of adrenergic and serotonergic stimulation on glial fibrillary acidic protein (GFAP) gene expression in rat C6 glioma cells was first examined as an in vitro model experiment for investigating the neuronal regulation of glial cell differentiation. Stimulation of these cells with isoproterenol and serotonin elevated GFAP mRNA levels followed by an increase in its protein contents, thus suggesting that both adrenergic and serotonergic stimulation might induce the differentiation of the glioma cells. In addition, progesterone and its 5alpha-reduced metabolite dihydroprogesterone also elevated GFAP mRNA levels in rat C6 glioma cells, consistent with their stimulatory actions on GFAP gene expression observed in rat astrocytes. Further studies showed that the elevation of GFAP mRNA levels induced by isoproterenol and serotonin as well as progesterone was abolished by pretreatment of the glioma cells with finasteride, an inhibitor of 5alpha-reduced steroid production. Moreover, the stimulatory actions of isoproterenol and serotonin on GFAP gene expression were inhibited by pretreatment with a GABA(A) receptor antagonist bicuculline and a progesterone receptor antagonist RU486. These findings suggest that both adrenergic and serotonergic stimulation may indirectly activate GFAP gene expression probably through the production of 5alpha-reduced steroid metabolites in rat C6 glioma cells, proposing the possibility that 5alpha-reduced neurosteroids may play a potential role in the neuronal regulation of glial cell differentiation.     

Dexamethasone in the presence of desipramine enhances MAPK/ERK1/2 signaling possibly via its interference with β-arrestin

Antidepressant medication is the standard treatment for major depression disorder (MDD). However, the response to these treatments is often incomplete and many patients remain refractory. In the present study, we show that the glucocorticoid receptor (GR) agonist dexamethasone (DEX) increased MAPK/ERK1/2 signaling in the presence of the noradrenergic antidepressant, desipramine (DMI), while no such effect was induced by DEX or DMI alone in human neuroblastoma SH-SY5Y cells. This enhancement was dependent on the activation of both α₂ adrenergic receptors (AR) and GR. The timing of MAPK/ERK1/2 activation as well as DEX-induced reduction in membranous α₂ AR suggests the involvement of a β-arrestin-dependent mechanism. In line with the latter, DEX increased cytosolic and decreased membranous levels of β-arrestin. Concomitantly, DEX induced a time-dependent increase in cytosolic α₂ AR-β-arrestin interaction and a decrease in β-arrestin interaction with Mdm2 E3 ubiquitin ligase. All of these effects of DEX were prevented by the GR antagonist RU486. Our data suggest an additional intracellular role for DEX, in which activation of GR interferes with the trafficking and degradation of β-arrestin-α2c-AR complex. We suggest that such an interaction in the presence of DMI can enhance MAPK/ERK1/2 signaling, a key player in neural plasticity and neurogenesis processes, which is impaired in MDD, while stimulated by antidepressants.     

Glucocorticoid-induced apoptosis in CNS microvascular pericytes

Pericyte loss or migration from its vascular location may be an important step in microvascular remodeling. Decreased pericyte to endothelial ratios are characteristics of newly formed vessels as well as microvessels undergoing regression, and may be due to selective degeneration via necrotic cell death or via programmed cell death. In this study, we have examined glucocorticoid-induced apoptosis in primary rat CNS pericytes. Characterization of apoptosis was determined using five independent criteria: (1) the translocation of receptors for annexin V from the inner to the outer surface of the plasma membrane, (2) the translocation of cytochrome C from the mitochondria to the cytosol, (3) the induction of DNA fragmentation, (4) the induction of classic changes in cell morphology, and (5) the appearance of TUNEL-positive cells. Incubation of CNS pericytes with dexamethasone induced the appearance of apoptotic cells in a time- and dose-dependent manner. Pericytes express immunologically detectable glucocorticoid receptors, and addition of the glucocorticoid receptor antagonist mifepristone inhibited dexamethasone-induced pericyte apoptosis. That pericytes undergo apoptosis in response to dexamethasone suggests that the regulatory function of this steroid may be important in vascular development and that pericyte apoptotic cell death may accompany vascular regression. Deregulation of pericyte involvement in vascular homeostasis and hemostasis may result in clinical disease.