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

Brain Research 923 (2001) 163–171 www.elsevier.com / locate / bres

Research report

Glucocorticoids exacerbate peroxynitrite mediated potentiation of glucose deprivation-induced death of rat primary astrocytes Chan Young Shin a , Ji-Woong Choi a , Eun Sook Jang a , Jong Hoon Ryu b , Won-Ki Kim c , d a, Hyoung-Chun Kim , Kwang Ho Ko * a

Department of Pharmacology, College of Pharmacy, Seoul National University, San 56 -1, Shillim-Dong, Kwanak-Gu, Seoul 151 -742, South Korea b Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, South Korea c Department of Pharmacology, College of Medicine, Ewha Womans University, South Korea d College of Pharmacy, Kang Won National University, South Korea Accepted 5 October 2001

Abstract 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 mM), 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.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Dexamethasone; Astrocyte; Peroxynitrite; Glucose-deprivation; ATP; Mitochondrial transmembrane potential

1. Introduction Glucocorticoids are hormones secreted from adrenal glands in response to various physical and psychological stresses. They serve as regulatory factors of gene expression through interaction with their cytoplasmic receptors and also regulate glucose transport and metabolism. Clinically, they have been widely used as anti-inflammatory agents. *Corresponding author. Tel.: 182-2-880-7848; fax: 182-2-885-8211. E-mail address: [email protected] (K.H. Ko).

Several researchers have reported that glucocorticoids exacerbate neuronal damage induced by several types of neuronal insults including hypoxia–ischemia, seizures, treatment of antimetabolites, or hypoglycemia [1–7]. In many cases, these endangerments correlate with the disruption of energy metabolism [1,8–11]. The disruption of energy metabolism may deteriorate cellular defense mechanisms, such as antioxidant pathways, which has been suggested as a general mechanism of glucocorticoid-stimulated neuronal toxicity in the instance of brain insults [12,13]. Recently, we have reported that rat cortical primary

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03212-7

164

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

astrocytes showed potentiation of glucose-deprivation-induced cell death upon induction of iNOS by the cotreatment with a lipopolysaccharide (LPS) and interferon-g (IFN-g) [14]. In this process, peroxynitrite (ONOO 2), which is produced from the reaction between superoxide anions and nitric oxide, plays an essential role [15–17]. Several researchers including us, have suggested that NO and ONOO 2 affect both mitochondrial function and the electron transport chain [17,18 and references therein]. Brookes et al. reported that NO, and presumably ONOO 2 , inhibit ATP synthesis from isolated brain mitochondria [19]. In addition, Tatsumi et al. have reported that stimulation of cardiac myocytes with IL-1b increased NO production with a concomitant decrease in the cellular ATP level [20], which coincides with our results obtained from LPS / IFN-g-stimulated rat primary cortical astrocytes [21]. The above-mentioned reports suggest that glucocorticoids might aggravate a glucose-deprivation induced death of immunostimulated rat primary astrocytes by a mechanism involving the synergistic deterioration of the intracellular energy status. In this study, we investigated the effects of glucocorticoids on the glucose deprivationinduced cell death of immunostimulated rat primary astrocytes. The results showed that glucocorticoids synergistically increased peroxynitrite / glucose deprivation-induced depolarization of mitochondrial transmembrane potential and the depletion of intracellular ATP, which leads to the accelerated cell death of rat primary astrocytes.

immunostimulation, rat primary astrocytes were pretreated for 48 h with IFN-g (100 U / ml) and LPS (1 mg / ml). After the immunostimulation, glucose-deprivation was achieved by repeated rinsing and incubation in glucose-free Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL), The glucose-free medium was not supplemented with a serum, which interfered with the LDH assay. Glucocorticoids and other drugs were added during the 6 h of glucose deprivation. In some cases, glucocorticoids were used to pretreat for 48 h during the immunostimulation period. In these cases, glucocorticoids were omitted during the glucosedeprivation period. To investigate the effect of glucocorticoid treatment on exogenous peroxynitrite-potentiated death of C6 glioma cells, the cells without immunostimulation were treated with 20 mM of SIN-1 during the 6-h glucose deprivation period.

2.3. Measurement of NO NO production was determined by measuring nitrite, a stable oxidation product of NO, as described previously [23]. In brief, nitrite levels were determined by adding the Greiss reagent (mixing equal volumes of 0.1% napthylethylenediamine dihydrochlroride and 1% sulfanilamide in 5% phosphoric acid). After 10 min, the absorbance at 550 nm was determined using an UV spectrophotometer (Beckman DU-650, Fullerton, CA, USA).

2.4. Measurement of cell death 2. Methods

2.1. Materials Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO, USA). Recombinant rat IFN-g, DMEM / F12, glucose-free DMEM and fetal bovine serum were obtained from Gibco BRL (Grand Island, NY, USA). Dexamethasone, hydrocortisone and mifepristone (RU486) were acquired from Sigma. Stock solutions (10 mM) of those steroids were prepared in DMSO and stored at 2708C. Stock solutions were diluted in DMSO before use and adjusted to final 0.1% DMSO in incubation media. 3-Morpholinosydnonimine (SIN-1) was obtained from Calbiochem (La Jolla, CA, USA).

2.2. Primary rat astrocyte cell culture Rat primary astrocytes were cultured from the prefrontal cortices of 2- to 4-day-old Sprague–Dawley rat pups as previously described [22]. Briefly, the prefrontal cortex was dissected out and digested with trypsin for 10 min at 378C. After trituation, cells were seeded onto poly-L-lysine coated plates and were cultured in DMEM / F12 with 10% heat inactivated fetal bovine serum (FBS). In most cases, 12–13 days-old cells were used for this study. For the

Cell death was assessed by morphological examination of cells using phase-contrast microscopy and quantified by measuring LDH release into the medium at various time points after starting glucose deprivation. The LDH amount, corresponding to complete glial damage / death, was measured in sister cultures treated with 0.1% Triton X-100 for 30 min at 378C. Basal LDH levels (generally less than 10% of total LDH release) were determined in sister cultures subjected to sham wash with 5 mM glucose containing DMEM and subtracted from the levels in experimental conditions to yield the LDH signal specific to the experimental injury.

2.5. Measurements of mitochondrial transmembrane potential The MTP was measured according to the previous report by Reers et al. [24] with minor modifications [16]. In brief, astrocytes were cultured on glass slides and were loaded for 30 min at 378C with JC-1 (1 mg / ml) in a culture medium. Depolarization of MTP was assessed by measuring the fluorescence intensities at an excitation wavelength of 485 nm and emission wavelengths of 530 and 590 nm, using a confocal microscope (a Zeiss Axiovert 135 inverted microscope equipped with a X20 Neoflur objective and Zeiss LSM410 confocal attachment). Fluorescence

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

165

intensity was observed at indicated times with identical parameters, such as contrast and brightness, for all samples. Four groups of 20 cells were randomly selected from the image. The average fluorescence intensity for each group was calculated from the fluorescence intensity per cell and averages were taken from groups of four.

2.6. Measurement of ATP contents The level of intracellular ATP was measured by the methods previously described with slight modifications [21,25]. Briefly, cells were harvested with 10% trichloroacetic acid (TCA) and sonicated for 15 s on ice. The lysates were supplemented with 2 mM EDTA and 2 mg / ml BSA. After centrifugation, the supernatants were adjusted to neutrality with 4 M KOH and the ATP content was determined using a commercial assay kit (ATP luminescence detection kit, Molecular Probe, Eugene, OR, USA).

2.7. Statistical analysis Data are expressed as the mean6standard error of mean (S.E.M) and analyzed for statistical significance by using one-way analysis of variance (ANOVA) followed by Newman–Keuls test as a posthoc test and a P value ,0.01 was considered significant.

3. Results As reported previously, rat primary astrocytes immunostimulated with IFN-g (100 U / ml) plus LPS (1 mg / ml) were vulnerable to glucose deprivation-induced toxicity, otherwise relatively non-toxic up to 10 h of glucosedeprivation [data not shown and 14]. Immunostimulated cells produced 142.34618.38 mmol / mg protein of nitric oxide as compared with 11.5161.12 mmol / mg protein from control cells. Treatment of NOS inhibitor N G -nitro-Larginine (NNA, 800 mM) or L-N v -nitro-L-arginine methyl ester ( L-NAME, 1 mM) during the immunostimulation period prevented the increase in NO production from the rat primary astrocytes and inhibited the glucose deprivation induced cell death. In this study, 5 h glucose-deprivation caused significant LDH release (19.162.88% of total LDH releasable from the cells) from immunostimulated rat primary astrocytes (Fig. 1). In contrast, in incubation media supplemented with 5 mM glucose, immunostimulated rat primary astrocytes did not show LDH release (Fig. 1). Co-treatment of dexamethasone during the glucose deprivation period, dose-dependently increased the LDH release from immunostimulated rat primary astrocytes (Fig. 1). The increase in LDH release was evident even at 1 nM, the lowest concentration examined. Addition of dexamethasone to 5 mM glucose supplemented rat primary astrocytes did not show toxicity (Fig. 1).

Fig. 1. Effects of dexamethasone on the glucose deprivation-induced cell death of immunostimulated rat primary astrocytes. Primary rat cortical astrocytes were immunostimulated for 48 h with IFN-g (100 U / ml) plus LPS (1 mg / ml) in DMEM / F12. Immunostimulated cells were incubated in the presence (black bar) or absence (gray bar) of 5 mM glucose with varying concentrations of dexamethasone. The amount of LDH released into the media after 6 h of glucose deprivation was determined as described in Methods. Each bar indicates the mean6S.E.M. (n54). *, Significant difference from each preceding dose unit of dexamethasone (*, P,0.01).

We previously reported that the vulnerability of immunostimulated rat astrocytes to glucose-deprivation was largely attributable to the increased production of peroxynitrite in immunostimulated cells [15–17]. An exogenous peroxynitrite generator SIN-1 mimicked the effect of immunostimulation upon glucose-deprivation induced cell death in rat primary astrocytes [14]. In this study, it was also observed that dexamethasone and another glucocorticoid hydrocortisone dose-dependently increased SIN-1 / glucose deprivation-induced cell death of rat primary astrocytes (data not shown). The LDH release induced by 100 nM hydrocortisone was 73.461.3% of total cellular LDH, which was comparable to the effect of 100 nM dexamethasone (71.765.5%). Because the cell death was dependent on NO / ONOO production, we investigated the effect of dexamethasone on NO production from the immunostimulated rat astrocyte. As shown in Fig. 2 4 h cotreatment of dexamethasone during the glucose-deprivation period did not affect NO production from the immunostimulated rat primary astrocyte, which indicates that increased cell death is not mediated by the increased production of NO / ONOO2. Glucocorticoids act as general immunosuppressants and it has been reported that longterm treatment (more than 24 h) with glucocorticoids inhibits iNOS induction from glial cells. In this study, it was also observed that longterm (48 h) treatment with dexamethasone during the immunostimulation period inhibited NO production from rat primary astrocytes (Fig. 2, inset). To investigate whether the effect of dexamethasone is

166

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

Fig. 2. Effects of dexamethasone on the NO production from immunostimulated rat primary astrocytes. Primary rat cortical astrocytes were immunostimulated for 48 h with IFN-g (100 U / ml) plus LPS (1 mg / ml) in DMEM / F12. Immunostimulated cells were deprived of glucose as described with varying concentrations of dexamethasone. After 4 h, NO production was determined as described in the Methods. Note that dexamethasone co-treatment during the glucose deprivation period did not affect NO production from the cell. Each points indicates the mean6S.E.M. (n54). Inset: in contrast, long-term pretreatment of dexamethasone (48 h) with IFN-g plus LPS during the immunostimulation period dose dependently inhibited NO production from the cells. *, Significant difference from 0 nM dexamethasone (n54, P,0.01).

mediated by the glucocorticoid receptors we used RU-486, a synthetic glucocorticoid receptor antagonist. As shown in Fig. 3, the increased cell death induced by dexamethasone was inhibited by cotreatment with RU-486 (Fig. 3). We previously reported that immunostimulation of rat primary astrocytes decreased the intracellular ATP levels [21] and glucose-deprivation in immunostimulated cells synergistically depleted intracellular ATP levels (unpublished data) and deteriorated mitochondrial transmembrane potentials, which leads to the synergistic cell death [17]. In this study, cotreatment with dexamethasone during the glucose-deprivation period dose-dependently depleted intracellular ATP levels from immunostimulated rat primary astrocytes (Fig. 4). It was also observed that SIN-1 / glucose-deprivation caused depolarization of mitochondrial transmembrane potential in rat primary astrocytes, which was determined by JC-1 fluorescence (Fig. 5). Cotreatment of dexamethasone further deteriorated the mitochondrial transmembrane potential, which was reversed by RU-486 (Fig. 5).

4. Discussion In the present study, the glucocorticoid dexamethasone exacerbated peroxynitrite-dependent, glucose-deprivation induced cell death of rat primary astrocytes. In various brain insult conditions such as ischemia, seizures and neurodegenerative diseases, glial cells are activated by cytokines released from infiltrating leukocytes as well as activated glial cells themselves. Several cytotoxic molecules, including NO and ROS released from activated glial cells, have been implicated in the increased neurotoxicity. In this condition, we found that intracellular ATP levels in glial cells decreased significantly, which is dependent on the production of NO from the glial cells [21]. These results suggest that activated glial cells may be vulnerable to secondary insults including hypoxia, glucose deprivation and overproduction of ROS [14 and our unpublished results]. Considering the essential homeostatic function of glial cells in the brain, it may underlie the even more devastating brain damage observed in secondary ischemia,

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

Fig. 3. Inhibition of dexamethasone-induced potentiation of SIN-1 / glucose deprivation-induced cell death of rat primary astrocytes by RU-486. Primary rat cortical astrocytes were incubated in glucose-free DMEM containing 20 mM SIN-1 with or without 100 nM dexamethasone. RU-486 (1 mM) was added to the incubation media and the amount of LDH released into the media at 6 h after starting glucose deprivation was determined as described in Methods. Each bar indicates the mean6S.E.M. (n54). *, Significant difference from 5 mM glucose supplemented control (P,0.01); †, significant difference from SIN-1 / glucose deprivation (P,0.01); ¶, significant difference from SIN-1 / glucose deprivation1dexamethasone (P,0.01).

Fig. 4. Potentiation of glucose deprivation-induced ATP depletion by dexamethasone in immunostimulated rat primary astrocytes. Primary rat cortical astrocytes were immunostimulated for 48 h with IFN-g (100 U / ml) plus LPS (1 mg / ml) in DMEM / F12. The immunostimulated cells were incubated in glucose-free DMEM with varying concentrations of dexamethasone. After 4 h, intracellular levels of ATP (line and black circle) and the amount of LDH released into the media (gray bar) were determined as described in Methods. Each data point indicates the mean6S.E.M. (n54). *, Significant difference from 0 mM dexamethasone (P,0.01). Note that glucose-deprivation slightly decreased intracellular ATP levels as compared to glucose supplemented cells (G1), and the addition of dexamethasone to glucose deprived cells dose-dependently potentiated the ATP depletion.

167

recurrent seizures and the later stages of neurodegenerative diseases. In this study, cotreatment of a glucocorticoid dexamethasone during the glucose-deprivation period significantly and dose-dependently depleted intracellular ATP levels and deteriorated mitochondrial transmembrane potentials, which implies that glucocorticoids increased astrocytic cell death primarily via perturbation of the intracellular energy metabolism. Several researchers have suggested the negative effects of glucocorticoids on intracellular energy metabolism. Behl and co-workers reported that glucocorticoids exacerbate amyloid b peptide or glutamate-induced neuronal cell death in hippocampus [11,26]. The potentiation of the toxicity was observed only in low glucose media, showing the involvement of energy depletion in this process. A direct glucocorticoid regulation on cytochrome c oxidase has been already suggested [27]. These authors have reported that low doses of corticosterone administered to rats acted synergistically with sodium azide, an inhibitor of mitochondrial respiration, to inhibit cytochrome c oxidase activity. Furthermore they showed that chronic corticosterone and sodium azide treatments might worsen diseases involving metabolic insults. This is coincident with the results of Simon et al., who showed that prednisolone could worsen the kidney mitochondrial toxicity induced by cyclosporin A [28]. Also, it has been reported that methyl prednisolone inhibits state 3 respiratory rate in skeletal muscle cells in culture [29], although the inhibitory effects required relatively high concentrations of the glucocorticoid (0.1 mM). Among the various mitochondrial components, glucocorticoids have been reported to selectively inhibit cytochrome c oxidase (complex IV) and NO has been reported to reversibly inhibit cytochrome c oxidase by competing with oxygen [30–32]. The simultaneous inhibition of cytochrome c oxidase by NO (peroxynitrite) and a glucocorticoid may accelerate the decrease of cellular ATP level below threshold levels, which may underlie the observed glucocorticoid-mediated potentiation of astrocytic death in this study. It has been reported that NO / ONOO 2 can inhibit components of the electron transport chain [33], and damage to the mitochondrial electron transport system has been implicated in several neurodegenerative disorders [18,34]. It has been reported that NO reacts directly with ubiquinol [35] and ONOO2 reacts directly with complex II [36]. These results suggest that NO / ONOO2 can inhibit complex II–III activity. In addition, NO / ONOO2 can inhibit complex IV activity both reversibly and irreversibly [37]. Furthermore, we reported that immunostimulation of rat primary astrocytes with LPS plus IFN-g decreased intracellular ATP levels, which is inversely proportional to the increase in NO production from the cell [21]. These results suggest that glucocorticoids may synergistically deplete intracellular ATP levels in immunostimulated rat primary astrocytes, accelerating energy crisis-induced cell death.

168

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

Fig. 5. Effects of dexamethasone on SIN-1 / glucose deprivation-induced deterioration of mitochondrial transmembrane potential (MTP) in rat primary astrocytes. Primary rat cortical astrocytes were loaded with JC-1 as described in Methods and incubated in glucose-free DMEM containing 20 mM SIN-1. To the incubation media, 100 nM dexamethasone was added with or without 1 mM RU-486. After 2 h, JC-1 fluorescence was monitored using a fluorescence confocal microscope; (a) 5 mM glucose (b) SIN-1 / 5 mM glucose (c) glucose-deprivation (d) SIN-1 / glucose-deprivation (e) SIN-1 / glucosedeprivation1dexamethasone (f) SIN-1 / glucose-deprivation1dexamethasone1RU486. Note that SIN-1 / glucose-deprivation caused fluorescence change to yellowish-green color indicating loss of MTP. The loss of MTP is more pronounced with the addition of dexamethasone (e), which was reversed by cotreatment of RU-486 (f). The fluorescence intensity was quantified and shown in B). In (B), each bar indicates the mean6S.E.M. (n54). *, Significant difference from 5 mM glucose supplemented control (P,0.01); †, significant difference from SIN-1 / glucose deprivation (P,0.01); ¶, significant difference from SIN-1 / glucose deprivation1dexamethasone (P,0.01).

Many researchers have reported the damaging properties of glucocorticoids during ischemic injury. It has been reported that glucocorticoids may exacerbate hepatocyte injury induced by hypoxia when cytochrome c oxidase activity is inhibited [38]. Endogenous corticosterone contributed to the basal level of brain injury resulting from cerebral ischemia and excitotoxic seizure activity and metapyrone, an inhibitor of glucocorticoid production, reduced the brain injury induced by focal and global ischemia and seizure [39]. In a model of myocardial ischemia reperfusion, Scheuer and Miffine have shown that the corticosterone-treated rats had an infarct size significantly higher compared to the control [40]. They concluded that chronic elevations in plasma corticosterone concentrations could contribute to the increased risk of cardiovascular disease in clinical conditions associated with elevated glucocorticoid levels. Glucocorticoids have also been shown to exacerbate injury both in neuron and

astrocyte following hypoxia or cyanide treatment [25,41]. In these studies, it has been reported that 24 h pretreatment of glucocorticoid accelerated hypoxia- or cyanide-induced ATP loss without affecting normal cellular ATP level, which is similar to the results obtained from the present study. In hippocampal astrocytes, glucocorticoids have been reported to inhibit glucose and glutamate transport, which is related to the augmentation of hippocampal neuronal damage by glucocorticoids [42]. However, these authors reported that the action is specific only to hippocampal astrocytes, but not to cerebellar or cortical astrocytes. In addition, we omitted glucose during dexamethasone exposure. Therefore the effect of dexamethasone cannot be attributed to the changes in glucose transport. These results suggest that the inhibition of glucose transport is not the direct cause of the observed exacerbation of astrocyte death in this study.

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

169

Fig. 5. (continued)

Impairment of intracellular energy homeostasis may deteriorate mitochondrial transmembrane potentials, which eventually can lead to the opening of the mitochondrial permeability transition (MPT) pore. The opening of MPT pore has been implicated in both necrotic and apoptotic cell death [for a review, see 43]. In this study, glucosedeprivation in immunostimulated rat primary astrocytes synergistically deteriorated mitochondrial transmembrane potential as reported previously [16,17]. In this condition, dexamethasone further deteriorated the mitochondrial transmembrane potential, which was reversed by cotreatment with RU-486 (Fig. 5). These results suggest that synergistic ATP depletion by dexamethasone, in energetically challenged rat primary astrocytes, causes disruption of the mitochondrial transmembrane potential, which eventually leads to increased cell death. Glucocorticoids generally act as immunosuppressants and it has been reported that dexamethasone inhibit NO accumulation in LPS or cytokine-treated cells either by inhibiting iNOS induction or by limiting cofactor and / or substrate availability, which requires at least 24 h coincubation with LPS to exert their genomic effects [44–46]. In this situation, it would be expected that dexamethasone may decrease the cell death of immunostimulated rat primary astrocytes by inhibiting the NO production. In

fact, this was the case when we added dexamethasone during the 48-h immunostimulation period (data not shown). Longterm incubation of dexamethasone during the immunostimulation period (48 h) inhibited iNOS induction and hence the NO production from rat primary astrocyte (Fig. 2) preventing glucose deprivation induced cell death (data not shown). However, in this study, we investigated only the short-term effect (2–6 h) of dexamethasone by adding it only during the glucose-deprivation period. In separate experiments, we identified that at least a 12-h incubation with dexamethasone, during the immunostimulation period, was required to inhibit iNOS induction. The addition of dexamethasone only, during the glucosedeprivation period in immunostimulated rat primary astrocytes, had no effect on the activation process of astrocytes including NO production during that period (Fig. 2). In this study, we observed that a 4-h coincubation of dexamethasone was already sufficient to reveal significant toxic effects. The time interval seems to be rather insufficient for a glucocorticoid to exert genomic effects. In addition, synergistic disruption of MTP and depletion of ATP by dexamethasone were already observed at 2 and 4 h, respectively. Most importantly, pretreatment of actinomycin D (1 mg / ml) or cycloheximide (10 mg / ml) did not abrogate the cell death promoting effect of dexa-

170

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

methasone. These results suggest that the effect of dexamethasone is rather direct, but not mediated by their genomic effects, although the exact target molecules remain to be determined. Brooke et al. reported that glucocorticoid increases gp120-mediated hippocampal neuronal cell death via the mechanism involving cellular ATP depletion [47]. The results from the present study suggest that glucocorticoidinduced ATP depletion from astrocytes is responsible at least in part for the glucocorticoid mediated exacerbation of neuronal death by gp120 or other neurotoxicants including amyloid b peptide. Considering the inhibitory effects of NO / ONOO 2 on cellular energy metabolism, it would be also very interesting to investigate whether NO or peroxynitrite exacerbate gp120- or Ab-induced neuronal or astrocytic toxicity. In summary, glucocorticoids exacerbated peroxynitritedependent cell death of rat primary astrocytes in energetically-challenged situations. The physiological relevance of this finding should be investigated further in conditions such as ischemia and neurodegenerative diseases, where activation of glial cells was observed.

Acknowledgements This work was supported in part by a grant of the good health R&D project.

References [1] R.M. Sapolsky, Glucocorticoid toxicity in the hippocampus: reversal by supplementation with brain fuels, J. Neurosci. 6 (1986) 2240– 2244. [2] R.M. Sapolsky, W.A. Pulsinelli, Glucocorticoids potentiate ischemic injury to neurons: therapeutic implications, Science 229 (1985) 1397–1400. [3] T. Koide, T.W. Wieloch, B.K. Siesjo, Chronic dexamethasone pretreatment aggravates ischemic neuronal necrosis, J. Cereb. Blood Flow Metab. 6 (1986) 395–404. [4] R.M. Sapolsky, L.C. Krey, B.S. McEwen, Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging, J. Neurosci. 5 (1985) 1222–1227. [5] R.M. Sapolsky, D.R. Packan, W.W. Vale, Glucocorticoid toxicity in the hippocampus: in vitro demonstration, Brain Res. 453 (1988) 367–371. [6] J.K. Morse, J.N. Davis, Regulation of ischemic hippocampal damage in the gerbil: adrenalectomy alters the rate of CA1 cell disappearance, Exp. Neurol. 110 (1990) 86–92. [7] G.C. Tombaugh, R.M. Sapolsky, Mechanistic distinctions between excitotoxic and acidotic hippocampal damage in an in vitro model of ischemia, J. Cereb. Blood Flow Metab. 10 (1990) 527–535. [8] H.C. Horner, D.R. Packan, R.M. Sapolsky, Glucocorticoids inhibit glucose transport in cultured hippocampal neurons and glia, Neuroendocrinology 52 (1990) 57–64. [9] M.S. Lawrence, R.M. Sapolsky, Glucocorticoids accelerate ATP loss following metabolic insults in cultured hippocampal neurons, Brain Res. 646 (1994) 303–306.

[10] R.M. Sapolsky, Stress, glucocorticoids and damage to the nervous system: the current state of confusion, Stress 1 (1996) 1–19. [11] C. Behl, F. Lezoualc’h, T. Trapp, M. Widmann, T. Skutella, F. Holsboer, Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro, Endocrinology 138 (1997) 101–106. [12] L.J. McIntosh, K.E. Hong, R.M. Sapolsky, Glucocorticoids may alter antioxidant enzyme capacity in the brain: baseline studies, Brain Res. 791 (1998) 209–214. [13] L.J. McIntosh, K.M. Cortopassi, R.M. Sapolsky, Glucocorticoids may alter antioxidant enzyme capacity in the brain: kainic acid studies, Brain Res. 791 (1998) 215–222. [14] J.J. Choi, W.K. Kim, Potentiated glucose deprivation-induced death of astrocytes after induction of iNOS, J. Neurosci. Res. 54 (1998) 870–875. [15] I.Y. Choi, S.J. Lee, C. Ju, W. Nam, H.C. Kim, K.H. Ko, W.K. Kim, Protection by a manganese porphyrin of endogenous peroxynitriteinduced death of glial cells via inhibition of mitochondrial transmembrane potential decrease, Glia 31 (2000) 155–164. [16] I.Y. Choi, S.J. Lee, W. Nam, J.S. Park, K.H. Ko, H.C. Kim, C.Y. Shin, J.H. Chung, S.K. Noh, C.R. Choi, D.H. Shin, W.K. Kim, Augmented death in immunostimulated astrocytes deprived of glucose: inhibition by an iron porphyrin FeTMPyP, J. Neuroimmunol. 112 (2001) 55–62. [17] C. Ju, K.N. Yoon, Y.K. Oh, H.C. Kim, C.Y. Shin, J.R. Ryu, K.H. Ko, W.K. Kim, Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death, J. Neurochem. 74 (2000) 1989–1998. [18] S.J. Heales, J.P. Bolanos, V.C. Stewart, P.S. Brookes, J.M. Land, J.B. Clark, Nitric oxide, mitochondria and neurological disease, Biochim. Biophys. Acta 1410 (1999) 215–228. [19] P.S. Brookes, J.P. Bolanos, S.J. Heales, The assumption that nitric oxide inhibits mitochondrial ATP synthesis is correct, FEBS Lett. 446 (1999) 261–263. [20] T. Tatsumi, S. Matoba, A. Kawahara, N. Keira, J. Shiraishi, K. Akashi, M. Kobara, T. Tanaka, M. Katamura, C. Nakagawa, B. Ohta, T. Shirayama, K. Takeda, J. Asayama, H. Fliss, M. Nakagawa, Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes, J. Am. Coll. Cardiol. 35 (2000) 1338–1346. [21] C.Y. Shin, J.W. Choi, J.R. Ryu, J.H. Ryu, W.K. Kim, H.C. Kim, K.H. Ko, Immunostimulation of rat primary astrocytes decreases intracellular ATP level, Brain Res. 902 (2001) 198–204. [22] W.K. Kim, D.O. Seo, J.J. Choi, K.H. Ko, Immunostimulated glial cells potentiate glucose deprivation-induced death of cultured rat cerebellar granule cells, J. Neurotrauma 16 (1999) 415–424. [23] S.J. Green, S. Mellouk, S.L. Hoffman, M.S. Meltzer, C.A. Nacy, Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes, Immunol. Lett. 25 (1990) 15–19. [24] M. Reers, T.W. Smith, L.B. Chen, J-Aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential, Biochemistry 30 (1991) 4480–4486. [25] G.C. Tombaugh, R.M. Sapolsky, Corticosterone accelerates hypoxia- and cyanide-induced ATP loss in cultured hippocampal astrocytes, Brain Res. 588 (1992) 154–158. [26] C. Behl, T. Trapp, T. Skutella, F. Holsboer, Protection against oxidative stress-induced neuronal cell death — a novel role for RU486, Eur. J. Neurosci. 9 (1997) 912–920. [27] M.C. Bennett, G.W. Mlady, M. Fleshner, G.M. Rose, Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity, Proc. Natl. acad. Sci. USA 96 (1996) 1330–1334. [28] N. Simon, R. Zini, C. Morin, F. Bree, J.P. Tillement, Prednisolone and azathioprine worsen the cyclosporin A-induced oxidative phos-

C.Y. Shin et al. / Brain Research 923 (2001) 163 – 171

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

phorylation decrease of kidney mitochondria, Life Sci. 61 (1997) 659–666. M.E. Martens, P.L. Peterson, C.P. Lee, In vitro effects of glucocorticoid on mitochondrial energy metabolism, Biochim. Biophys. Acta 1058 (1991) 152–160. N. Simon, P. Jolliet, C. Morin, R. Zini, S. Urien, J.P. Tillement, Glucocorticoids decrease cytochrome c oxidase activity of isolated rat kidney mitochondria, FEBS Lett. 435 (1998) 25–28. G.C. Brown, Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase, FEBS Lett. 369 (1995) 136–139. V. Borutaite, G.C. Brown, Rapid reduction of nitric oxide by mitochondria, and reversible inhibition of mitochondrial respiration by nitric oxide, Biochem. J. 315 (1996) 295–299. J.P. Bolanos, S. Peuchen, S.J. Heales, J.M. Land, J.B. Clark, Nitric oxide-mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes, J. Neurochem. 63 (1994) 910–916. M.F. Beal, B.T. Hyman, W. Koroshetz, Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases?, Trends Neurosci. 16 (1993) 125–131. J.J. Poderoso, M.C. Carreras, F. Shopfer, C. Lisdero, N. Riobo, P. Evelson, A. Boveris, Ubiquinol-2 reacts with nitric oxide, Second International Conference on the Biochemistry and Molecular Biology of Nitric Oxide, Los Angeles, CA, USA, Abstract C24 (1996) 94. P.S. Brookes, J.M. Land, J.B. Clark, S.J. Heales, Peroxynitrite and brain mitochondria: evidence for increased proton leak, J. Neurochem. 70 (1998) 2195–2202. G.C. Brown, J.P. Bolanos, S.J. Heales, J.B. Clark, Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration, Neurosci. Lett. 193 (1995) 201–204. N. Chandel, G.R.S. Budinger, R.A. Kemp, P.T. Schmaker, Inhibition of cytochrome-c oxidase activity during prolonged hypoxia, Am. J. Physiol. (Lung Cell Mol. Physiol.) 268 (1995) L918–L925. V. Smith-Swintosky, C. Pettigrew, R.M. Sapolsky, C. Phares, S.D.

[40]

[41]

[42]

[43] [44] [45]

[46]

[47]

171

Craddock, S.M. Brooke, M.P. Mattson, Metyrapone, an inhibitor of glucocorticoid production, reduces brain injury induced by focal and global ischemia and seizures, J. Cereb. Blood Flow Metab. 16 (1996) 585–598. D.A. Scheuer, S.W. Mifflin, Chronic corticosterone treatment increases myocardial infarct size in rats with ischemia–reperfusion injury, Am. J. Physiol. 272 (1997) R2017–R2024. G.C. Tombaugh, S.H. Yang, R.A. Swanson, R.M. Sapolsky, Glucocorticoids exacerbate hypoxic and hypoglycemic hippocampal injury in vitro: biochemical correlates and a role for astrocytes, J. Neurochem. 59 (1992) 137–146. C.E. Virgin Jr., T.P. Ha, D.R. Packan, G.C. Tombaugh, S.H. Yang, H.C. Horner, R.M. Sapolsky, Glucocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity, J. Neurochem. 57 (1991) 1422– 1428. M. Crompton, Mitochondrial intermembrane junctional complexes and their role in cell death, J. Physiol. 529 (2000) 11–21. P.D. Drew, J.A. Chavis, Inhibition of microglial cell activation by cortisol, Brain Res. Bull. 52 (2000) 391–396. J.L. Balligand, D. Ungureanu-Longrois, W.W. Simmons, D. Pimental, T.A. Malinski, M. Kapturczak, Z. Taha, C.J. Lowenstein, A.J. Davidoff, R.A. Kelly, T.W. Smith, T. Michel, Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro, J. Biol. Chem. 269 (1994) 27580–27588. W.W. Simmons, D. Ungureanu-Longrois, G.K. Smith, T.W. Smith, R.A. Kelly, Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and L-arginine transport, J. Biol. Chem. 271 (1996) 23928–23937. S.M. Brooke, S.A. Howard, R.M. Sapolsky, Energy dependency of glucocorticoid exacerbation of gp120 neurotoxicity, J. Neurochem. 71 (1998) 1187–1193.