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Keywords:

  • Astrocytes;
  • Brain inflammation;
  • Nitric oxide;
  • γ-Glutamyl transpeptidase;
  • Glutathione;
  • Vitamin D

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

Abstract : Astrocytes play a pivotal role in CNS detoxification pathways, where glutathione (GSH) is involved in the elimination of oxygen and nitrogen reactive species such as nitric oxide. We have previously demonstrated that the specific activity of γ-glutamyl transpeptidase (γ-GT), an enzyme of central significance in GSH metabolism, is regulated in vivo in astrocytes by 1,25-dihydroxyvitamin D3 (1,25-D3). The aim of the present work was to investigate, in primary cultures of newborn rat astrocytes, the effects of this hormone on γ-GT synthesis and on GSH and nitrite levels after lipopolysaccharide (LPS) treatment. This study demonstrates that both γ-GT gene expression and specific activity, induced by LPS, are potentiated by 1,25-D3. In contrast, 1,25-D3 does not regulate the expression of other enzymes involved in astrocyte detoxification processes, such as superoxide dismutase or GSH peroxidase. In parallel, 1,25-D3 enhanced intracellular GSH pools and significantly reduced nitrite production induced by LPS. Taken together, these results suggest that γ-GT, GSH, and 1,25-D3 play a fundamental role in astrocyte detoxification pathways.

Reactive oxygen and nitrogen intermediates, such as hydrogen peroxide or nitric oxide (NO), are metabolic products that are synthesized in increased amounts during brain inflammation. From these precursor species, more reactive intermediates such as peroxynitrite (ONOO-) and the free radicals hydroxyl (OH) or nitrogen dioxide (NO2) are produced. In the CNS, they may cause the death of neurons or oligodendrocytes by inducing lipid peroxidation, DNA breaks, and enzyme inactivation (Halliwell, 1992 ; Merrill et al., 1993 ; Mitrovic et al., 1995 ; Dawson and Dawson, 1996). The CNS responds to the injury by a broad spectrum of lesion-induced events associated with modification of CNS cell behavior to maintain or restore its integrity. Astrocytes play a major role in antioxidative and detoxification processes in the brain. These cells can respond to a sublethal oxidative stress by up-regulating their intracellular glutathione (GSH) pool. Such an increased GSH concentration is likely to be protective against further oxidative challenge (Pellmar et al., 1992 ; Damier et al., 1993 ; Makar et al., 1994 ; Bains and Shaw, 1997 ; Peuchen et al., 1997 ; Wilson, 1997).

γ-Glutamyl transpeptidase (γ-GT), a membrane-bound enzyme, provides the only enzyme activity able to achieve the hydrolysis of extracellular GSH. This favors the cellular reutilization of its constituent amino acids and allows the generation of intracellular GSH. γ-GT synthesis or activity is regulated by a large variety of effectors, including ethanol, growth factors, or hormones such as glucocorticoids, retinoic acids, or thyroid hormones (for review, see Laperche et al., 1990). We have also reported that the specific activity of γ-GT can be enhanced in the rat CNS by another hormone, 1,25-dihydroxyvitamin D3 (1,25-D3), the biologically active form of vitamin D (Garcion et al., 1996).

There are presently several lines of evidence indicating that 1,25-D3 is active in the CNS. Indeed, the vitamin D receptor has been identified in various populations of neurons, as well as in nonneuronal cells (Stumpf and O'Brien, 1987 ; Bidmon et al., 1991). The hormone is available in the brain, because it can both cross the blood-brain barrier (Gascon-Barré and Huet, 1983 ; Pardridge et al., 1985) and be synthesized by activated microglial cells (Neveu et al., 1994a). Besides its effect on γ-GT, 1,25-D3 has been reported to elicit various actions in the CNS, such as the up-regulation of choline acetyltransferase, nerve growth factor, and calbindin 28K (Sonnenberg et al., 1986 ; Neveu et al., 1994b ; Saporito et al., 1994 ; Alexianu et al., 1998). Moreover, 1,25-D3 also exerts immunosuppressive effects in the CNS, for instance, by modulating the expression of major histocompatibility complex class II molecules, CD4, and inducible or type II NO synthase (NOS II) during experimental allergic encephalomyelitis (EAE) (Nataf et al., 1996 ; Garcion et al., 1997) and by down-regulating the expression of NOS II in a brain inflammatory reaction induced by a local injection of bacterial lipopolysaccharide (LPS) (Garcion et al., 1998). These data suggest that 1,25-D3 participates in the antioxidant machinery that controls brain homeostasis. Astrocytes are one of the targets of 1,25-D3, because they express the vitamin D receptor gene (Neveu et al., 1994b).

Therefore, the aim of the present work was to explore the potential ability of 1,25-D3 to modulate CNS detoxification pathways, in particular under inflammatory conditions. For this purpose, we have analyzed the effects of 1,25-D3 on γ-GT expression, GSH levels, and nitrite (NO2-) accumulation in rat primary astrocytes, after an inflammatory stress induced by LPS treatment.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

Materials

Culture media and fetal calf serum were purchased from GIBCO (Cergy-Pontoise, France). Cell culture dishes were from Nunc (Ballerup, Denmark). 1,25-D3 was obtained from Leo Pharmaceutical Products (Ballerup). Radioactive [α-33P]dCTP and the random priming labeling kit were purchased from Amersham (Little Chalfont, U.K.). Anti-glial fibrillary acidic protein (anti-GFAP) antibodies were purchased from Dakopatts (Glostrup, Denmark). MRC-OX42 directed against rat complement receptor 3 was from Serotec (Kidlington, U.K.). Anti-carbonic anhydrase type II was a kind gift of Dr. M. S. Ghandour (Centre de Neurochimie, CNRS, Strasbourg, France). LPS (Escherichia coli serotype O55 : B5) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Cell culture

The procedure used to obtain rat primary astrocyte cultures has been described previously (Neveu et al., 1992). Cells were plated at a density of 3 × 104 cells/cm2 (on 57-cm2 dishes for γ-GT specific activity assay and northern blot analysis ; on 9-cm2 dishes for all the other experiments) and grown for 3 weeks in a 1:1 (vol/vol) mixture of Dulbecco's modified Eagle's medium/Ham's F12 medium supplemented with 10% (vol/vol) fetal calf serum, 33 mmol/L glucose, and 5 mmol/L HEPES buffer. After the 3-week growth period, the confluent cells were shown to consist of >95% astrocytes by immunostaining with anti-GFAP antibodies. Cultures contained ~2-3% of carbonic anhydrase type II-positive cells (oligodendrocytes) and <1% of complement receptor 3-positive cells (microglial cells).

Before experiments, the medium was replaced by a serum-free medium (M2) consisting of a 1:1 (vol/vol) mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with 33 mmol/L glucose, 5 mmol/L HEPES buffer (pH 7.2), 25 mg/L insulin, 50 mg/L human transferrin, 20 nmol/L progesterone, 60 μmol/L putrescine, and 30 nmol/L sodium selenite. The cells were maintained in serum-free conditions for 3 days, with the medium being changed 24 h before addition of the required concentrations (see below) of 1,25-D3, LPS, or solvent (absolute ethanol for 1,25-D3 and water for LPS) in the case of controls. LPS was used at 500 ng/ml. In dose-response experiments performed with 10-10-10-6 mol/L 1,25-D3, an effect on astrocytes was only obtained between 10-8 and 10-6 mol/L.

Northern blot analysis

The levels of γ-GT, NOS II, Cu/Zn superoxide dismutase (SOD Cu/Zn), and GSH peroxidase mRNAs were estimated using northern blot analysis. RNA extraction was performed using the LiCl/urea method (Auffray and Rougeon, 1980). Fifty micrograms of glyoxal-treated total RNAs was separated on 1.4% (wt/vol) agarose gels and blotted onto a nylon membrane, according to well-described procedures (Maniatis et al., 1982). After UV irradiation or baking to cross-link the RNAs to the membrane, the blots were hybridized sequentially with rat γ-GT cDNA (a kind gift from Dr. Y. Laperche, INSERM, Créteil, France), rat astrocyte NOS II cDNA (a kind gift from Dr. D. Feinstein, New York Hospital-Cornell Medical Center, New York, NY, U.S.A.), rat SOD Cu/Zn cDNA, and rat GSH peroxidase cDNA (kind gifts from Dr. Y. Ho, Duke University Medical Center, Durham, NC, U.S.A.). Random priming was used to label the probes with [α-32P]dCTP (Amersham, U.K.). The blots were prehybridized for 4-6 h and hybridized for 15 h at 42°C in 50% (vol/vol) formamide, 5× standard saline citrate (SSC ; 1× SSC corresponds to 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7), 0.5% (wt/vol) sodium dodecyl sulfate (SDS), 5 mg/L heat-denatured herring sperm DNA, and 5× Denhardt's reagent (1 g/L Ficoll, 1 g/L polyvinylpyrrolidone, and 1 g/L bovine serum albumin fraction V). Blots were further washed four times for 5 min each at room temperature in 2× SSC containing 0.5% SDS and then four times for 20 min each at 62°C in 0.1× SSC containing 0.5% SDS. They were then autoradiographed at -80°C. The mRNA loading was controlled by hybridization with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (Fort et al., 1985). Autoradiograms were analyzed after various exposure times by densitometric scanning. All experiments were performed at least in triplicate using independent cultures.

γ-GT activity assay

Cultured cells were dissociated with phosphate-buffered saline containing 0.1% (wt/vol) trypsin and 1 mmol/L EDTA and then resuspended in the M2 culture medium containing 10% (vol/vol) fetal calf serum. After centrifugation at 1,800 g for 5 min, the pellet was dissolved in 400 μl of phosphate-buffered saline containing 1% (vol/vol) Triton X-100 and protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Nα-p-tosyl-l-lysine chloromethyl ketone, and 25 mg/L aprotinin). The samples were then stored at -80°C until the enzyme assays.

γ-GT activity was assayed using the kit from Boehringer Mannheim (Mannheim, Germany). It consists of l-γ-glutamyl-3-carboxy-4-nitroanilide as a substrate of γ-GT hydrolytic activity and of glycylglycine as a glutamate acceptor for the transpeptidation reaction. The production of 5-amino-2-nitro-benzoate was measured by spectrophotometry at 405 nm. One milliunit of γ-GT represents the formation of 1 nmol of this product/min at 30°C. For γ-GT specific activity calculations, protein concentration was determined for each sample by the Bradford method using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and bovine serum albumin as standard.

NO2-release

The NO2- concentration was measured in cell supernatant according to a previously described microassay (Ding et al., 1988). In brief, an equal volume of Griess reagent [1% (wt/vol) sulfanylamide, 0.1% (wt/vol) N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% (vol/vol) H3PO4] was incubated with the cell supernatants for 10 min at room temperature ; absorbance was measured at 550 nm in a micro-ELISA reader (Titertek Multiskan ; Labsystem, Cergy-Pontoise, France). The NO2- concentration was determined using NaNO2 as a standard and the serum-free medium used for the culture as a blank. To express NO2- release in nanomoles per milligram of protein, protein concentration was determined in parallel by the Bradford method (Bio-Rad protein assay) on the cell pellet resuspended in 150 mmol/L NaCl, 1% (vol/vol) Nonidet P-40, and 50 mmol/L Tris buffer, pH 8.

GSH assay

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

Cultured cells were collected with 1.5% (vol/vol) perchloric acid and then sonicated for 15 s at maximal frequency. After centrifugation at 14,000 g for 3 min, the protein pellet was removed, and the total glutathione (reduced and oxidized) content was determined by the method of Tietze (1969) with minor modifications. The incubation mixture consisted of 25 μl of protein-free supernatant of the cell lysate, 100 μl of 100 mmol/L sodium phosphate buffer (pH 7.4) containing 1 mmol/L Tris-EDTA (pH 7.4), 35 μl of 1 mmol/L NADPH containing 2 U/ml type 3 GSH reductase, and 15 μl of 0.1 mmol/L 5,5′-dithio-bis(2-nitrobenzoic acid). All reagents were prepared in 100 mmol/L sodium phosphate buffer (pH 7.4) containing 1 mmol/L EDTA. The production of 2-nitro-5-thiobenzoic acid was monitored over time by measuring the absorbance of the incubation mixture at 412 nm on a COBAS-MIRA automated recording spectrophotometer (Roche, Neuilly-sur-Seine, France). For calculation of specific GSH levels, protein concentration was determined on the protein pellet resuspended in 0.5 mol/L NaOH by the Bradford method (Bio-Rad protein assay).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

γ-GT synthesis and concomitant events

To investigate the potential ability of 1,25-D3 to modulate astrocyte behavior under inflammatory conditions, 3-week-old confluent cultures of newborn rat astrocytes were exposed to 1,25-D3 (10-10-10-6 mol/L) or to 500 ng/ml LPS, with increasing concentrations of 1,25-D3. After 6 h of treatment, the cell content of γ-GT mRNAs, which was barely detectable, was not changed (Fig. 1). In contrast, after 24 h, the treatment with 500 ng/ml LPS induced a marked accumulation of at least two distinct γ-GT mRNAs, as demonstrated by the two observed bands on the autoradiography (10-fold over control ; Fig. 2), and 1,25-D3 potentiated this effect in a dose-dependent manner (43-, 32-, and 20-fold increases at 10-6, 10-7, and 10-8 mol/L, respectively ; Fig. 2).

image

Figure 1. Northern blot analysis of γ-GT, NOS II, SOD, and GSH peroxidase transcripts in rat primary astrocytes. Total RNA was extracted from untreated cells (C) or cells treated for 6 h with 10-6-10-10 mol/L 1,25-D3 (-6 to -10, respectively) in combination or not with LPS. Identical amounts of total RNA were fractionated and blotted. Blots were serially hybridized with the different probes. Loading was checked with a GAPDH probe.

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image

Figure 2. Northern blot analysis of γ-GT, NOS II, SOD, and GSH peroxidase transcripts in rat primary astrocytes. Total RNA was extracted from untreated cells (C) or cells treated for 24 h with 10-6-10-10 mol/L 1,25-D3 (-6 to -10, respectively) in combination or not with LPS. Identical amounts of total RNA were fractionated and blotted. Blots were serially hybridized with the different probes. Loading was checked with a GAPDH probe. The time exposure for autoradiography was the same for each probe matched between the two northern blot analyses at 6 and 24 h.

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We then measured the specific activity of γ-GT in our primary astrocytes. After 24 h in culture we found (Fig. 3) both an increase by the LPS treatment (37%) and a potentiating effect of 1,25-D3 (95%), suggesting an effect of both compounds on the enzyme synthesis.

image

Figure 3. γ-GT specific activity in rat primary astrocyte cultures after a 24-h treatment by vehicle (control), 10-8 mol/L 1,25-D3, and 500 ng/ml LPS, alone or in combination (LPS + 1,25-D3). Data are mean ± SE (bars) values and were obtained from one representative of three experiments done using three independent astrocyte primary cultures. Significant differences between control, 1,25-D3, LPS, and LPS + 1,25-D3 were confirmed with a one-way ANOVA. Significance was assessed by a paired Student's t test, and the criterion for significance was defined as a p value : oop < 0.01, ooop < 0.001 for comparisons with control ; ***p < 0.001 for comparisons between LPS and LPS + 1,25-D3.

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The up-regulation of γ-GT synthesis by LPS was preceded by an accumulation of mRNA encoding the inducible form of NOS (NOS II), as has previously been shown in this experimental model (Galea et al., 1994). In contrast to γ-GT transcripts, NOS II mRNA levels were maximal 6 h after the onset of the treatment and were more reduced after 24 h (Figs. 1 and 2). This induction was unaffected by 1,25-D3 treatment (Figs. 1 and 2). Moreover, the survey of the transcripts encoding two other important enzymes in astrocyte detoxification mechanisms, SOD Cu/Zn and GSH peroxidase, did not reveal any regulatory effect of either the LPS or 1,25-D3 treatment (Figs. 1 and 2).

NO2- accumulation

To explore the activity of NOS II, we then measured the amounts of NO2- in the culture medium, because it constitutes a stable derivative of NO. Indeed, NO and NO2- are both derived from l-arginine via the action of NOS II, the major form of NOS in astrocytes, and the amounts of NO2- are dependent on both NO production and NO consumption.

We found that NO2- production was only weakly detectable in unstimulated control cells, as well as after 1,25-D3 treatment alone (Fig. 4). In contrast, NO2- concentration dramatically increased after 24 h of culture in the presence of LPS and reached a plateau from 48 to 96 h after the onset of LPS treatment (Fig. 4). At this latter time, 1,25-D3 significantly reduced the accumulation of NO2- both in control cells and in LPS-treated cells, thus lowering to some extent the dramatic action of the bacterial endotoxin (Fig. 4).

image

Figure 4. NO2- concentration in rat primary astrocyte cultures, 24, 48, and 96 h after treatment by vehicle (control), 10-7 mol/L 1,25-D3, and 500 ng/ml LPS, alone or in combination (LPS + 1,25-D3). Data are mean ± SE (bars) values and were obtained from one representative of four experiments done using four independent astrocyte primary cultures. Significant differences between control, 1,25-D3 LPS, and LPS + 1,25-D3 were confirmed with a one-way ANOVA. Significance was assessed by a paired Student's t test, and the criterion for significance was defined as a p value : oop < 0.01 for comparisons with control ; *p < 0.02 for comparisons between LPS alone and LPS + 1,25-D3.

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GSH levels

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

To establish a physiological relevance between the observed effects of 1,25-D3 on γ-GT gene expression and specific activity and on NO2- amounts, we then measured the intracellular levels of GSH.

The basal level of intracellular GSH in astrocyte cultures was ~30 nmol/mg of protein (Fig. 5), in agreement with previous data (Martin and White, 1991 ; Devesa et al., 1993 ; O'Connor et al., 1995 ; Dringen et al., 1998).

image

Figure 5. GSH levels in rat astrocyte primary cultures, 24, 48, and 96 h after treatment by vehicle (control), 10-7 mol/L 1,25-D3, and 500 ng/ml LPS, alone or in combination (LPS + 1,25-D3). Data are mean ± SE (bars) values and were obtained from one representative of four experiments done using four independent astrocyte primary cultures. Significant differences between control, 1,25-D3, LPS, and LPS + 1,25-D3 were confirmed with a one-way ANOVA. Significance was assessed by a paired Student's t test, and the criterion for significance was defined as a p value : °p < 0.05, °°p < 0.01, °°°p < 0.001 for comparisons with control.

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Treatment with 1,25-D3 alone significantly increased GSH concentration up to ~40 nmol/mg of protein at 24 and 48 h (Fig. 5). In contrast, treatment with LPS alone reduced the intracellular GSH amounts after 48 and 96 h, at which time its action was highly significant because the GSH level was <10 nmol/mg of protein (Fig. 5). However, 1,25-D3 had no effect on the reduction of intracellular GSH levels induced by LPS, even though, in the presence of LPS, the up-regulation of GSH levels was observed after 24 h of treatment by 1,25-D3 (Fig. 5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

In this study we have investigated the potential ability of 1,25-D3 to modulate astrocyte detoxification pathways under inflammatory conditions. We found that LPS acts as a potent inducer of the γ-GT gene in cultures of newborn rat astrocytes. The endotoxin treatment caused a marked increase in levels of γ-GT mRNAs expressed in primary astrocytes and increased γ-GT specific activity. 1,25-D3 alone had no effect on the expression of the γ-GT gene, but it potentiated the action of LPS and further enhanced the synthesis of this enzyme. This response indicates that the LPS treatment renders the γ-GT gene permissive to a regulatory action of 1,25-D3. It is noteworthy that in a similar experimental system, 1,25-D3 was shown to lower accumulation of macrophage-colony stimulating factor and tumor necrosis factor-α transcripts in LPS-treated astrocytes, while having no significant effect per se (Furman et al., 1996). Taken together, these data indicate that 1,25-D3 acts as an effector controlling the expression of various genes, when astrocytes are placed under proinflammatory conditions. This implies that 1,25-D3 has a dual action in these cells. First, the hormone alone can influence the expression of certain genes in unstimulated astrocytes, such as those encoding its own receptor, nerve growth factor or neurotrophin-3 neurotrophic factor, or the catabolizing enzyme 1,25-D3-24-hydroxylase (Naveilhan et al., 1993 ; Neveu et al., 1994b,c). Second, 1,25-D3 can modulate the expression of genes such as γ-GT, whose expression is triggered by LPS.

The absence of effect of 1,25-D3 on unstimulated astrocytes seems to be in contradiction with our previous data reporting that treatment with 1,25-D3 increased γ-GT activity in astrocyte cultures (Garcion et al., 1996). An explanation of this apparent discrepancy is that in the former study the cultures were not plated at the same cell density (Dehouck et al., 1990). At low cell density, rat microglial cells develop more easily (E.G. et al., unpublished data) and could constitute a source of proinflammatory cytokines able to activate the astrocytes, making them permissive to the action of 1,25-D3. Moreover, the basal specific activity of γ-GT determined for the astrocyte cultures is severalfold higher than that reported by previous studies (Shine et al., 1981 ; Makar et al., 1994). This disparity could be explained by the heterogeneity of astrocyte cells that develop from the precursors originally plated and therefore by the specific procedure and timing of the culture conditions (Morgenstern et al., 1992 ; Devesa et al., 1993 ; McKhann et al., 1997).

Through its action on γ-GT, 1,25-D3 participates in the control of the antioxidant protective mechanisms of astrocytes. This enzyme is the initiator of the cycle of GSH and is thus pivotal in regulating its intracellular supply (Ahmad et al., 1987 ; Meister, 1988 ; Prezioso et al., 1994) (Fig. 6), even if recent studies have demonstrated the existence of specific transport systems for the uptake of extracellular GSH in brain (Kannan et al., 1996 ; Favilli et al., 1997). GSH displays a wide range of protective effects on CNS cells, e.g., astrocytes, motor neurons, and oligodendrocytes (Makar et al., 1994 ; Back et al., 1998 ; Lucas et al., 1998). It is the substrate of GSH peroxidase, a key enzyme of cellular detoxification (Perry and Wee Yong, 1986 ; Jain et al., 1991 ; Damier et al., 1993) (Fig. 6, pathway 1), and can also mediate by itself some vital chemical reactions leading to the elimination of reactive oxygen or nitrogen compounds such as NO (Hogg et al., 1996) (Fig. 6, pathway 2) or electrophic xenobiotics (Huang and Philbert, 1995 ; Cooper and Kristal, 1997). However, the wider spectrum of γ-GT functions in the brain remains to be elucidated (Wolff et al., 1998), and some alternative functions need to be considered, such as the involvement as an enzymatic barrier for vasoactive leukotrienes in brain (Black et al., 1994) or in metabolite trafficking of substrate for GSH synthesis between astrocytes and neurons (Dringen et al., 1999).

image

Figure 6. Astrocyte oxygen and nitrogen detoxification pathways using GSH as a substrate. Intracellular GSH could be provided from extracellular GSH by serial enzyme steps from which γ-GT is the first one. GSH can be used by GSH peroxidase (pathway 1) to convert hydrogen peroxide (H2O2, produced from the superoxide anion O2·-) into H2O, which prevents the formation of the hydroxyl radical OH·. Moreover, GSH can react with NO (pathway 2), thus generating S-nitrosoglutathione (GSNOH), then nitrous oxide (N2O) and H2O ; this metabolic pathway prevents the formation of the peroxynitrite anion ONOO- and of the hydroxyl OH· and nitrogen dioxide NO2· radicals. NO2- and NO3- (nitrate) are stable NO derivatives produced from NO2·, NO, and H2O. The most toxic reactive species are underlined. The positive [+] or negative [-] effects of 1,25-D3 on the production on the enzymes and compounds studied are noted under their names. ext.cell., extracellular ; int.cell., intracellular.

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An intriguing observation resulting from the present study is that, although 1,25-D3 per se had no effect on the specific activity of γ-GT, SOD, or GSH peroxidase transcripts, the hormone enhanced by ~25% the pool of GSH, as compared with untreated cells. One interpretation of this phenomenon is that 1,25-D3 could modulate GSH intracellular levels by several different mechanisms, either by acting on GSH constitutive dipeptides or amino acid transport systems (Virgin et al., 1991 ; Schmidlin and Wiesinger, 1995 ; Dringen et al., 1997, 1998) or by stimulating some intracellular steps of GSH neosynthesis (Okonkwo et al., 1974). This might be by acting on astrocyte γ-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of GSH, as has been demonstrated in hepatocytes for other hormones (Cai et al., 1997 ; Kang et al., 1997). A last hypothesis is that 1,25-D3 treatment could decrease the production of reactive oxygen species, which are generated by the cell metabolism and which are scavenged by GSH, which could result in an increase in the GSH intracellular concentration.

However, in the presence of LPS, 1,25-D3 seems to have no effect on GSH levels. This observation should be considered with the fact that activation of astrocytes by LPS leads to the production of large amounts of NO2-, which can be correlated with the strong induction of NOS II mRNA. However, 1,25-D3 treatment decreases the concentration of NO2- generated after LPS stimulation, whereas this hormone had no effect, in the same conditions, on the expression of NOS II mRNA. An attractive hypothesis is that, whereas the production of NO (via NOS II) is the same, the NO generated is trapped by GSH, which is produced in larger amounts in the presence of 1,25-D3. The subsequent formation of S-nitrosoglutathione, then nitrous oxide and water, occurs instead of the classical metabolic pathway leading to NO2- generation, which lowers the overall amount of NO2- in the extracellular medium (Do et al., 1996 ; Hogg et al., 1996) (Fig. 6).

It is surprising that our data show that 1,25-D3 had no effect on the accumulation of NOS II mRNA, which is rapidly induced by a LPS treatment. This result is puzzling, because we observed in vivo that 1,25-D3 down-regulated the level of NOS II mRNA and immunoreactivity in the rat CNS, both during EAE (Garcion et al., 1997) and after an inflammatory reaction induced by LPS injection into rat hippocampus (Garcion et al., 1998). However, expression of the NOS II gene in astrocytes appears to be very different in vitro and in vivo. Indeed, our present in vitro data, which show that the maximal accumulation of NOS II transcript occurs 6 h after cell stimulation, support the original observation of Galea et al. (1994), who reported that LPS is a rapid inducer of the NOS II gene, but contrast with in vivo observations demonstrating that NOS II expression in astrocytes is a late event in both EAE and focal brain inflammation (Matsumoto et al., 1992 ; Garcion et al., 1997, 1998). Therefore, the mechanisms of NOS II gene regulation by 1,25-D3 are probably different in sustained events such as those occurring during EAE when compared with the very rapid and controlled activation induced by LPS in rat astrocyte primary cultures. Indeed, the down-regulation of NOS II expression by 1,25-D3, observed during EAE, may be accounted for by indirect phenomena, which cannot take place in pure astrocyte cultures ; for instance, the balance between pro- and antiinflammatory cytokines may be influenced by 1,25-D3. In support of this hypothesis, it has been shown that this hormone is able to enhance transforming growth factor-β production both in human breast carcinoma cells (Mercier et al., 1996) and during mouse EAE (Cantorna et al., 1998).

Taken together, the data resulting from this study demonstrate that the treatment of astrocytes by 1,25-D3, a hormone active in the CNS and that can be produced in situ under inflammatory conditions by microglial cells, does the following : (a) potentiates the synthesis of γ-GT induced by LPS ; (b) increases intracellular GSH pools ; and (c) reduces the production of reactive nitrogen species triggered by proinflammatory stimuli. These points therefore suggest that γ-GT, GSH, and 1,25-D3 together constitute a regulatory system involved in astrocyte detoxification pathways. As such, they may influence the progression of CNS diseases associated with the production of reactive species, for example, in multiple sclerosis. Another example is Parkinson's disease, in which the GSH pool drops only in the substantia nigra, whereas γ-GT specific activity, unlike that of other enzymes involved in GSH metabolism, is increased in the same structure (Sian et al., 1994).

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSH assay
  5. RESULTS
  6. GSH levels
  7. DISCUSSION
  8. Acknowledgements

We thank Dr. R. Milner from the Wellcome/CRC Institute of Cambridge (U.K.) for critical reading of the manuscript. We are also grateful to Dr. Y. Laperche from the Unité INSERM 99, Créteil (France), Dr. X. T. Do from the Laboratory of Biochemistry of the Faculty of Pharmacy of Angers (France), and Prof. P. Allain from the Laboratory of Toxicology-Pharmacology of the Centre Hospitalier Universitaire of Angers (France). Funds were provided by the Institut National de la Santé et de la Recherche Médicale (INSERM). E.G. was a postdoctoral fellow supported by the Association pour la Recherche sur la Sclérose en Plaques (ARSEP).

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