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Address correspondence and reprint requests to George I. Henderson, PhD, Department of Medicine, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA. E-mail: email@example.com
Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron γ-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of γ-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrosoyte up to 517% (p < 0.05). Ethanol increased both γ-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the γ-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.
Glutathione (l- γ-glutamyl-l-cysteinylglycine) (GSH) is an intracellular thiol that plays a key role in scavenging reactive oxygen species (ROS) and mitigating oxidative stress. GSH serves multiple functions critical to cell survival, including detoxifying electrophiles, maintaining the essential thiol status of proteins and providing a reservoir for cysteine (Meister and Anderson 1983; DeLeve and Kaplowitz 1990; Suthanthiran et al. 1990).
Cortical astrocytes possess an active, highly regulated GSH homeostasis machinery that typically generates cell GSH content far in excess of that in neurons (Cooper 1997; Schulz et al. 2000). Additionally, astrocytes are in high abundance (Bignami 1991), they establish numerous small contacts with neurons (Rohlmann and Wolff 1996) and they play an important role in maintaining neuron GSH. The initial step in maintenance of neuron GSH homeostasis is efflux of GSH by the astrocyte (Wang and Cynader 2000), with the GSH subsequently hydrolyzed to the CysGly dipeptide by the astrocyte membrane-bound enzyme, γ-glutamyl transpeptidase (γGT) (Dringen et al. 1997). CysGly readily diffuses to adjacent neurons where it is cleaved to its two constituent amino acids at the neuronal surface by the dipeptidase, aminopeptidase N (APN) (Dringen et al. 2000; Dringen et al. 2001). This generates cysteine, which is transported into the neuron by the sodium-dependent alanine-serine-cysteine (ASC) system where its availability is an important determinant of GSH synthesis (Kranich et al. 1996; Wang and Cynader 2000).
A practical consequence of astrocyte-mediated control of neuron GSH homeostasis is protection of the neuron from oxidative damage by reactive oxygen and nitrogen species (Tanaka et al. 1999; Gegg et al. 2003). Reports from numerous laboratories have illustrated that ethanol elicits apoptotic death of neurons in the developing brain (Ramachandran et al. 2001; Olney et al. 2002) and in neuron cultures (de la Monte and Wands 2001; Ramachandran et al. 2003), and there is compelling evidence that ethanol-induced oxidative stress may be an important player in this process (Heaton et al. 2002; Ramachandran et al. 2003). Importantly, augmentation of neuron GSH content can prevent ethanol-mediated oxidative stress in cultured fetal cortical neurons and the ensuing increase in apoptotic death (Ramachandran et al. 2003). Recent studies in our laboratory have illustrated that co-culturing astrocytes with fetal cortical neurons likewise blocks ethanol-mediated oxidative stress, normalizes neuron GSH homeostasis and decreases subsequent apoptotic death (Watts et al. 2005). This strongly supports the concept that the astrocyte-related neuroprotection occurs by maintenance of neuron GSH homeostasis. The following studies extend these observations to delineate the regulatory elements that are activated by this pro-oxidant setting, thereby enhancing the neuroprotective potential of astrocytes. These experiments detail three key points in the γ-glutamyl cycle that are up-regulated by exposure to this pro-oxidant: increased astrocyte GSH efflux, increased activity and expression of astrocyte γGT, and increased activity and expression of neuron APN.
Materials and methods
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum and tissue culture plastics were purchased from Life Technologies (Carlsbad, CA, USA). Monochlorobimane, orthophthalaldehyde, γ-glutamyl cysteine, l-cysteine, l-glutamic acid, glycine, glutathione, glutathione-S-transferase, γ-glutamyl transpeptidase kits, acivicin, bestatin, alanine-p nitroanilide and 4-nitroaniline were from Sigma Chemicals (St Louis, MO, USA). Antibodies towards γGT (light subunit) and CD13 were polyclonals from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). The kit for protein estimation was from Bio-Rad (Hercules, CA, USA).
Astrocytes were prepared from the cerebral hemispheres of 2-day-old Sprague Dawley rat neonates as described by McCarthy and de Vellis (1980). In brief, brain cortices were removed, tissue was disrupted, treated with 2.5% trypsin and DNase, filtered through a 0.25 µm sieve and seeded in 75 cm2 plastic flasks at a density of 6 × 106 cells. The cultures were grown to confluency in DMEM with 10% fetal bovine serum (FBS) and antibiotics. The medium was changed every 3 days and cultures were split on day 6 into the desired plates (100 mm, 60 mm or 6-well). The astrocytes were grown to confluency and were identified as more than 95% pure by staining with glial fibrillary acidic protein.
Co-culture and cortical neurons
For co-cultures, cortical astrocytes were likewise prepared from 2-day-old Sprague Dawley rat cortices (McCarthy and de Vellis 1980). The dissociated cells were washed, suspended in a culture medium consisting of Eagle's minimal essential medium (MEM) with 10% fetal bovine serum and glutamine (2 mm), and plated in 75 cm2 cell culture flasks. At confluency, astrocytes were lightly trypsinized and replated onto cell culture inserts (Fisher-Costar Brand) Fisher Scientific, Pittsburgh, PA, USA at 2.5 × 105 cells/well. The astrocytes were used for co-cultures 7 days after re-plating, at which time they formed a confluent layer across the surface of the insert. Primary cortical neuron cultures were prepared from embryonic day 16–17 rats as described by Dutton (1990). The neurons were suspended in glial conditioned medium (MEM containing 10% horse serum). They were plated into 6-well culture plates (1.2 × 106 cells/well) previously coated with poly d-lysine. On the following day, neurons were treated with mitotic inhibitors to prevent astrocyte contamination. On the third day in vitro, inserts containing confluent astrocyte cultures were placed in wells containing neurons.
Astrocytes (for 1, 2, 4 and 6 days) and neurons (for 1, 2, 6 and 24 h) were treated with ethanol (E, 2.5 and 4 mg/mL). An ethanol-filled beaker was used in incubators to maintain Ethanol concentrations in the culture media (Heitman et al. 1987; Devi et al. 1993). The 2.5 mg/mL ethanol concentration used is consistent with blood alcohol levels in rat models which we have previously found to adversely affect fetal growth, elicit brain oxidative stress, increase apoptotic cell death and generate mitochondrial dysfunction (Henderson et al. 1979, 1995; Ramachandran et al. 2001). The 4 mg/mL ethanol concentration is at the high end, but is at or below that reported in mouse models which expressed striking brain apoptotic responses (Olney et al. 2002).
Cell content of GSH
The fluorometric GSH assays were based on those published by Chung et al. (2000), which used a quantization approach described by Fernandez-Checa and Kaplowitz (1990). These fluorometer-based measures were confirmed by the latter HPLC approach. Confluent astrocyte monolayers were washed with phosphate-buffered saline (PBS), then scraped and incubated with 100 µm monochlorobimane (MCB) for 10 min at 37°C. The cells were lysed with triton X-100 (0.02%) and centrifuged at 13 000 g. Fluorescence of the MCB–GSH complex in the supernatant fluid was measured [360 excitation (Ex), 460 emission (Em)] on a Perkin Elmer HTS 7000 BioAssay Reader (Perkin Elmer, Wellesley, MA, USA). GSH content was calculated with a standard curve prepared from GSH incubated with MCB and glutathione-S-transferase (0.1 U). GSH was expressed as mmoles/mg DNA.
Measurement of ROS generation using DCF
Generation of reactive oxygen species (ROS) was estimated with the fluorescent probe, 2,7′-dichlorofluoroscein diacetate (DCF-DA) as previously described by Jung et al. (2001). Cells were harvested and washed with PBS and re-suspended at 1 × 106 cells/mL. DCF-DA was added to a final concentration of 20 µm and incubated for 30 min at 37°C. Following incubation, the cells were washed with PBS and re-suspended in PBS. ROS generation was measured by fluorescence intensity (505 nm Ex, 535 nm Em) of 10 000 cells with a FACS flow cytometer.
Annexin-V binding was used as a measure of apoptotic cell death. Following treatment, cells (floating and adherent) were harvested and stained with Annexin-V fluorescein isothiocyanate (Molecular Probes, Eugene, OR, USA), 1 µg/mL suspension, for 15 min in the dark in PBS containing 1% bovine serum albumin. Cell suspensions were adjusted to 1 × 106 cells/mL. Acquisition and analysis was performed on a FACS flow cytometer (495 nm Ex, 519 nm Em).
Glutathione reductase activity
Samples were prepared as described by Beutler (1975). Enzyme activity was measured as described by Savvides and Karplus (1996). Confluent cells were washed with PBS, then solubilized with 0.2% Triton X100 for 1 h. The assay was carried out in 1 mL of the following solution: 0.2 m KCl, 0.1 m K2HPO4, pH 7.0, 1 mm EDTA, 0.1 mm NADPH, 1 mm GSSG and 35 nm bovine serum albumin. Enzyme activity was spectrophotometrically monitored for the rate of conversion of NADPH to NADP (ε340 =6.2 mm/cm). Linear absorbance of NADPH in the reaction mixture was measured over a period of 1 min.
Astrocytes were washed with PBS, scraped, then homogenized in 0.25 m sucrose containing 1 mm EDTA, 20 mm Tris-HCL (pH 7.4) and protease inhibitors. The homogenate was centrifuged at 4°C, 3000 g, for 10 min, then the supernatant fluid was again centrifuged for 20 min. The final spin at 105 000 g yielded the cytosolic fraction, which was dialyzed for 3 h using porous membrane tubing (mol.wt cut-off: 12–14 000; Spectrum Medical Industries, Los Angeles, CA, USA) in 100× PBS to remove endogenous GSH, γ-glutamylcysteine (γGC) and cysteine (Huang et al. 2000). The cytosolic γGC was assayed based on the protocol of Yan and Huxtable (1995). Assay was by fluorescence HPLC using a Beckman Ultra sphere ODS (250 × 4.6 mm i.d., 5 µm particle size) (Beckman Coulter, Fullerton, CA, USA) reverse phase stainless steel column maintained at room temperature 24°C (Ex 340 nm, Em 450 nm) (Mopper and Delmas 1984). The pre-column derivatization of γGC was with orthophthalaldehyde (OPA) and 2-mercaptoethanol (Tsikas et al. 1999).
Glutathione synthetase activity
Assays of glutathione synthesis capacity utilized the fluorescence HPLC method as stated above. The dialyzed cytosolic fraction was used for the assay. Cell extract (0.5 mg protein) was added to the cuvette containing 100 mm Tris-HCL, 150 mm KCL, 2 mm EDTA (pH 7.3), 0.05 mmγ-glutamylcysteine + dithiothreitol, 10 mm glycine, 3 mm ATP and 20 mm Mg2+ in a final volume of 1.0 mL at 37°C (Lu et al. 1999). The sample was derivatized as above and injected into the HPLC column. The amount of GSH formed by glutathione synthetase was calculated from a standard GSH curve.
GSH efflux was measured as described by Lu et al. (1996). Cells were washed with Krebs Henseleit buffer (Sigma, St Louis, MO, USA) supplemented with 12.5 mm HEPES (pH 7.4), then incubated in 5 mL Krebs buffer at 37°C for 60 min. Aliquots of supernatant fluid were removed and GSH measured (Fernandez-Checa and Kaplowitz 1990). Acivicin (5 mm), a γGT inhibitor, was added to the medium (Kannan et al. 1999).
Samples were prepared as described by Garcion et al. (1999). γGT activity was assayed using a kit from Sigma, which used l-γ-glutamyl-3-carboxy-4 nitroanilide as the substrate and glycylglycine as the glutamate acceptor for the transpeptidation reaction. The formation of 5-amino-2 nitrobenzoate was measured spectrophotometrically at 405 nm. One milliunit of γGT represented the formation of 1 nanomole of this product/minute/milligram protein at 30°C. Experiments in which γGT activity was inhibited used two approaches. One was to pre-incubate astrocytes with antibodies to γGT (5F10 and 2A10, each at 50 µg/mL) (Bluvshtein et al. 1999) for 4 h prior to, and during a 2 h treatment period. The 2A10 antibody inhibits hydrolysis and thus, the ensuing transpeptidation cannot occur. The other was to incubate cells with 0.5 mm acivicin for 1 h and subsequently, during the 2 h treatment period.
Western blot analysis for γGT protein
Samples were lysed with RIPA buffer at 4°C for 30 min, and 50 µg cytosolic protein were separated on a 12.5% sodium dodecyl sulfate (SDS) gel. The proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) (polytran) membrane, followed by blocking with 5% non-fat dry milk in TBST (50 mm Tris-HCL pH 7.4, 150 mm NaCl, 0.2% Tween 20) for 1 h at room temperature. The membrane was incubated overnight with primary antibody-rabbit polyclonal IgG-γGT ½ (1 : 250 for γGGT2), and secondary antibody. The antigen-antibody complex was detected using the enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA).
Membrane samples were prepared according to Hemmings and Storey (1999). Briefly, cells were homogenized in 0.32 m sucrose and centrifuged at 2200 g (4°C) for 10 min, and the supernatant fluid was centrifuged at 20 000 g (4°C) for 1 h. The resulting supernatant fluid was discarded, and the pellet was dissolved in 50 mm Tris HCl (pH 7.5) and centrifuged at 20 000 g for 1 h. The pellet was dissolved in 10 mm Tris HCl and 0.5 mm dithiothreitol (DTT) (protein concentration was 1–2 mg/mL). Then, 100 µg protein were incubated with 0.5 mL 2 mm alanine-p-nitroanilide at 37°C for 1 h. Incubation was stopped by placing the tubes on ice and subsequently placing them in boiling water for 5 min. Samples were then centrifuged at 14 000 g for 7 min, and the formation of p-nitroaniline was read at 405 nm. The enzyme activity was calculated from the standard curve with p-nitroaniline (Gillespie et al. 1992). Bestatin 0.5 mm was used for inhibition of APN activity.
Western blot for APN protein
Samples were lysed with RIPA buffer at 4°C for 30 min, and 20 µg cytosolic protein were separated on a 7.5% SDS gel. The membrane was incubated overnight with primary antibody-rabbit polyclonal IgG-CD13 (1 : 300 for CD13 and 1 : 5000 for actin). The antigen-antibody complex was detected using the ECL kit (Pierce).
DNA was assayed using a kit from Trevigen (Trevingen Inc., Gaithersburg, MD, USA). Cells were washed with PBS and re-suspended in serum-free medium. To 150 µL cell suspension, 50 µL diluted Hoechst dye solution were added and incubated at 37°C for 1 h. The fluorescence was recorded at excitation 350 nm and emission 460 nm. DNA content of the sample was calculated from a standard curve which used DNA from salmon testes (Aldrich, Milwaukee, WI, USA).
Protein was estimated by the method of Bradford (1976), using a kit from Bio-Rad (Biorad Laboratories, Hercules, CA, USA). Bovine serum albumin was the standard.
Results are expressed as mean ± SEM. Two-way analysis of variance followed by Student's Newman Kuels test was used for comparisons between the control and treated groups. A value of p < 0.05 was considered statistically significant.
Astrocyte maintenance of neuron GSH homeostasis in the presence of ethanol
Ethanol exposure decreased the GSH content of cultured fetal rat cortical neurons. Figure 1 shows that within 1 h of treatment with 2.5 or 4.0 mg/mL ethanol, neuron GSH content was reduced by 30% and 26%, respectively (p < 0.05). A 2 h treatment with 2.5 or 4.0 mg/mL ethanol likewise reduced GSH by 36% and 44% of the corresponding control. When the neurons were co-cultured with astrocytes, this response to ethanol was completely mitigated (Fig. 1).
Astrocytes protect neurons from an ethanol-mediated increase in ROS and cell death
Previous studies have illustrated that maintenance of neuron GSH content by pre-treatment with N-acetylcysteine prevents ethanol-mediated oxidative stress and subsequent apoptotic death (Ramachandran et al. 2003). Figure 2 shows that in the absence of astrocytes when neuronal GSH was reduced, 2.5 mg/mL and 4.0 mg/mL ethanol increased ROS by 23% and 66%, respectively, within 1 h (p < 0.05). Within 2 h, ROS content likewise exceeded control values by 60% and 68% with 2.5 mg/mL and 4.0 mg/mL ethanol, respectively. Co-culturing the neurons with astrocytes, which was previously shown to normalize neuron GSH (Fig. 1), prevented the increase in ROS (Fig. 2). A 24 h treatment with either 2.5 mg/mL or 4.0 mg/mL ethanol increased Annexin-V binding (as a measure of apoptosis) by 87% and 126%, respectively (p < 0.05) (Fig. 3), and this response was also prevented by the presence of astrocytes (Fig. 3).
Ethanol effects on astrocyte GSH homeostasis
Ethanol (2.5 and 4.0 mg/mL) did not alter astrocyte GSH content. While GSH appeared to be depressed with an initial 24 h exposure, this did not reach statistical significance (Fig. 4). With further ethanol exposures of up to 6 days, there was clearly no effect on GSH content (p < 0.05). One possible mechanism underlying this stability of GSH homeostasis could be an enhancement of glutathione reductase. Figure 5 shows that at the 24 h exposure point, activity of this enzyme is increased by 25% and 39% by 2.5 mg/mL and 4.0 mg/mL ethanol, respectively (p < 0.05). These activities remained at approximately the same levels throughout a 6 day ethanol exposure. There was also an upward creep of glutathione reductase activities in control astrocytes, ranging from a 25% increase over the 1 day value by day 2, to a 34% increase after a total of 6 days in culture (p < 0.05). By 4 days in culture, ethanol treatment had no effect compared with the corresponding controls. There are two enzymes which synthesize GSH, γ-glutamyl cysteine ligase (γGCL) and glutathione synthetase (GS), with the former being the rate-limiting reaction. Ethanol effects on these two enzymes were variable. Exposure to 2.5 mg/mL ethanol had no effect on either enzyme activity, regardless of exposure duration, while 4.0 mg/mL ethanol treatments for 2 days and 4 days decreased γGCL by 15% and 35%, respectively (p < 0.05) (Fig. 6). However, there were no ethanol effects on γGCL with the 1 day and 6 day exposure periods (p < 0.05) (Fig. 6). GS activity was significantly reduced (by 23%, 4.0 mg/mL ethanol) only at the 2 day exposure (p < 0.05), and no changes occurred at the other two time points (Fig. 7). In addition to stability of GSH homeostasis in the presence of ethanol, the drug had little effect on confluent astrocyte viability (measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test and trypan blue exclusion; data not shown). In summary, astrocyte GSH homeostasis was highly resistant to ethanol, with only inconsistent effects on GSH synthesis machinery.
Increased GSH efflux from astrocytes
GSH efflux from astrocytes is an initial step in maintenance of neuron GSH homeostasis. Figure 8 illustrates a steady baseline GSH efflux over a 6-day period, and some striking increases in this process with ethanol exposure. At 4 mg/mL, ethanol increased efflux by 204% (54.8 ± 6.70 µmoles), 175% (57.14 ± 5.62 µmoles), 306% (61.81 ± 4.46 µmoles) and 517% (90.25 ± 8.16 µmoles) (p < 0.001) with 1, 2, 4 and 6 days of treatment, respectively. At 2.5 mg/mL, ethanol had no significant effect on efflux by astrocytes until they had been exposed to ethanol for 6 days, at which time it stimulated efflux by 462% above the corresponding control (p < 0.05).
γGT and APN activities are required for protection by astrocytes
The second step in astrocyte-mediated enhancement of neuron GSH synthesis is hydrolysis of the extruded tripeptide to the CysGly dipeptide by γGT on the astrocyte plasma membrane. The third step is further hydrolysis of the dipeptide to its constituent amino acids by APN on the surface of the neuron. Figure 9 illustrates the importance of these two systems to astrocyte protection of neurons from ethanol-mediated oxidative stress, as well as confirming the role of the γ-glutamyl cycle in this protection. In these experiments, co-cultures were treated with 50 µg/mL antibody 2A10 (to inhibit the hydrolysis of γGT) and 50 µg antibody 5F10 (to inhibit transpeptidation of γGT) for 4 h prior to a 2 h exposure to ethanol (4 mg/mL). Also included was addition of acivicin to astrocytes (for 1 h prior to ethanol and during ethanol exposure) with the antibody, or bestatin to neurons (the same regimen). Acivicin inhibits astrocyte γGT activity by approximately 60% (data not shown) and bestatin inhibits neuron APN by greater than 70% (data not shown). In the absence of astrocytes, a 2 h exposure to ethanol increased DCF fluorescence by 65% (p < 0.05), while in a 2 h co-culture, this increase was completely blocked (Fig. 9). When γGT was inhibited by the antibodies (Ab) or by Ab combined with acivicin, astrocyte protection was prevented (p > 0.05). The presence of Ab or Ab plus acivicin increased the ethanol-related DCF fluorescence to values above that in ethanol-exposed neurons not co-cultured, but the difference between the two treatments did not reach significance (p > 0.05). Inhibition of APN with bestatin likewise blocked the protective effect of astrocytes (p < 0.05).
Up-regulated activity and expression of astrocyte γGT
Ethanol exposure increased activity of γGT. Activity of the enzyme in controls remained relatively constant (p > 0.05) over 6 days of culture, although there was a slight elevation by treatment day 6 (Fig. 10a). Treatment with 4.0 mg/mL ethanol increased γGT activity by 40%, 35% and 41% (p < 0.05) of the corresponding controls at the 1, 2 and 4 day exposure times, respectively (Fig. 10a). Likewise, 2.5 mg/mL ethanol increased γGT activity at the 2 and 4 day treatment times by 31% and 23% (p < 0.05), respectively. Activities were also increased at the 1 and 6 day points, but they did not reach statistical significance (p > 0.05). The increased activity of γGT reflected a parallel increase in its expression within 1 day of ethanol exposure (Fig. 10b). For the western blot analysis of γGT protein expression, ethanol treatment regimens were staggered so that all cells were harvested on the same day of culture. Figure 10(b) is an example of one of the four experiments utilizing an antibody to the γGT light subunit which is a measure of the whole enzyme.
Up-regulated activity and expression of neuron APN
Once the extruded GSH has been cleaved to the CysGly dipeptide, the latter compound is further hydrolyzed to cys by the neuronal membrane-bound enzyme, APN. Ethanol rapidly increased activity of APN in neurons. A 2.5 mg/mL concentration of ethanol increased APN activity by 62% within 1 h of exposure (p < 0.05), and by 76% and 35% within 6 and 24 h, respectively (p < 0.05). A 4.0 mg/mL ethanol concentration, increased activity in a similar fashion by 55%, 57% and 41% at 1, 6 and 24 h, respectively (p < 0.05) (Fig. 11a). There was also a parallel increase in expression of the APN protein at 6 h and 24 h in neurons, as shown by western blot (Fig. 11b). This is one of four western blots presenting the same up-regulation.
Alcohol consumption during pregnancy impairs brain development. These neurotoxic effects may be on astrocytes (Aschner and Allen 2000), as well as on the size and distribution of neuronal populations, and neuronal differentiation and migration (West et al. 1984; Little et al. 1989; Goodlett et al. 1991; Miller 1995). Earlier studies in our laboratory (Ramachandran et al. 2001) illustrated that in utero ethanol exposure enhanced apoptotic cell death in the fetal brain, which might be mediated by oxidative stress and 4-hydroxynonnenal formation in the mitochondria. These studies demonstrated that maternal ethanol intake can accelerate neuronal apoptotic cell death in whole fetal brain, a setting with a low astrocyte content. Subsequent research has shown that in cultured cortical neurons, ethanol rapidly elicits oxidative stress-mediated apoptotic death (Figs 2 and 3), and that this can be mitigated by GSH supplementation (Ramachandran et al. 2003) and by co-culture with astrocytes (Watts et al. 2005). The current experiments detail an astrocyte-mediated system, which maintains neuronal GSH homeostasis in the presence of ethanol, and they flag key components of the system that are up-regulated by this drug. Elimination of this protective capacity by inhibition of γGT or APN illustrates that it is this system which provides protection of neurons from the ethanol-mediated oxidative challenge (Fig. 9). Significant to the developmental neurotoxicity of ethanol is that the vast majority of cortical astrocytes do not appear until the first postnatal month in rats and mice, and they can only be detected around embryonic day 16 (Qian et al. 2000). Thus, the emergence of astrocytes in the developing brain may be an important determinant of effects of duration of ethanol exposure and timing of dosage on neurotoxic responses to this compound (Watts et al. 2005).
Ethanol and GSH synthesis machinery in the astrocyte
Central to this neuroprotective role of astrocytes is stability of the cells' GSH-generating machinery. In our studies, ethanol had little effect on astrocyte GSH content (Fig. 4), even with 6 days exposure to 4 mg/mL ethanol. This is unlike ethanol effects on neuronal GSH content (Fig. 1) and it likely reflects a highly stable and resilient astrocytic GSH homeostasis machinery. These studies have found astrocytes to be much more resistant to E-mediated effects than neurons, a phenomenon which may be due to the higher GSH content and subsequent protection from oxidative stress-mediated damage (Ramachandran et al. 2003). This is supported by the lower baseline content of GSH in neurons in our culture setting, typically ranging from 2.5 to 3.7 mmoles/mg DNA, while in astrocytes it is between 5.5 and 6 mmoles/mg DNA (Fig. 4). A rather remarkable finding in the current studies is that, when exposed to the mild pro-oxidant, ethanol, astrocyte GSH content remains stable, even in the presence of striking increases in GSH efflux (Fig. 9). This reflects either a prodigious baseline capacity to synthesize the compound and/or compensatory systems that up-regulate synthesis. We found no consistent evidence of an ethanol-related up-regulation of the two enzymes which synthesize GSH. Rather, γ-glutamyl cysteine ligase and glutathione synthetase activities either remained unchanged or were decreased (Figs 6 and 7), suggesting little or no change in availability of substrates for the synthesis of GSH. γ-Glutamyl cysteine ligase is the rate-limiting enzyme in the synthesis of GSH and is subject to multiple post-translational regulatory events, including feedback inhibition by GSH and availability of the substrate, cysteine (Huang et al. 1993; Soltaninassab et al. 2000). The above experiments illustrate that, even when γ-glutamyl cysteine ligase activity is reduced by almost 40% (Fig. 6) by 4 mg/mL ethanol at 4 days of exposure, GSH efflux is strongly enhanced (Fig. 8). One final factor in the maintenance of net astrocyte GSH content is the increase in glutathione reductase in response to ethanol (Fig. 5). Compared with control values, these were statistically significant for up to 2 days of ethanol exposure, increasing their maximum activity within 1 day of treatment. These elevated activities remained constant for the entire 6 day regimen, although there was also a time-dependent increase in control samples. The mechanism underlying the latter phenomenon is unclear. Thus, the maintenance of astrocyte GSH content in the presence of such an elevated efflux may reflect both a high baseline GSH synthesis capacity and an up-regulation of glutathione reductase activity.
Regulatory responses to ethanol
Increased GSH efflux
A predominant fraction of GSH in brain is in astroglial cells, and these cells appear to provide extracellular antioxidant protection through its efflux (Dringen et al. 1999b; Shroeter et al. 1999). Figure 8 shows that ethanol can strikingly increase GSH release, especially at the highest ethanol concentration. Such a setting is not without precedent, as Sagara et al. (1996) have reported that efflux of GSH can be increased in astrocytes by oxidative stress. Ethanol is a pro-oxidant and its up-regulation of GSH efflux may be an important mechanism by which astrocytes protect neurons from ethanol-related oxidative damage. How this increased efflux occurs remains to be determined, but it could reflect an effect of ethanol and/or oxidative stress on the transporter protein, MRP1. Hirrlinger et al. (2002) have reported that the ATP-dependent, multidrug-resistant protein 1 (MRP1) expressed in astrocytes mediates export of GSH, GSSG and glutathione conjugates. Also, it is possible that ethanol elicited some degree of non-specific damage to the astrocyte plasma membrane that resulted in leakage of GSH. However, these ethanol regimens do not decrease trypan blue exclusion (Watts et al. 2005), nor do they increase Lactate dehydrogenase (LDH) leakage (data not shown). A final point is that astrocyte protection of neurons occurs within 24 h of exposure, a period during which 2.5 mg/mL ethanol did not enhance astrocyte GSH efflux. This suggests that, at least at the lower level of ethanol, baseline efflux is sufficient to provide neuroprotection.
Up-regulation of γGT
In addition to astrocyte GSH efflux, two other systems are in play that provide the neuron with the rate-controlling GSH precursor, cysteine (Dringen et al. 1999a). The first step in this ‘γ-glutamyl cycle’ is the extracellular degradation of GSH to the CysGly dipeptide by γGT (Meister and Anderson 1983; Hanigan 1998). γGT is a membrane-bound enzyme with its active site on the extracellular surface of the plasma membrane (Hammond et al. 2001). It is highly expressed in the central nervous system, largely on astrocytic end feet (Cambier et al. 2000). Concomitant with GSH efflux from astrocytes, there is a rapid up-regulation of astrocyte γGT activity and expression of its protein (Figs 10a and b). The increase in enzyme activity corresponds with increased expression of the γGT light chain (Fig. 10b) and the rapidity of this up-regulation is conspicuous. The latter occurs within 1 h of exposure to 4 mg/mL ethanol (data not shown) and it continues through at least 4 days of ethanol treatment (Fig. 10b). As with the increased GSH efflux, ethanol-generated oxidative stress may be a key player in this up-regulation. Oxidative stress increases γGT activity and its protein and mRNA (Kugelman et al. 1994). Its synthesis and activity are altered by a variety of agents, including ethanol, growth factors and hormones such as glucocorticoids, retinoic acid and thyroid hormones (Garcion et al. 1999). This step of the γ-glutamyl cycle may be a vital one with respect to maintenance of neuron GSH homeostasis, since its inhibition can prevent astrocyte-induced enhancement of neuron GSH content (Dringen et al. 1999a). Interestingly, γGT appears to play an important role in development, since mice deficient in this enzyme, while appearing normal at birth, grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels are elevated sixfold and 2500-fold, while GSH levels are markedly reduced in eye, liver and pancreas, and plasma cysteine levels are reduced by 80% (Lieberman et al. 1996).
Up-regulation of APN
A third point of ethanol up-regulation is an increase of activity and expression of APN on the neuronal plasma membrane. The CysGly dipeptide generated from extruded GSH by γGT is a precursor for neuron GSH (Dringen et al. 1999a). Recent reports have illustrated that in neurons, this occurs not directly but subsequent to CysGly hydrolysis to its constituent amino acids by APN (Dringen et al. 2001). This reaction is mediated by APN on the outer surface of the neuron plasma membrane and generates cysteine that is imported as a rate-limiting precursor for GSH synthesis (Dringen 2000). Effects of ethanol on this ectopeptidase is an unexplored area. Membrane-bound proteases are widely distributed in cell systems and their expression is highly regulated. These roles are varied and include immune function, embryonic development, regulation of synaptic plasticity, cell growth regulation and regulation of apoptosis (Sedo and Malik 2001). In the brain, APN, which possesses cysteinylglycinase activity, has been localized in microglia, astrocytes and neurons in a variety of brain structures, including the cerebral cortex (Barnes et al. 1994; Lucius et al. 1995; Noble et al. 2001). The current experiments illustrate that exposure of neurons to ethanol increased both the APN enzyme activity and its expression on the neuron plasma membranes (Figs 11a and b). Clearly, such an up-regulation should enhance the production of cysteine and subsequent synthesis of GSH.
The experiments detailed above demonstrate the presence of an effective system by which astrocytes and a neuronal ectopeptidase can act in consort to maintain neuron GSH homeostasis when challenged by the pro-oxidant, ethanol. Experiments using inhibitors of γGT and APN confirm that it is this system that protects cultured neurons from ethanol-mediated oxidative stress. An important finding is the ability of ethanol, and possibly other pro-oxidants, to bolster this system by up-regulation at least at three connected levels. In response to ethanol, GSH efflux by astrocytes is strikingly increased in parallel with up-regulation of γGT on the astrocyte outer membrane and APN on neurons. The enhanced production of cysteine provides the rate-controlling precursor for GSH synthesis in the neuron. We propose that this is an important mechanism by which astrocytes may protect neurons from ethanol-mediated oxidative stress and apoptotic death.
This work was supported by R21 AA013431 and RO1 AA010114.