R. Dringen, Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany. Fax: +49 7071 295360, Tel.: +49 7071 2973334, E-mail: firstname.lastname@example.org
The cells of the adult human brain consume ≈ 20% of the oxygen utilized by the body although the brain comprises only 2% of the body weight. Reactive oxygen species, which are produced continuously during oxidative metabolism, are generated at high rates within the brain. Therefore, the defense against the toxic effects of reactive oxygen species is an essential task within the brain. An important component of the cellular detoxification of reactive oxygen species is the antioxidant glutathione. The main focus of this short review is recent results on glutathione metabolism of brain astrocytes and neurons in culture. These two types of cell prefer different extracellular precursors for glutathione. Glutathione is involved in the disposal of exogenous peroxides by astrocytes and neurons. In coculture astrocytes protect neurons against the toxicity of reactive oxygen species. One mechanism of this interaction is the supply by astrocytes of glutathione precursors to neurons.
Reactive oxygen species (ROS) are generated continuously during oxidative metabolism. ROS include inorganic molecules, such as the superoxide radical anion, hydrogen peroxide (H2O2) and hydroxyl radicals, as well as organic molecules such as alkoxyl and peroxyl radicals. In order to avoid damage caused by ROS, such as DNA strand breaks, lipid peroxidation and protein modification, mechanisms have been developed during evolution which dispose of, or prevent the generation, of ROS. For example, the removal of H2O2 and superoxide prevents the generation of highly reactive hydroxyl radicals, which are formed by the iron-catalyzed Fenton reaction or by the Haber–Weiss reaction . Increased production of ROS and/or a decrease in the antioxidative capacity of cells causes oxidative stress which can compromize essential cellular functions.
Compared with other organs, the brain appears to be especially endangered with regard to the generation and detoxification of ROS. The cells of the human brain utilize 20% of the oxygen consumed by the body although this organ comprises only 2% of the body weight . This indicates the generation of a large quantity of ROS during oxidative phosphorylation in brain. In addition, a high iron content has been reported for some brain areas , which is able to catalyze the generation of ROS. In contrast, the brain might be especially vulnerable to ROS, because it is rich in lipids with unsaturated fatty acids, the targets of lipid peroxidation. In addition, the brain contains only low to moderate superoxide dismutase, catalase and glutathione peroxidase (GPx) activity compared with kidney or liver . Such disadvantages of the brain have to be considered in view of the fact that a loss of neurons in adult brain cannot be compensated for by the generation of new neurons. Nevertheless, the brain is able to function during a long human life, indicating the presence of an effective antioxidant system in brain. However, the balance between the generation of ROS and antioxidative processes may become disturbed as reported for several neurological disorders. The best evidence of an altered glutathione metabolism as an important factor contributing to the pathogenesis of a neurodegenerative disease has been found in Parkinson's disease. The involvement of a compromised glutathione system in a neurological disorders is reviewed in this issue by Schulz et al. . In this context, insufficient mitochondrial functions are considered to play an important role . The knowledge on glutathione and nitric oxide in respect to mitochondrial function has recently been summarized .
Metabolism and functions of glutathione
The tripeptide glutathione (GSH; γ-l-glutamyl-l-cysteinylglycine) is the most abundant thiol present in mammalian cells with concentrations of up to 12 mm. GSH is synthesized in vivo by the consecutive action of two enzymes (Fig. 1). γ-Glutamylcysteine (γGluCys) synthetase uses glutamate and cysteine as substrates and forms the dipeptide γGluCys, which is combined with glycine in a reaction catalyzed by glutathione synthetase to generate GSH. ATP is a cosubstrate for both enzymes. The balance of cellular synthesis and consumption of GSH is regulated by feedback inhibition of the γGluCys synthetase reaction by the endproduct GSH .
GSH has important functions as an antioxidant, is a transport and storage form of cysteine, is a reaction partner for the detoxification of xenobiotica, and is a cofactor in isomerization reactions [4,9]. In addition, GSH maintains the thiol redox potential in cells keeping sulfhydryl groups of cytosolic proteins in the reduced form. Recent results suggest that GSH also plays a role in the regulation of apoptosis .
The glutathione system is especially important for cellular defense against ROS. GSH reacts directly with radicals in nonenzymatic reactions and is the electron donor in the reduction of peroxides catalyzed by GPx (Fig. 2). The product of the oxidation of GSH is glutathione disulfide (GSSG). GSH is regenerated from GSSG within cells in a reaction catalyzed by the flavoenzyme glutathione reductase (GR). This enzyme regenerates GSH by transferring reduction equivalent from NADPH to GSSG (Fig. 2).
During the course of the reactions catalyzed by GPx and GR, glutathione is recycled (Fig. 2). In contrast, GSH is consumed during the generation of glutathione-S-conjugates by glutathione-S-transferases  or by the release of glutathione from cells [12,13]. Both processes lower the level of total intracellular glutathione. Therefore, in order to maintain a constant intracellular GSH concentration the GSH consumed has to be replaced by resynthesis from its constituent amino acids. Extracellular GSH and glutathione conjugates are substrates for the ectoenzyme γ-glutamyl transpeptidase (γGT). This enzyme catalyzes the transfer of a γ-glutamyl moiety from GSH or a glutathione conjugate onto an acceptor molecule (Fig. 1). Products are a γ-glutamyl compound and the dipeptide cysteinylglycine (CysGly) or the CysGly conjugate . Peptidases hydrolyze CysGly to cysteine and glycine. These amino acids can subsequently serve again as substrates for cellular GSH synthesis (Fig. 1).
Glutathione metabolism of astrocytes and neurons
The available data on the glutathione content of various brain areas and the localization in brain of GSH and enzymes of glutathione metabolism have recently been reviewed [4,15]. Astrocytes appear to contain higher GSH levels than neurons both in vivo and in culture [4,15]. In recent years, glutathione metabolism of brain cells has been studied predominantly in primary cultures enriched for one type of brain cell. From experiments performed on such cultures ample information is available regarding glutathione metabolism in astrocytes and neurons. In contrast, little is known about GSH metabolism in oligodendroglial and microglial cells .
The GSH content of cultured astroglial cells can be modulated by a variety of treatments . For example, GSH levels decrease as soon as the synthesis of GSH is inhibited by buthionine sulfoximine or if cells are incubated in the presence of reagents such as dimethyl maleate or ethacrynic acid, which react with the thiol group of GSH. In contrast, GSH levels increase after application of GSH precursors . Depletion of GSH by amino acid deprivation and the subsequent refeeding of putative GSH precursors revealed that a variety of exogenously applied amino acids, sulfur-containing compounds and peptides can be used by cultured astrocytes as precursors for GSH synthesis . The best exogenous precursor of the cysteine necessary for GSH synthesis in astroglial cells is the amino acid cystine . This amino acid is transported in a sodium-independent manner across the astroglial cell membrane in exchange for glutamate via the transport system X−C.
Astroglial cultures release GSH [19–21] which is used as a substrate for the ectoenzyme γGT. Within 1 h astroglial cultures release ≈ 10% of their intracellular glutathione . Simultaneously, GSH is resynthesized in order to compensate for the GSH released and to maintain a constant cellular concentration of GSH. This release of GSH from astroglial cells is quantitatively the most important process consuming intracellular GSH. The rate of release of GSH from astroglial cells depends on the intracellular GSH concentration and follows apparent Michaelis–Menten kinetics . Extracellular GSH serves as substrate for the astroglial ectoenzym γGT . The dipeptide CysGly, the product of the γGT reaction, is reused by cultured astroglial cells for GSH synthesis. The peptide transporter PepT2 is expressed in these cultures and is responsible for the uptake of CysGly . After intracellular hydrolysis of CysGly, the cysteine and glycine generated serve as substrates for astroglial glutathione synthesis .
The GSH levels reported for cultured neurons vary greatly which has been attributed to different preparation techniques, to species differences or to different culture conditions . With regard to the culture conditions, the content of cysteine or cysteine precursors in the medium in particular determines the GSH level in neurons, because neurons are not able to use the cystine present in most culture media and rely on the availability of cysteine for their glutathione synthesis [24,25]. In contrast to cysteine, the availability of the glutamate precursor glutamine or of glycine does not limit neuronal glutathione synthesis . In addition to cysteine, brain neurons are able to use the cysteine donors CysGly, γGluCys and N-acetylcysteine as precursors for GSH [26,27]. Other compounds and treatments which modulate GSH levels in cultured neurons have recently been summarized .
Among exogenous precursors of neuronal GSH, the dipeptide CysGly may be the most important because it is generated from extracellular GSH in the γGT reaction. CysGly in micromolar concentrations is efficiently utilized by neurons . The concentration of CysGly leading to a half-maximal GSH level is lower in neurons  than in astroglial cells , indicating that neurons are more efficient in using this peptide than astroglial cells. To date, the mechanism by which CysGly is utilized by neurons has not been elucidated completely. This peptide might be taken up into neurons by a peptide transporter, as described for astroglial cells . Alternatively, the dipeptide might be hydrolyzed by a neuronal ectopeptidase generating amino acids, which are subsequently taken up as precursors for GSH synthesis. Evidence obtained recently in our laboratory strongly suggests the involvement of a neuronal ectopepetidase in the utilization of CysGly by neurons (R. Dringen, unpublished results). The cysteine and glycine liberated by the hydrolysis of CysGly serve as precursors for neuronal GSH . These amino acids are taken up into brain cells via sodium-dependent transport processes [18,28,29].
Glutathione and the disposal of peroxides by astrocytes and neurons
Cultured astroglial cells dispose of exogenous H2O2[30,31] and organic hydroperoxides such as tertiary butyl hydroperoxide or cumene hydroperoxide [32,33] very efficiently. These peroxides are substrates of GPx. Indeed, rapid oxidation of GSH was found after application of peroxides to astroglial cultures [31–33]. Inhibition of catalase, the second cellular enzyme involved in H2O2 disposal, reduced at best marginally the clearance rate for H2O2 as long as the astroglial glutathione system was not compromised. In contrast, inhibition of both catalase and GPx strongly reduced the capability of astroglial cells to dispose of H2O2. These findings demonstrate that the glutathione system of astroglial cultures can substitute for the function of catalase in H2O2 clearance. Catalase does not accept organic hydroperoxides as substrates. Therefore, the glutathione system is responsible and sufficient for the rapid disposal of organic hydroperoxides by astroglial cultures [32,33].
Cultured neurons have also been reported to dispose of exogenous H2O2. Evidence has been presented that the neuronal defense against H2O2 is mediated primarily by the glutathione system . Indeed, application of H2O2 to neurons causes rapid oxidation of GSH. Removal of the peroxide is followed by an almost complete regeneration of the original GSH to GSSG ratio within minutes . Apparently, astroglial cultures have a higher capacity than neurons to detoxify H2O2[30,34]. However, it must be stressed that for such a comparison the differences in cell numbers between confluent astroglial cultures and cultured postmitotic neurons have to be considered. If the differences in protein content of the cultures are taken into consideration, the rate of H2O2 clearance by the cells in primary neuronal and astroglial cultures is almost identical . This indicates that, at least in culture, both types of brain cell are equally able to detoxify exogenous H2O2. However, for the rapid clearance of H2O2 by neurons both GPx and catalase are essential and, in contrast to the situation in astroglial cultures , the glutathione system in neurons cannot functionally compensate for loss of the catalase reaction . The lower efficiency of the neuronal glutathione system of peroxide detoxification compared with that of astroglial cells is confirmed by the reduced ability of cultured neurons to dispose of the organic peroxide cumene hydroperoxide from the medium .
Interaction between astrocytes and neurons in glutathione metabolism and the defense against ros
In vivo the different types of brain cell are in close contact with each other. Evidence is growing that, especially between astrocytes and neurons, an intensive metabolic exchange occurs. Such interactions also appear to be important regarding cerebral glutathione homeostasis and the protection of the brain against oxidative stress .
In coculture, astrocytes support other brain cell types in the defense against ROS. In the presence of astroglial cells neurons are protected against the ROS-induced toxicity of various compounds and treatments (Table 1). Because H2O2 is the peroxide generated in the highest quantity in the brain, the protection by astrocytes of neurons against the toxicity caused by H2O2 appears to be particularly important [30,35]. In coculture, neurons are protected against H2O2 toxicity even at a cellular ratio of 1 astroglial cell to 20 neurons . Neurons in culture become damaged by extracellular ROS  which are detoxified in the presence of astroglial cells. GSH is important for this function, because the protective effect of astroglial cells is diminished when these cells contain low levels of GSH .
Table 1. Protection by cocultured astroglial cells of neurons against the toxic effects of various compounds.l-Dopa, 2,4-dihydroxyphenylalanine.
For the synthesis of GSH, a metabolic interaction between neurons and astroglial cells takes place. Only the availability of cysteine determines the level of neuronal GSH . If neurons are cultured in the presence of astroglial cells, the GSH content of the neurons increases strongly indicating that, in the presence of astroglial cells, a cysteine precursor is provided from the astroglial cells to the neurons improving neuronal GSH synthesis. The dipeptide CysGly, which is generated from extracellular GSH by the γGT reaction , is utilized efficiently in micromolar concentrations as a precursor for neuronal GSH . Inhibition of γGT prevented totally the astroglia-induced effect on the GSH content in neurons  demonstrating that CysGly is most probably the GSH precursor provided by astroglial cells to neurons (Fig. 3).
Figure 3 shows our hypothesis for the metabolic interaction between astrocytes and neurons regarding glutathione metabolism. With the release of glutamine by astroglial cells  and the extracellular generation of CysGly from GSH, astroglial cells provide neurons with all three constituent amino acids of GSH. The hypothesis presented here for the metabolic interaction involved in GSH metabolism between astrocytes and neurons (Fig. 3) is supported by recent results obtained on brain slices  and in a microdialysis study . Following the onset of hypoxia, the concentration of cysteine in the superfusion solution of brain slices increased strongly, an effect which was prevented almost completely in the presence of the γGT-inhibitor acivicin . After microinfusion of 1-methyl-4-phenylpyridinium into rat brain a > 1000-fold transient increase in the concentration of GSH in the microdialysates was determined, which was followed by an increase in the extracellular cysteine concentration . The rate of disappearance of GSH and the subsequent increase in cysteine concentration was strongly affected by inhibition of γGT . These data demonstrate that the cysteine found in these experimental systems has most likely been generated from extracellular GSH by the consecutive reactions of γGT and a dipeptidase.
Coculture experiments have demonstrated convincingly that brain astrocytes and neurons strongly influence each other with regard to GSH metabolism and defense against ROS. The importance of astroglial cells for the defense of the brain against ROS and especially the function of astroglial GSH metabolism has become evident at least for cell culture models. Such results suggest that in vivo a compromised astroglial glutathione system may contribute to a lower defense capacity of the brain against ROS and subsequently to increased susceptibility to ROS of astrocytes themselves and of neighboring cells.
The authors would like to thank Dr Bernd Hamprecht for his continuous support, Dr Heinrich Wiesinger for critically reading the manuscript, and the Deutsche Forschungsgemeinschaft for financial support.