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.

Abstract:The ability of neurons to detoxify exogenously applied peroxides was analyzed using neuron-rich primary cultures derived from embryonic rat brain. Incubation of neurons with H 2 O 2 at an initial concentration of 100 M (300 nmol/3 ml) led to a decrease in the concentration of the peroxide, which depended strongly on the seeding density of the neurons. When 3 ϫ 10 6 viable cells were seeded per dish, the half-time for the clearance by neurons of H 2 O 2 from the incubation buffer was 15.1 min. Immediately after application of 100 M H 2 O 2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H 2 O 2 caused an almost complete regeneration of the original ratio of GSH to GSSG within 2.5 min. Compared with confluent astroglial cultures, neuron-rich cultures cleared H 2 O 2 more slowly from the incubation buffer. However, if the differences in protein content were taken into consideration, the ability of the cells to dispose of H 2 O 2 was identical in the two culture types. The clearance rate by neurons for H 2 O 2 was strongly reduced in the presence of the catalase inhibitor 3-aminotriazol, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H 2 O 2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein-normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells.

To obtain information on the glutathione metabolism of microglial cells, the content of glutathione and activities of enzymes involved in the defense against peroxides were determined for microglia-rich cultures from rat brain. These cultures contain approximately 90% microglia cells as determined by immunocytochemical staining for glial markers, by the phagocytosis activity of the cells and by the production of superoxide after stimulation of the cells with phorbolester. For these cultures, a glutathione content of 41.2 ± 11.2 nmol/mg protein and a specific activity of glutathione reductase of 15.2 ± 3.2 nmol/(min × mg protein) were determined. These values are significantly higher than those found for astroglial or neuronal cultures. In addition, with 68.7 ± 23.5 nmol/(min × mg protein), the specific activity of glutathione peroxidase in microglial cultures was 78% higher than in cultured neurons. The specific catalase activity of microglial cultures was less than 40% that of astroglial or neuronal cultures. Microglial cultures contain only marginal amounts of oxidized glutathione. However, on application of oxidative stress by incubation of microglial cultures with hydrogen peroxide or with the superoxide-producing hypoxanthine/xanthine oxidase system, cellular glutathione was rapidly oxidized. These results demonstrate that microglial cells have a prominent glutathione system, which is likely to reflect the necessity for self-protection against reactive oxygen species when produced by these or surrounding brain cells.

Neurons in culture rely on the supply of exogenous cysteine for their glutathione synthesis. After application of cysteine to neuron-rich primary cultures, the glutathione content was doubled after a 4-hr incubation. The dipeptide cysteinylglycine (CysGly) was able to substitute for cysteine as exogenous glutathione precursor. In kidneys, the ectopeptidase aminopeptidase N (ApN) has been reported to hydrolyze CysGly. Expression of mRNA of ApN in rat brain and cultured rat neurons was demonstrated by reverse transcriptase polymerase chain reaction and sequencing of the cDNA fragment obtained. In addition, the presence of ApN protein in cultured neurons was demonstrated by its immunocytochemical localization. In the presence of an activity-inhibiting antiserum against ApN the utilization of CysGly as neuronal glutathione precursor was completely prevented, whereas that of cysteine plus glycine was not affected. The data presented demonstrates that cultured rat neurons express ApN and that this ectopeptidase participates in the utilization of CysGly as precursor for neuronal glutathione.

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 lM), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 ± 30.4 nmol · min )1 · mg protein )1 and 27.5 ± 5.4 nmol · min )1 · mg protein )1 of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 ± 0.54 min )1 · mg protein )1 . Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells. Keywords: astrocytes, glutathione, microglia, neurones, oligodendrocytes, oxidative stress. In mammalian cells, reactive oxygen species (ROS) like peroxides and radicals are continuously generated during aerobic metabolism and, consequently, have to be detoxified continuously. These processes appear to be especially important for the brain, since oxidative stress has been connected with neurodegenerative diseases, i.e. Parkinson's disease and Alzheimer's disease (Bains and Shaw 1997;Schulz et al. 2000). Several studies demonstrate that the tripeptide glutathione (GSH) plays an important role in the detoxification of reactive oxygen species in brain and that neuronal damage induced by insults which have been discussed to act by generation of ROS is substantially increased if brain glutathione levels are reduced (for an overview see Bains and Shaw 1997;Cooper 1997;Dringen 2000;Schulz et al. 2000).During detoxification of radicals and peroxides, GSH is involved in two types of reactions (Halliwell and Gutteridge 1999; Dringen 2000): (i) GSH reacts nonenzymatically with radicals such as the superoxide radical anion, nitric oxide or the hydroxyl radical; and (ii) GSH is the electron donor for the reduction of peroxides in the reactions catalyzed by glutathione peroxidases (GPx). The final product...