Johannes Hirrlinger

The Glutathione System of Peroxide Detoxification Is Less Efficient in Neurons than in Astroglial Cells

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 H2O2 at an initial concentration of 100 μM (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 × 106 viable cells were seeded per dish, the half‐time for the clearance by neurons of H2O2 from the incubation buffer was 15.1 min. Immediately after application of 100 μM H2O2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H2O2 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 H2O2 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 H2O2 was identical in the two culture types. The clearance rate by neurons for H2O2 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 H2O2 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.

The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide‐induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline‐derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT‐PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1‐expressing brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.

Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones

Multidrug resistance proteins (Mrps) are ATP‐driven export pumps that mediate the export of organic anions from cells. So far only little information is available on expression and physiological functions of Mrps in brain. The expression of mRNAs of six Mrp paralogs in rat brain, as well as in rat cultures enriched for neurones, astrocytes, oligodendrocytes and microglial cells, was studied by qualitative and semiquantitative RT‐PCR analysis. In adult rat brain as well as in neural cell cultures the mRNAs coding for Mrp1, Mrp3, Mrp4 and Mrp5 were detected. Semiquantitative analysis revealed that the mRNAs coding for Mrp1 and Mrp5 were more abundant in the four cell culture types than mRNAs of the other Mrps. mRNAs coding for Mrp3 and Mrp4 were found at significant levels in cultured astrocytes and microglial cells, whereas cultures of neurones and oligodendrocytes contained only marginal quantities of these mRNAs. Putative physiological functions of Mrps in brain cells are discussed.

Glutathione release from cultured brain cells: Multidrug resistance protein 1 mediates the release of GSH from rat astroglial cells

To investigate the release of glutathione (GSH) from brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells derived from rat brain were studied. During incubation of astroglial cultures, GSH accumulated in the medium with a rate of 3.1 ± 0.6 nmol × h−1 × mg protein−1. In contrast, only marginal amounts of extracellular GSH were detectable in the media of the other brain cell cultures investigated. The mechanism of GSH release from astroglial cells, as yet, has not been reported. Multidrug resistance protein 1 (Mrp1), a transport protein known to mediate cellular export of glutathione disulfide and glutathione conjugates, is expressed in astroglial cultures. Inhibitors of Mrp1 were used to test for a function of this transporter in mediating GSH release from astroglial cells. The presence of the competitive Mrp1 inhibitor MK571 at a concentration of 50 μM inhibited the rate of GSH release by 63%. In contrast, the low concentration of 1 μM of MK571 increased the rate of GSH release by 83%. This bimodal concentration‐dependent effect of MK571 is in accord with literature data for the effects of Mrp1 substrates on GSH release from cells. In addition, the presence of cyclosporin A (10 μM) reduced the GSH release rate significantly and completely blocked the stimulating effect of 1 μM MK571 on the release of GSH from astroglial cells. In conclusion, the data presented are a strong indication that Mrp1 participates in the release of GSH from astroglial cells.

Aminopeptidase N mediates the utilization of the GSH precursor CysGly by cultured neurons.

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.

Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species.

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.

Oligodendroglial cells in culture effectively dispose of exogenous hydrogen peroxide: comparison with cultured neurones, astroglial and microglial cells

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 µm), 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.

Purification of Glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells

Abstract : Glutathione reductase (GR) is an essential enzyme for the glutathione‐mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000‐fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate‐polycrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double‐labeling immunocytochemical staining in astroglia‐rich and neuron‐rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astrogliarich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron‐rich primary cultures, GAP43‐positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.

Effects of dopamine on the glutathione metabolism of cultured astroglial cells: implications for Parkinson's disease

To investigate the effects of dopamine (DA) on the release of glutathione (GSH) from astrocytes, we used astroglia‐rich primary cultures from the brains of newborn rats. In the absence of DA, GSH accumulated in the medium of these cultures with a constant rate. In contrast, during incubation of the cells with 50 µm DA extracellular GSH was not detectable anymore. This disappearance of extracellular GSH was prevented by superoxide dismutase, indicating that DA does not affect GSH release but rather reacts with the released GSH in a superoxide‐dependent reaction. Incubation of astroglial cultures with 0.5 and 1 mm DA established almost constant extracellular concentrations of H2O2 of 5 µm and 15 µm, respectively. Under these conditions astroglial cultures release glutathione disulphide (GSSG). This GSSG export was blocked by catalase and by MK571, an inhibitor of the multidrug resistance protein 1. The effects of DA on the extracellular accumulations of GSH and GSSG were not modulated by inhibitors of DA receptors, DA transport, and monoamine oxidases. The other catecholamines adrenalineandnoradrenaline showed similar effects on the accumulation of GSH and GSSG in the medium compared with those obtained for DA. In conclusion, the data presented demonstrate that DA affects astroglial GSH metabolism by two mechanisms: (i) directly by chemical reaction with extracellular GSH, and (ii) indirectly by generation of hydrogen peroxide that leads to the efflux of GSSG from astroglial cells. These observations are discussed in the context of the brains GSH metabolism in Parkinsons disease.

Chemotherapy-induced cell death in primary cerebellar granule neurons but not in astrocytes: in vitro paradigm of differential neurotoxicity.

The nervous system is frequently the site of symptomatic toxicity of antineoplastic agents. However, there is limited information about the differential vulnerability of neurons, astrocytes and glioma cells. We have analyzed the effects of four chemotherapeutic drugs (lomustine, cisplatin, topotecan and vincristine) on primary cerebellar granule neurons and astrocytes derived from rats. All drugs led to cell death in cerebellar granule neurons in a concentration‐dependent manner. Comparison of the EC50 values for cerebellar neurons and astrocytes with the median EC50 values of 12 malignant glioma cell lines demonstrated a large therapeutic range for lomustin and cisplatin. Further, this comparison revealed a 100‐fold higher sensitivity of cerebellar neurons towards vincristine and 10‐fold higher sensitivity towards topotecan compared with glioma cells. Astrocytes were generally resistant to vincristine. In cerebellar granule neurons, vincristine and to a lesser extent topotecan induced caspase 3 and caspase 9 cleavage, and enhanced caspase activity and Akt‐dependent expression of phosphorylated BAD. zVAD‐fmk, a caspase inhibitor and brain‐derived neurotrophic factor (BDNF), but not MK‐801, a non‐competitive NMDA receptor antagonist, significantly reduced vincristine‐ or topotecan‐induced cell death.