The glutathione content of retinal Muller (glial) cells: effect of pathological conditions

Maintenance of isolated retinal Muller (glial) cells in glutamate-free solutions over 7 h causes a significant loss of their initial glutathione content; this loss is largely prevented by the blockade of glutamine synthesis using methionine sulfoximine (5 mM). Anoxia does not reduce the glutathione content of Muller cells when glucose (11 mM), glutamate and cystine (0.1 mM each) are present. In contrast, simulation of total ischemia (i.e., anoxia plus removal of glucose) decreases the glutathione levels dramatically, even in the presence of glutamate and cystine. Less severe effects are caused by high extracellular K+ (40 mM). Reactive oxygen species are generated in the retina under various conditions, such as anoxia, ischemia, and reperfusion. One of the crucial substances protecting the retina against reactive oxygen species is glutathione, a tripeptide constituted of glutamate, cysteine and glycine. It was recently shown that glutathione can be synthesized in retinal Muller glial cells and that glutamate is the rate-limiting substance. In this study, glutathione levels were determined in acutely isolated guinea-pig Muller cells using the glutathione-sensitive fluorescent dye monochlorobimane. The purpose was to find out how the glial glutathione content is affected by anoxia/ischemia and accompanying pathophysiological events such as depolarization of the cell membrane. Our results further strengthen the view that glutamate is rate-limiting for the glutathione synthesis in glial cells. During glutamate deficiency, as caused by e.g., impaired glutamate uptake, this amino acid is preferentially delivered to the glutamate-glutamine pathway, at the expense of glutathione. This mechanism may contribute to the finding that total ischemia (but not anoxia) causes a depletion of glial glutathione. In situ depletion may be accelerated by the ischemia-induced increase of extracellular K+, decreasing the driving force for glutamate uptake. The ischemia-induced lack of glutathione is particularly fatal considering the increased production of reactive oxygen species under this condition. Therefore the therapeutic application of exogenous free radical scavengers is greatly recommended. (C) 2000 Elsevier Science Ltd. All rights reserved.