GLUTATHIONE METABOLISM IN PRIMARY ASTROCYTE CULTURES - FLOW CYTOMETRIC EVIDENCE OF HETEROGENEOUS DISTRIBUTION OF GSH CONTENT

The time-course of intracellular glutathione (GSH) values after incubation with L-buthionine-(SR)-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, showed that glutathione turns over with a half-life of 5 h. Intracellular GSH was assayed by flow cytometry using three different methods. Astrocytes showed a narrow range of cellular size but a wide range of intracellular GSH. This heterogeneity was resolved into three distinct subpopulations which represent 20%, 35% and 45% of the total astrocyte number. The less abundant subpopulation had the lower GSH content, while the most abundant was the subpopulation with the higher content. Over 95% of astrocytes were in the G0/G1 phase of the cell cycle, the distribution of cytosolic pH was homogeneous and the number of viable cells at the time of the assay was 90%. These results show that several pools exist when astrocyte GSH is considered and these findings may be relevant to the understanding of brain GSH metabolism.