Key result
Human umbilical vein endothelial cells can be reversibly depleted of glutathione using sulfur-free medium, with cystine and cysteine acting as effective precursors for resynthesis.
The study provides a methodology for manipulating glutathione levels in human endothelial cells to study oxidant toxicity and sulfur dependence.
Offers reversible glutathione manipulation in endothelial cells for oxidant studies; leaves open in vivo or clinical translation.
Confluent human umbilical vein endothelial (HUVE) cells were readily (within 1 h) depleted of their glutathione (GSH) by diethylmaleate (0.1-1.0 mM), but dose-dependent cell detachment was noted. Buthionine sulfoximine (BSO, 25 microM) depleted cell GSH with sigmoidal kinetics, showing an initial half-life of depletion of 4-6 h and greater than 95% depletion by 48 h without morphological changes to the cells. However, BSO-dependent depletion of cell GSH was only partially reversible by cell washing and reincubation with complete medium. Likewise, incubation of the cells in sulfur-free medium depleted cell GSH again without morphological changes to the cells. However, unlike with BSO, these cells readily resynthesized GSH when resupplied with complete medium, fresh plasma, or whole blood, with a characteristic overloading of cell GSH (up to 200%) by 12 h. By use of the sulfur-free medium, it was shown that both cystine and cysteine are effective precursors to GSH synthesis in HUVE cells in culture and that cystine is the most likely precursor in vivo. During cystine-supported resynthesis of GSH, high levels of cysteine accumulated in the cells (up to 10% of total soluble free thiol). Physiologically relevant concentrations of extracellular GSH were not as effective as cystine or cysteine in stimulating GSH biosynthesis, whereas nonphysiologically high (mM) concentrations resulted in substantial elevation of GSH levels above those of control cells in a BSO-insensitive manner. These findings provide a simple methodology for the manipulation of HUVE cell GSH in studies of endothelial-specific oxidant toxicity and the sulfur dependence of the biochemistry and turnover of GSH in these human cells.
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Cotgreave et al. (1991) studied this question. Glutathione depletion and resynthesis manipulation was evaluated on Glutathione (GSH) depletion and resynthesis. Human umbilical vein endothelial cells can be reversibly depleted of glutathione using sulfur-free medium, with cystine and cysteine acting as effective precursors for resynthesis.
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