Cotgreave I A, Constantin-Teodosiu D, Moldéus P
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
J Appl Physiol (1985). 1991 Mar;70(3):1220-7. doi: 10.1152/jappl.1991.70.3.1220.
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.
汇合的人脐静脉内皮（HUVE）细胞可通过马来酸二乙酯（0.1 - 1.0 mM）迅速（1小时内）耗尽其谷胱甘肽（GSH），但观察到剂量依赖性的细胞脱离。丁硫氨酸亚砜胺（BSO，25 microM）以S形动力学耗尽细胞内GSH，显示出最初的耗尽半衰期为4 - 6小时，48小时后耗尽率大于95%，且细胞无形态变化。然而，通过细胞洗涤并用完全培养基再孵育，BSO依赖性的细胞GSH耗尽仅部分可逆。同样，将细胞在无硫培养基中孵育可再次耗尽细胞GSH，且细胞无形态变化。然而，与BSO不同的是，当重新供应完全培养基、新鲜血浆或全血时，这些细胞能迅速重新合成GSH，12小时时细胞GSH有特征性的过载（高达200%）。通过使用无硫培养基表明，胱氨酸和半胱氨酸都是培养的HUVE细胞中GSH合成的有效前体，且胱氨酸最可能是体内的前体。在胱氨酸支持的GSH重新合成过程中，细胞内积累了高水平的半胱氨酸（高达总可溶性游离硫醇的10%）。生理相关浓度的细胞外GSH在刺激GSH生物合成方面不如胱氨酸或半胱氨酸有效，而非生理高浓度（mM）则以BSO不敏感的方式导致GSH水平大幅高于对照细胞。这些发现为在内皮特异性氧化毒性研究中操纵HUVE细胞GSH以及这些人类细胞中GSH生物化学和周转的硫依赖性研究提供了一种简单方法。
