Anderson Corinna L, Iyer Smita S, Ziegler Thomas R, Jones Dean P
Department of Medicine/Pulmonary, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.
Am J Physiol Regul Integr Comp Physiol. 2007 Sep;293(3):R1069-75. doi: 10.1152/ajpregu.00195.2007. Epub 2007 Jun 13.
Human cell lines regulate the redox state (E(h)) of the cysteine/cystine (Cys/CySS) couple in culture medium to approximately -80 mV, a value similar to the average E(h) for Cys/CySS in human plasma. The mechanisms involved in regulation of extracellular E(h) of Cys/CySS are not known, but GSH is released from tissues at rates proportional to tissue GSH concentration, and this released GSH could react with CySS to contribute to maintenance of this balance. The present study was undertaken to determine whether depletion of cellular GSH alters regulation of extracellular Cys/CySS E(h). Decrease of GSH in HT-29 cells by inhibiting synthesis with l-buthionine-[S,R]-sulfoximine showed no effect on the rate of reduction of extracellular CySS to achieve a stable E(h) for Cys/CySS in the culture medium. Limiting Cys and CySS in the culture medium also substantially decreased cellular GSH but resulted in no significant effect on extracellular Cys/CySS E(h). Addition of CySS to these cells showed that extracellular Cys/CySS E(h) approached -80 mV at 4 h while cellular GSH and extracellular GSH/GSSG E(h) recovered more slowly. Together, these results show that HT-29 cells have the capacity to regulate the extracellular Cys/CySS E(h) by mechanisms that are independent of cellular GSH. The results suggest that transport systems for Cys and CySS and/or membranal oxidoreductases could be more important than cellular GSH in regulation of extracellular Cys/CySS E(h).
人类细胞系可将培养基中半胱氨酸/胱氨酸（Cys/CySS）对的氧化还原状态（E(h)）调节至约 -80 mV，该值与人类血浆中Cys/CySS的平均E(h)相似。调节细胞外Cys/CySS E(h)的机制尚不清楚，但谷胱甘肽（GSH）从组织中释放的速率与组织GSH浓度成正比，且这种释放的GSH可与CySS反应，有助于维持这种平衡。本研究旨在确定细胞内GSH的耗竭是否会改变细胞外Cys/CySS E(h)的调节。用L-丁硫氨酸-[S,R]-亚砜亚胺抑制合成来降低HT-29细胞中的GSH，对细胞外CySS还原速率无影响，无法在培养基中实现Cys/CySS稳定的E(h)。限制培养基中的Cys和CySS也会大幅降低细胞内GSH，但对细胞外Cys/CySS E(h)无显著影响。向这些细胞中添加CySS表明，细胞外Cys/CySS E(h)在4小时时接近 -80 mV，而细胞内GSH和细胞外GSH/GSSG E(h)恢复得更慢。总之，这些结果表明HT-29细胞有能力通过独立于细胞内GSH的机制调节细胞外Cys/CySS E(h)。结果表明，在调节细胞外Cys/CySS E(h)方面，Cys和CySS的转运系统和/或膜氧化还原酶可能比细胞内GSH更重要。
