Issels R D, Nagele A, Eckert K G, Wilmanns W
Gesellschaft für Strahlen- und Umweltforschung (GSF), Institut für Klinische Haematologie, Munich, Federal Republic of Germany.
Biochem Pharmacol. 1988 Mar 1;37(5):881-8. doi: 10.1016/0006-2952(88)90176-1.
Chinese hamster ovary (CHO) cells obtain a high capacity to utilize cystine from the growth medium by exposure to cysteamine (2-mercaptoethylamine, MEA) or N-acetylcysteine (NAC). For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM [35S]cystine was used. The uptake of cystine was dependent on the time of exposure (0-60 min) and the concentrations of MEA or NAC (0-8 mM). At high concentrations of MEA or NAC, the uptake of cystine became saturated. Half-maximal uptake of cysteine was observed at concentrations of 0.12 mM MEA and 0.66 mM NAC, respectively. Increase in temperature (37-44 degrees) or pH (6.0-8.0) during MEA or NAC exposure further increased the cystine uptake. The increased uptake of cystine was not affected in the presence of glutamate or homocysteate which both inhibited the cystine uptake of control cells. Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione showed a twofold increase in MEA- or NAC-treated CHO cells. DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis completely blocked the promotion of cystine uptake by MEA and NAC. By further analysis using reversed-phase HPLC of cell extracts, more than 90% of the [35S] radioactive cystine taken up by the cells could be recovered within the pool of GSH. The results demonstrate that exposure of CHO cells with MEA and NAC leads to a promoted uptake of cystine from the culture medium and its rapid utilization for cellular GSH biosynthesis.
中国仓鼠卵巢（CHO）细胞通过暴露于半胱胺（2-巯基乙胺，MEA）或N-乙酰半胱氨酸（NAC）获得从生长培养基中利用胱氨酸的高能力。对于摄取研究，使用补充有0.1 mM [35S]胱氨酸的改良McCoy's 5A培养基。胱氨酸的摄取取决于暴露时间（0 - 60分钟）以及MEA或NAC的浓度（0 - 8 mM）。在MEA或NAC的高浓度下，胱氨酸的摄取变得饱和。分别在0.12 mM MEA和0.66 mM NAC的浓度下观察到半最大摄取量的半胱氨酸。在MEA或NAC暴露期间温度升高（37 - 44摄氏度）或pH升高（6.0 - 8.0）进一步增加了胱氨酸的摄取。在存在谷氨酸或同型半胱氨酸的情况下，增加的胱氨酸摄取不受影响，而这两种物质均抑制对照细胞的胱氨酸摄取。对还原型（GSH）和氧化型（GSSG）细胞内谷胱甘肽的测定表明，经MEA或NAC处理的CHO细胞增加了两倍。DL-丁硫氨酸-S，R-亚砜亚胺（BSO），一种GSH生物合成抑制剂，完全阻断了MEA和NAC对胱氨酸摄取的促进作用。通过使用反相HPLC对细胞提取物进行进一步分析，细胞摄取的超过90%的[35S]放射性胱氨酸可以在GSH池中回收。结果表明，用MEA和NAC处理CHO细胞会导致从培养基中促进胱氨酸的摄取及其快速用于细胞GSH生物合成。
