van Klaveren R J, Hoet P H, Pype J L, Demedts M, Nemery B
Laboratory of Pneumology, K.U. Leuven, Belgium.
Free Radic Biol Med. 1997;22(3):525-34. doi: 10.1016/s0891-5849(96)00375-9.
The purpose of our study was to investigate the effect of oxidative stress or intracellular glutathione (GSH) depletion on gamma-glutamyltransferase (gamma-GT) activity in cultured type II pneumocytes. Twenty-four hours after isolation, primary cultures of rat type II pneumocytes were preincubated with one of four compounds: 15, 30, 60, 125, 250 microM L-buthionine-[SR]-sulfoximine (BSO) for 3 h; 100, 200, 400, 800 microM tertiary-butylhydroperoxide (t-BOOH) for 45 min; 10, 25, 50, 100 microM menadione for 15 min; 100, 1000 microM paraquat for 1 h. GSH levels, H2O2 and O2.- generation were measured immediately after the incubation, gamma-GT activity and GSH levels also up to 24 h or 48 h later. Exposure to BSO led to a persistent GSH depletion without increase in H2O2 or O2.- production, together with a dose and time-dependent increase (doubling) of gamma-GT activity with a nonsignificant increase in gamma-GT mRNA expression 24 h after exposure to BSO. Exposure to 100 microM menadione, which increased H2O2 production, decreased gamma-GT activity. t-BOOH or paraquat did not give rise to a measurable increase in H2O2 or O2.-. Paraquat did not affect initial GSH levels, but increased GSH and decreased gamma-GT activity 24 h later. t-BOOH (400 and 800 microM) initially decreased GSH, and tended to increase GSH 24 h later, 100 and 200 microM increased gamma-GT activity 24 h later, but 800 microM decreased it. Restoration of intracellular GSH levels by addition of GSH to the culture medium completely prevented the increase in gamma-GT activity by BSO, while the addition of catalase or DMTU had no effect. We conclude that at least two effects are operating upon gamma-GT activity: GSH depletion seems to increase gamma-GT activity, while exposure to compounds generating oxidative stress correlates with a decrease in gamma-GT activity.
我们研究的目的是调查氧化应激或细胞内谷胱甘肽（GSH）耗竭对培养的II型肺细胞中γ-谷氨酰转移酶（γ-GT）活性的影响。分离后24小时，将大鼠II型肺细胞原代培养物与四种化合物之一进行预孵育：15、30、60、125、250微摩尔L-丁硫氨酸-[SR]-亚砜亚胺（BSO），孵育3小时；100、200、400、800微摩尔叔丁基过氧化氢（t-BOOH），孵育45分钟；10、25、50、100微摩尔甲萘醌，孵育15分钟；100、1000微摩尔百草枯，孵育1小时。孵育后立即测量GSH水平、H2O2和超氧阴离子（O2.-）的生成量，γ-GT活性和GSH水平在孵育后24小时或48小时内也进行测量。暴露于BSO导致GSH持续耗竭，而H2O2或O2.-的生成量没有增加，同时γ-GT活性呈剂量和时间依赖性增加（翻倍），暴露于BSO 24小时后γ-GT mRNA表达无显著增加。暴露于100微摩尔甲萘醌可增加H2O2的生成，降低γ-GT活性。t-BOOH或百草枯不会导致可测量的H2O2或O2.-增加。百草枯不影响初始GSH水平，但24小时后GSH增加，γ-GT活性降低。t-BOOH（400和800微摩尔）最初降低GSH水平，24小时后GSH水平有升高趋势，100和200微摩尔在24小时后增加γ-GT活性，但800微摩尔降低γ-GT活性。向培养基中添加GSH恢复细胞内GSH水平可完全阻止BSO导致的γ-GT活性增加，而添加过氧化氢酶或二甲基硫脲（DMTU）则没有效果。我们得出结论，至少有两种效应作用于γ-GT活性：GSH耗竭似乎会增加γ-GT活性，而暴露于产生氧化应激的化合物与γ-GT活性降低相关。
