Fernandez-Checa J C, Ookhtens M, Kaplowitz N
Liver Research Laboratory, Wadsworth Veterans Administration Medical Center, Los Angeles, California 90073.
J Clin Invest. 1989 Apr;83(4):1247-52. doi: 10.1172/JCI114008.
Chronic ethanol feeding to rats increases the sinusoidal component of hepatic glutathione (GSH) efflux, despite a lower steady-state GSH pool size. In the present studies, no increase of biliary GSH efflux in vivo was found in chronic ethanol-fed cells. Studies were performed on ethanol-fed and pair-fed cells to identify the kinetic parameters of cellular GSH concentration-dependent efflux. The relationship between cytosolic GSH and the rate of efflux was modeled by the Hill equation, revealing a similar Vmax, 0.22 +/- 0.013 vs. 0.20 +/- 0.014 nmol/min per 10(6) cells for ethanol-fed and pair-fed cells, respectively, whereas the Km was significantly decreased (25.3 +/- 2.3 vs. 33.5 +/- 1.4 nmol/10(6) cells) in ethanol-fed cells. The difference in Km was larger when the data were corrected for the increased water content in ethanol-fed cells. We found a direct correlation between mitochondria and cytosolic GSH, revealing that mitochondria from ethanol-fed cells have less GSH at all cytosolic GSH values. The rate of resynthesis in depleted ethanol-fed cells in the presence of methionine and serine was similar to control cells and gamma-glutamylcysteine synthetase remained unaffected by chronic ethanol. However, the reaccumulation of mitochondrial GSH as the cytosolic pool increased was impaired in the ethanol cells. The earliest time change in GSH regulation was a 50% decrease in the mitochondrial GSH at 2 wk.
给大鼠长期喂食乙醇会增加肝脏谷胱甘肽（GSH）流出的窦状隙成分，尽管稳态GSH池大小较低。在本研究中，未发现长期喂食乙醇的细胞在体内胆汁GSH流出增加。对喂食乙醇和配对喂食的细胞进行了研究，以确定细胞GSH浓度依赖性流出的动力学参数。通过希尔方程对胞质GSH与流出速率之间的关系进行建模，结果显示，喂食乙醇的细胞和配对喂食的细胞的Vmax相似，分别为每10⁶个细胞0.22±0.013和0.20±0.014 nmol/分钟，而喂食乙醇的细胞的Km显著降低（25.3±2.3对33.5±1.4 nmol/10⁶个细胞）。当对喂食乙醇的细胞中增加的水分含量进行数据校正时，Km的差异更大。我们发现线粒体和胞质GSH之间存在直接相关性，表明在所有胞质GSH值下，喂食乙醇的细胞中的线粒体GSH含量都较低。在蛋氨酸和丝氨酸存在的情况下，耗尽的喂食乙醇的细胞中的再合成速率与对照细胞相似，γ-谷氨酰半胱氨酸合成酶不受长期乙醇的影响。然而，在乙醇细胞中，随着胞质池增加，线粒体GSH的再积累受到损害。GSH调节的最早时间变化是在2周时线粒体GSH下降50%。
