García-Ruiz C, Fernández-Checa J C, Kaplowitz N
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033.
J Biol Chem. 1992 Nov 5;267(31):22256-64.
We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.
我们测定了细胞外还原型谷胱甘肽（GSH）对新鲜分离的大鼠肝细胞内源性标记GSH流出速率的反式作用。培养基中存在GSH（10 mM）显著刺激了[35S]GSH的流出分数速率，从5.2%/15分钟增加到12.6%/15分钟（p < 0.01）。这种作用具有浓度依赖性，呈S型动力学（半数有效浓度为0.32 mM），并且在去除外部GSH后是可逆的。用5 mM氧化型谷胱甘肽（GSSG）和眼酸也观察到了反式刺激（逆向转运）（[35S]GSH流出分数：分别在15分钟内为13.4%±4.1和8.8%±2.3，而对照组为4.7±2.5/15分钟）。Krebs缓冲液中的溴磺酞谷胱甘肽（BSP - GSH，5 mM）抑制了[35S]GSH的流出分数（1.1%/15分钟），而在无Cl(-)缓冲液中，与对照组相比，GSH流出受到刺激（14.2%/15分钟）。反式刺激与氯离子无关。BSP - GSH顺式抑制并反式刺激基底外侧富集膜囊泡（bLPM）中GSH转运的初始速率，但对胆小管富集膜囊泡（cLPM）无此作用。γ - 谷氨酰化合物也顺式抑制并反式刺激bLPM囊泡中的GSH转运。用10 mM [35S]GSH孵育的GSH耗竭肝细胞比补充细胞积累了更多的GSH，但补充细胞中放射性摄取的初始速率更快。相反，用示踪剂或50 μM [35S]GSH孵育的补充肝细胞不摄取GSH。因此，肝血窦膜GSH转运体对GSH摄取和流出的反式刺激均表现出具有S型特征的低亲和力动力学，这解释了在低生理细胞外浓度下GSH缺乏摄取的现象。所以，我们的发现支持并解释了广泛认可的观点，即在生理条件下GSH转运是单向的。然而，GSH的流出也可能以交换有机阴离子和γ - 谷氨酰化合物摄取的方式发生。
