Ookhtens M, Lyon I, Fernandez-Checa J, Kaplowitz N
Liver Research Laboratory, Veterans Administration Wadsworth Medical Center, Los Angeles, California 90073.
J Clin Invest. 1988 Aug;82(2):608-16. doi: 10.1172/JCI113639.
Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.
利用离体、原位、单通道灌注大鼠肝脏、新鲜分离的肝细胞培养物以及富含窦状隙膜的囊泡，我们和其他研究人员已证明肝脏谷胱甘肽（GSH）转运（外排）的饱和性。这些观察结果提示存在一种载体机制。我们目前的研究旨在通过证明肝细胞摄取的肝脏特异性配体的竞争性作用，为肝脏GSH外排的载体机制提供进一步证据。在用不同物质灌注肝脏时，我们发现：（a）磺溴酞钠 - GSH（BSP - GSH）对GSH外排具有剂量依赖性且完全可逆的抑制作用，而单独的GSH则无任何作用；（b）牛磺胆酸盐无抑制作用；（c）所研究的所有有机阴离子，即BSP、孟加拉玫瑰红、吲哚菁绿和未结合胆红素（UCB），均表现出强效的、剂量依赖性抑制作用，UCB无毒性作用且抑制作用完全可逆。提高（氯化钴诱导的）肝脏GSH浓度可部分克服UCB的抑制作用。鉴于UCB的生理重要性，我们在UCB循环浓度范围内，对其在分离的肝细胞模型中的抑制动力学进行了详细研究。使用无氯培养基抑制肝细胞摄取UCB的研究表明，UCB对GSH外排的抑制作用显然来自细胞内部。这一点通过以下实验得到证实：只有当胆红素负载细胞清除胆红素（与5%牛血清白蛋白孵育）时，抑制作用才会被克服。使用冈恩大鼠肝细胞以及纯化的胆红素单葡萄糖醛酸酯和双葡萄糖醛酸酯，我们发现UCB及其葡萄糖醛酸酯形式的胆红素均以剂量依赖性方式抑制GSH外排。我们得出结论，有机阴离子虽然通过一种独立于GSH的机制被摄取，但可能从肝细胞内部竞争性抑制GSH外排的载体。
