Fernández-Checa J C, Takikawa H, Horie T, Ookhtens M, Kaplowitz N
Division of Gastrointestinal and Liver Diseases, School of Medicine, University of Southern California, Los Angeles 90033.
J Biol Chem. 1992 Jan 25;267(3):1667-73.
We have characterized the transport of GSH and the mechanism for impaired GSH transport in mutant Eisai hyperbilirubinemic rats (EHBR) using isolated canalicular membrane-enriched vesicles (cLPM). In control animals, the transport of GSH is an electrogenic process and is trans-stimulated by preloading the vesicles with GSH and is not enhanced in the presence of ATP. GSH transport in cLPM is saturable with a single component having a Km of approximately 16 mM and a Vmax of 6.7 nmol/mg/15 s. EHBR is a Sprague-Dawley rat with hyperbilirubinemia due to impaired bile secretion of organic anions by the ATP-dependent organic anion/GSH-conjugate transporter. In cLPM from EHBR we confirmed the defective stimulation by ATP of the transport of LTC4 and GSSG. In the mutant cLPM, the characteristics and kinetics of GSH transport were the same as in the controls. 2,4-(dinitrophenyl)-glutathione (DNP-GSH), which is a substrate for the ATP-dependent canalicular organic anion carrier, in the absence of ATP, cis-inhibited the transport of GSH into cLPM vesicles; however, when the vesicles were preloaded with DNP-GSH, there was a dose-dependent trans-stimulation of GSH transport. In contrast, in the presence of ATP, DNP-GSH enhanced GSH transport in cLPM vesicles; at 0.25 mM DNP-GSH, a concentration which did not cis-inhibit GSH, addition of ATP resulted in accelerated GSH transport; at 1.0 mM DNP-GSH, cis-inhibition was completely reversed by the addition of ATP despite a negligible fall in the medium DNP-GSH. Interestingly, sulfobromophthalein-glutathione (BSP-GSH) neither cis-inhibited nor trans-stimulated GSH transport in cLPM. This contrasts with bLPM where BSP-GSH interacts with the GSH carrier. Therefore, GSH is transported into bile by a multispecific low affinity electrogenic carrier which is distinct from the multispecific high affinity ATP-driven organic anion transporter. Although both carriers have overlapping specificities, BSP-GSH and GSH are uniquely specific for only one of the carriers. The near absence of GSH in the bile of mutant rats can be best explained as a secondary defect due to cis-inhibition from retained substrates for the defective carrier and/or loss of trans-stimulation by these same substrates which normally are concentratively transported into the bile. Other possibilities such as change in GSH carrier activity upon isolation or loss of a negative protein regulator during membrane isolation, although theoretical alternatives are less easily reconciled with the defect in the ATP-driven organic anion transporter.
我们利用分离的富含胆小管膜的囊泡（cLPM），对谷胱甘肽（GSH）的转运以及突变型卫材高胆红素血症大鼠（EHBR）中GSH转运受损的机制进行了表征。在对照动物中，GSH的转运是一个电生成过程，通过向囊泡中预加载GSH可对其进行反式刺激，且在ATP存在时不会增强。cLPM中GSH的转运具有饱和性，由单一成分介导，其Km约为16 mM，Vmax为6.7 nmol/mg/15 s。EHBR是一种斯普拉格-道利大鼠，由于ATP依赖性有机阴离子/GSH共轭转运体对有机阴离子的胆汁分泌受损而患有高胆红素血症。在来自EHBR的cLPM中，我们证实了ATP对LTC4和谷胱甘肽二硫化物（GSSG）转运的刺激存在缺陷。在突变型cLPM中，GSH转运的特征和动力学与对照相同。2,4-二硝基苯基谷胱甘肽（DNP-GSH）是ATP依赖性胆小管有机阴离子载体的底物，在无ATP时，顺式抑制GSH进入cLPM囊泡的转运；然而，当囊泡用DNP-GSH预加载时，存在剂量依赖性的GSH转运反式刺激。相反，在ATP存在时，DNP-GSH增强了cLPM囊泡中GSH的转运；在0.25 mM DNP-GSH（该浓度不会顺式抑制GSH）时，添加ATP导致GSH转运加速；在1.0 mM DNP-GSH时，尽管培养基中DNP-GSH的下降可忽略不计，但添加ATP可完全逆转顺式抑制。有趣的是，磺溴酞谷胱甘肽（BSP-GSH）在cLPM中既不产生顺式抑制也不产生反式刺激GSH转运。这与基底外侧质膜（bLPM）形成对比，在bLPM中BSP-GSH与GSH载体相互作用。因此，GSH通过一种多特异性低亲和力电生成载体转运到胆汁中，该载体不同于多特异性高亲和力ATP驱动的有机阴离子转运体。尽管两种载体具有重叠的特异性，但BSP-GSH和GSH仅对其中一种载体具有独特的特异性。突变大鼠胆汁中几乎不存在GSH，这最好解释为由于缺陷载体的保留底物的顺式抑制和/或这些相同底物的反式刺激丧失导致的继发性缺陷，这些底物通常被浓缩转运到胆汁中。其他可能性，如分离后GSH载体活性的变化或膜分离过程中负性蛋白质调节剂的丧失，尽管从理论上说是其他选择，但不太容易与ATP驱动的有机阴离子转运体的缺陷相协调。
