Functions of the Hepatic Gamma-Glutamyl Cycle

描述（由申请人提供）：还原型谷胱甘肽（GSH）在许多生化过程中起着关键作用，其体内平衡的紊乱与许多疾病的病因学和进展有关。在所有哺乳动物细胞中，这种三肽周转的初始步骤是其跨质膜转运到细胞外空间;然而，介导GSH外排的转运系统仍然不清楚。在肝脏中，GSH合成的主要部位，GSH以高速率释放到血浆和胆汁中。GSH转运到胆汁中作为胆汁分泌的驱动力，并在药物、金属和其他内源性和外源性活性化合物的肝脏解毒中起重要作用。GSH也通过窦状膜释放到血浆中，用于递送到其他组织。我们最近的研究已经确定了一个关键作用的一些成员的Oatp和Mrp家族的转运蛋白GSH出口从细胞。拟议的研究的总体目标是表征GSH运输的机制，并深入了解这些运输过程的生理意义。我们的具体目标是：（1）建立在非洲爪蟾卵母细胞中表达的大鼠Oatp 1上GSH转运的动力学和特异性，并测试人OATP-C（一种主要的人正弦有机溶质转运蛋白）是否也介导GSH/有机溶质交换。(2)表征大鼠Mrp 2的GSH转运机制，将该机制与Mrp 2的酵母直向同源物Ycf 1 p的GSH转运机制进行比较，并检验巯基尿酸（N-乙酰半胱氨酸S-缀合物）是Mrp 2底物的假设。(3)测试在经历细胞凋亡的细胞中观察到的GSH流出是否由Oatp或Mrp蛋白介导，或由其他GSH输出机制介导;和（4）通过在S.啤酒。由于GSH跨细胞膜转运是其体内平衡的关键步骤，并与其生物学功能密切相关，因此拟议的研究应提供预防和治疗氧化剂和反应性亲电化学品引起的细胞和组织损伤的关键信息。

DESCRIPTION (provided by applicant): Reduced glutathione (GSH) plays a critical role in a multitude of biochemical processes, and disturbances in its homeostasis are implicated in the etiology and progression of a number of diseases. The initial step in the turnover of this tripeptide in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the transport systems that mediate GSH efflux remain poorly defined. In the liver, a major site of GSH synthesis, GSH is released at high rates into both blood plasma and bile. GSH transport into bile functions as a driving force for bile secretion and plays an important role in hepatic detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin. GSH is also released across the sinusoidal membrane into blood plasma for delivery to other tissues. Our recent studies have identified a key role for some members of the Oatp and Mrp families of transport proteins in GSH export from cells. The overall goals of the proposed studies are to characterize the mechanisms of GSH transport, and to gain insight into the physiological significance of these transport processes. Our specific aims are: (1) Establish the kinetics and specificity of GSH transport on rat Oatp1 expressed in Xenopus laevis oocytes, and test whether human OATP-C, a major human sinusoidal organic solute transporter, also mediates GSH/organic solute exchange. (2) Characterize the mechanism of GSH transport by rat Mrp2, compare this mechanism with that for GSH transport by Ycf1p, the yeast orthologue of Mrp2, and test the hypothesis that mercapturic acids (N-acetylcysteine S-conjugates) are substrates for Mrp2. (3) Test whether the GSH efflux that is observed in cells undergoing apoptosis is mediated by the Oatp or Mrp proteins, or by other GSH export mechanisms; and (4) Identify additional bi-directional GSH transporters by expression cloning in S. cerevisiae. Because GSH transport across cell membranes is a key step in its homeostasis and is intimately linked to its biological functions, the proposed studies should provide information critical for the prevention and treatment of cell and tissue injury produced by oxidants and reactive electrophilic chemicals.