Functions of the Hepatic Gamma-Glutamyl Cycle

描述(由申请人提供)：还原型谷胱甘肽(GSH)在多种生化过程中起关键作用，其稳态失调与许多疾病的病因和进展有关。这种三肽在所有哺乳动物细胞中周转的第一步是它穿过质膜进入细胞外空间；然而，介导GSH外流的运输系统仍然没有得到很好的确定。在肝脏中，GSH合成的主要部位，GSH被高速释放到血浆和胆汁中。GSH转运到胆汁中是胆汁分泌的驱动力，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发挥着重要作用。谷胱甘肽也会通过肝窦膜释放到血浆中，然后输送到其他组织。我们最近的研究已经确定了OATP和MRP家族中的一些成员在细胞中GSH输出中的关键作用。这项研究的总体目标是描述谷胱甘肽转运的机制，并深入了解这些转运过程的生理意义。我们的具体目标是：(1)建立非洲爪哇卵母细胞表达的大鼠Oatp1转运GSH的动力学和特异性，并检测人类主要的正弦有机溶质转运体OATP-C是否也介导GSH/有机溶质交换。(2)研究大鼠MRP2转运GSH的机制，并与其酵母同源物YCF1P转运GSH的机制进行比较，验证硫代尿酸(N-乙酰半胱氨酸S结合物)是MRP2转运底物的假设。(3)检测细胞中观察到的GSH外流是否由OATP或MRP蛋白或其他GSH输出机制介导；(4)通过在酿酒酵母中的表达克隆来鉴定更多的双向GSH转运体。由于谷胱甘肽跨细胞膜的转运是其动态平衡的关键步骤，并且与其生物学功能密切相关，因此，拟议的研究将为预防和治疗氧化剂和活性亲电化学物质造成的细胞和组织损伤提供关键信息。

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.