Functions of the Hepatic Gamma-Glutamyl Cycle

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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.