Glutathione (GSH) plays a critical role in cellular detoxification in the liver and other organs. Sinusoidal export of hepatic GSH is a major determinant of the turnover of hepatic GSH and its interorgan homeostasis. The efflux of GSH, characterized in the perfused rat liver and isolated rat hepatocytes, and its uptake in sinusoidal membrane vesicles, exhibit features of carrier-mediated transport. Our purpose is to better understand the mechanism, regulation and significance of GSH transport in the liver. We wish to define if GSH transport is: facilitative or active; electrogenic or electroneutral; and, mediated by a uniport symport or antiport.
The specific aims are: (1) delineation of the kinetics, driving forces, and regulation of GSH efflux in rat hepatocytes. We will determine the kinetics of efflux in relation to the free cytosolic GSH concentration and examine GSH uptake in depolarized hepatocytes; we will assess the role of membrane potential and intracellular pH by ion substitutions, the inhibition of efflux by K+ channel blockers, and the effect of ATP depletion on the driving forces for GSH efflux. The hormonal and intracellular messenger-mediator-dependent regulation of GSH transport will be determined and related to their effects on the driving forces for GSH transport; (2) characterization of the driving forces and mechanism of GSH transport in sinusoidal membrane vesicles. We will examine the electrogenic nature of GSH transport by the relationship between GSH transport and proton or potassium conductance and the ability of GSH to generate a membrane potential, using pH and voltage sensitive fluorescent dyes; we will also assess the ATP dependency of GSH uptake or efflux from vesicles; (3) determination of the mechanism of inhibition of GSH transport by organic anions in perfused liver, isolated hepatocytes and sinusoidal membrane vesicles. We will characterize inhibition of GSH transport by bilirubin, the molecular form of bilirubin which inhibits transport and determine if GSH and organic anions share a carrier for efflux; (4) Identification and purification of the GSH transport protein and its relationship to the organic anion transporter. We will use affinity chromatography, photoaffinity labeling and/or labeling with N-ethylmaleimide to identify and purify the sinusoidal membrane protein which binds GSH. Understanding of the process of sinusoidal efflux of GSH will permit future determination of the influence of liver disease on the homeostasis of this vital substance.
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