The liver is quantitatively the most important site of glutathione synthesis. It plays a central role in providing glutathione for cellular protection against reactive metabolic intermediates and pollutant xenobiotics, and in supplying plasma glutathione for inter-organ glutathione metabolism. Hepatic glutathione turnover is accounted for almost exclusively by the efflux of reduced glutathione (GSH), Of glutathione disulfide (GSSG), and of glutathione conjugates (GSH-thioethers and thioesters) into bile and plasma. Recent studies demonstrate that greater than 50% of the glutathione released by the rat liver is secreted into canalicular bile. However, within the biliary tree, glutathione is extensively degraded by the enzymes gamma-glutamyl transferase and peptidases, so that only a small fraction of the glutathione secreted by liver cells is found in its intact form in excreted bile. Some of glutathione's catabolic products are reabsorbed from bile. The functions of this intrahepatic cycle are unknown. The overall objectives of the proposed studies are to characterize the mechanisms of transport, and the physiological functions f the hepatobiliary metabolism of glutathione. Using purified rat liver cancalicular and basolateral plasma membrane vesicles, the specific aims are to 1) establish the criteria for GSH transport in these two membrane domains, 2) examine whether specific transport processes exist on canalicular membranes for the transport of cysteine-S-conjugates, and 3) evaluate the role of GSH in protecting liver plasma membranes from oxidant challenge. Using the isolated perfused rat liver preparation, 1) identify the factors that modulate the GSH/GSSG ratio in bile, and the rate of glutathione efflux into the perfusate, 2) examine whether the glutathione redox status in bile is a determinant of the permeability of the biliary tree, 3) test the hypothesis that secretion of GSH into bile is a primary driving force in the formation of canalicular bile, and 4) determine the role of membrane potential in the transport of GSH into bile and plasma. The cellular and subcellular distribution of hepatic gamma- glutamyl transferase will be examined immunohistochemically using two heterologous antibodies to the enzyme, under a series of conditions that are known to modulate hepatic gamma-glutamyl transferase activity.
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