Transport of reduced glutathione (GSH) into the extracellular space is the initial and perhaps limiting step in the turnover of the tripeptide in all mammalian cells; however, the transport system or systems that mediate GSH efflux remain obscure. In the liver, a major site of GSH synthesis, GSH is released at high rates into both blood plasma and bile. Nearly half of the GSH released by rat hepatocytes is transported across the canalicular membrane into bile, with biliary GSH concentrations reaching 8 to 10 mM. 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. The remainder of the GSH is released across the sinusoidal membrane into blood plasma for delivery to other tissues. The molecular mechanisms of GSH efflux have not been identified for any cell type, although recent studies provide important insight into possible mechanisms. In particular, oatp1, the sinusoidal organic solute transporter, was recently shown to function as a GSH/organic solute exchanger. This finding identifies both the energy coupling mechanism for oatp1 and a pathway for GSH release into blood plasma. However, oatp probably only accounts for a fraction of the total GSH released into sinusoidal blood. A candidate canalicular GSH transport mechanism has also recently been described. Canalicular GSH efflux may be mediated by the adenosine 5'-triphosphate (ATP)-dependent organic solute transport protein MRP2 (also termed cMOAT or cMRP). MRP2 is a member of the multidrug resistance-associated family of proteins (MRP) whose preferred substrates include glutathione S-conjugates. Recent studies suggest that MRP can also transport GSH itself. This report summarizes the evidence documenting a role for oatp1 and MRP2 in GSH efflux from hepatocytes, and their possible contribution to hepatic GSH homeostasis.

The transport systems involved in the export of cellular reduced glutathione (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including MRP1 and MRP2. The present study examined the hypothesis that the yeast orthologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [3 H]GSH transport was measured in vacuolar membrane vesicles isolated from a control strain of Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that lacks a functional Ycf1p, and in DTY167 transformed with a 2-m plasmid vector containing YCF1. GSH transport in control vacuolar membrane vesicles was mediated largely by an ATP-dependent, low affinity pathway (K m ‫؍‬ 15 ؎ 4 mM). ATP-dependent [ 3 H]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 and MRP2, but was minimally affected by membrane potential or pH gradient uncouplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a functional Ycf1p but was restored to near wild-type levels in the DTY167 strain transformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the other hand, expression and functional activity of a bile acid transporter, Bat1p, and of the V-type ATPase were similar in all three yeast strains. These results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functional homology between Ycf1p and MRP1 and MRP2, these data support the hypothesis that GSH efflux from mammalian cells is mediated by these membrane proteins.Turnover of cellular GSH is initiated by export across the plasma membrane for delivery to the catabolic ectoenzymes ␥-glutamyl transpeptidase and dipeptidase (1, 2). Intracellular GSH concentrations are normally 1-10 mM, whereas blood plasma concentrations are 1-20 M. The mechanisms of GSH transport are not well understood, although recent studies provide some insight (3-7). One mechanism of GSH release involves the organic anion-transporting polypeptide, Oatp1 (5). Li and co-workers (5) recently demonstrated that Oatp1 functions as a GSH/organic solute exchanger. Cellular release of GSH down its large electrochemical gradient apparently energizes uptake of organic solutes via the Oatp1 transporter. Oatp1 is a member of a growing family of organic solute transporters whose driving force has not been identified, although the work of Li et al. (5) suggests that GSH exchange may be both a common mechanism of energy coupling and a mechanism of GSH release from cells.An additional mechanism of GSH release may involve the MRP 1 family of membrane transporters (6). Studies in intact cells indicate that GSH is released by ATP-dependent mechanisms, including possibly the MRP1 and MRP2 proteins; however, none of these studies have directly documented AT...

Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19+/-5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2, 4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18+/-2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.

Drug interactions with P-glycoprotein (Pgp) were quantitatively assessed using ATPase assay. Two experimental systems were used, (i) plasma membranes isolated from a multidrug-resistant cell line, which contained 30% Pgp as fraction of total membrane protein, and (ii) purified reconstituted Pgp. The cardioactive drugs verapamil, quinidine, diltiazem, nifedipine, and a series of digitalis analogs, interacted directly with Pgp as shown on ATPase in both systems. Apparent affinities of drug binding were calculated. Direct competition was shown between digitoxin and verapamil. Drug-drug interaction in vivo at the level of Pgp is expected from the results. This approach seems well-suited for empirical determination of drug interactions with Pgp, and prediction of drug-drug interactions.