Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

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Abstract

The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions.

Introduction

Reduced glutathione (GSH) plays a central role in a multitude of biochemical processes, and disturbances in its homeostasis are implicated in the etiology and progression of a number of diseases. GSH is required for proper protein and DNA synthesis, cell cycle regulation, thermotolerance, exocrine secretion, maintenance, and regulation of the thiol-redox status of the cell, protection against oxidative damage, detoxification of endogenous and exogenous reactive metals and electrophiles, biosynthesis of mercapturic acids (S-substituted N-acetyl-l-cysteines), and storage and transport of cysteine (DeLeve and Kaplowitz, 1990, Meister, 1984, Meister and Anderson, 1983, Wang and Ballatori, 1998). Additional important roles for this tripeptide include the regulation of gene expression, apoptosis, and membrane transport of both endogenous and exogenous molecules (Hammond et al., 2001).

The synthesis and catabolism of GSH and its adducts occurs by a regulated series of enzymatic and plasma membrane transport steps that are collectively referred to as the γ-glutamyl cycle (Meister and Anderson, 1983, Meister and Tate, 1976). GSH is synthesized in every cell of the body, but the liver is quantitatively the major site of synthesis (DeLeve and Kaplowitz, 1990, Hahn et al., 1978, Lauterburg et al., 1984). GSH is synthesized intracellularly from its precursor amino acids by the ATP-requiring cytosolic enzymes γ-glutamylcysteine synthetase and GSH synthetase. Within the cell, it exists mainly (>98%) in the thiol-reduced form (GSH), but some are also present in the thiol-oxidized (GSSG), thioether, mercaptide, or other thioester forms (glutathione S-conjugates). After its synthesis, GSH is delivered to other intracellular compartments, including mitochondria and endoplasmic reticulum, and to the extracellular space (e.g., blood plasma and bile) for utilization by other cells and tissues.

In contrast to GSH synthesis which occurs intracellularly, GSH degradation occurs exclusively in the extracellular space, and only on the surface of cells that express the ectoenzyme γ-glutamyl transpeptidase (also called γ-glutamyl transferase or γGT). γ-Glutamyl transpeptidase, which is abundant on the apical surface of most transporting epithelia, including liver canalicular and bile ductular membranes, is the only enzyme that can initiate catabolism of GSH, glutathione S-conjugates, and glutathione-complexes under physiological conditions. Because γ-glutamyl transpeptidase is a plasma membrane-bound enzyme with its active site on the extracellular surface of the membrane, export of GSH and its adducts into the extracellular space is the initial, and presumably regulated step in their turnover in all mammalian cells.

Despite the importance of this transport step, relatively little is known at the molecular level about GSH transporters. The paucity of information is explained in large part by a number of practical and theoretical limitations that have hampered the functional and molecular characterization of GSH transporters. The first factor is the relative difficulty of the study of efflux transporters when compared to the study of uptake transporters. To date, we know relatively little about the proteins that mediate export out of cells (other than the ABC proteins), whereas we know a great deal about uptake transporters (Borst and Elferink, 2002, Gao and Meier, 2001, Meier and Stieger, 2002, Sekine et al., 2000, Suzuki and Sugiyama, 2000). The major limitations of efflux studies are that it is often impossible to determine intracellular concentrations (or more precisely, chemical activities) of substrates and inhibitors, and that it is difficult to load or alter intracellular substrate or inhibitor concentrations without affecting other cellular functions. These problems are particularly acute in the characterization of GSH transport, given the many roles that this tripeptide plays in cellular functions. Thus, it is difficult to manipulate GSH levels without indirectly affecting a myriad of other biochemical pathways and physiological functions that are regulated by this tripeptide. The use of isolated plasma membrane vesicles overcomes some of the limitations; however, directionality of transport is lost for all secondary-active transporters when using vesicle systems. Moreover, membrane vesicle preparations usually contain both inside-out and right side-out vesicles, further confounding interpretation of results.

Second, GSH transporters exhibit low catalytic efficiency (Ballatori and Dutczak, 1994, Paulusma et al., 1999, Rebbeor et al., 1998a, Rebbeor et al., 1998b, Rebbeor et al., 2000, Rebbeor et al., 2002). That is, the apparent affinity values (Km values) for GSH exporters are relatively high and the transport velocities (Vmax values) are only moderate, leading to a low Vmax/Km ratio, a measure of catalytic efficiency. The high Km values are not unexpected given that GSH is present in high concentrations within cells (1–10 mM), but they pose a severe problem in measuring transport rates in intact cells or in membrane vesicles.

Third, is the fact that all cells have endogenous GSH export mechanisms (Meister and Anderson, 1983). This high background rate of GSH transport is a major confounding variable in GSH transport measurements. The ubiquitous nature of GSH transport also severely limits the choice of cells with which this process can be studied. For example, the possibility of using Xenopus laevis oocytes for expression cloning of GSH transporters is essentially precluded by the high endogenous GSH efflux rates observed in these cells (Ballatori et al., 1996).

Fourth, GSH is chemically reactive and is present in several chemical forms (glutathione-thioethers, -thioesters, and -mercaptides). GSH participates in nucleophilic displacement reactions, in thiol–redox reactions, and forms coordinate-covalent adducts with several transition metals (Ballatori, 1994, Ballatori, 2002). These adducts can form non-enzymatically, and many of them are chemically unstable, present in low concentrations, and difficult to detect and quantify. Because GSH adducts are generally transported across cell membranes more efficiently than the parent GSH molecule (via the MRP proteins), this may give misleading results. In addition to the sulfhydryl-dependent reactions, GSH and GSH-containing molecules are also subject to degradation by the ectoenzyme γ-glutamyl transpeptidase. Catabolism of GSH and GSH-containing molecules can usually be minimized by the use of acivicin, a γ-glutamyl transpeptidase inhibitor; however, the extent of inhibition is never complete and the residual GSH catabolism may also confound interpretation of experimental results.

Fifth, GSH transporters are present both on the plasma membrane and on intracellular membranes. Thus, in studies with subcellular membrane fractions, these intracellular GSH transporters may contribute to the overall transport rate. Moreover, as indicated above, membrane vesicle preparations generally have both inside-out and right side-out vesicles, which can make the results difficult to interpret. The inability of many researchers to demonstrate ATP-dependent GSH transport using liver, kidney, or intestinal plasma membrane vesicles has formed the basis for much of the skepticism over whether the MRP proteins can transport GSH. However, Rebbeor et al. (2002) recently discovered that dithiothreitol and other reducing agents that are normally added to membrane vesicle incubation solutions in order to keep GSH in the reduced state, actually inhibit GSH transport. As the concentration of these reducing agents was lowered, direct transport of GSH on Mrp2 was demonstrated (Rebbeor et al., 2002).

Sixth, at present, there are no specific GSH transport inhibitors. The only inhibitors that are available are relatively non-specific organic anion transport inhibitors (Lee et al., 1997). As such, it is impossible to selectively inhibit GSH transport in any tissue or cell.

Finally, the search for GSH transporters was significantly hampered by the publication of two papers that claimed to have identified cDNA clones for low-affinity canalicular (RcGshT) and sinusoidal (RsGshT) GSH transporters, and that claimed to have functionally characterized these transporters (Yi et al., 1994, Yi et al., 1995). However, studies in our laboratory demonstrated that these putative gene products not only have no GSH transport activity, but are not even of mammalian origin (Li et al., 1997). Xenopus laevis oocytes injected with either rat liver mRNA, the cRNA for RcGshT, or the cRNA for RsGshT did not transport GSH at a higher rate than water-injected oocytes, when measured either as [3H]GSH uptake or efflux, at low or high GSH concentrations, or in the presence or absence of acivicin to inhibit γ-glutamyl transpeptidase activity (Li et al., 1997). Sequencing of RcGshT indicated that it is nearly identical to a sequence found in the Escherichia coli K-12 genome, suggesting that RcGshT may be a cloning artifact (Li et al., 1997). Similarly, the published RsGshT cDNA sequence also has high sequence homology with E. coli K-12, although with a different region of its genome (Li et al., 1997), indicating that RsGshT is also a cloning artifact.

Despite these difficulties in measuring GSH export, two transporter families in mammals have now been implicated in GSH efflux, namely the MRP/CFTR family and the OATP family, as well as three yeast proteins (Fig. 1) (Ballatori and Rebbeor, 1998). However, it is likely that additional GSH transport systems exist but have not yet been identified at the molecular level.

Section snippets

The multidrug resistance-associated protein (MRP) family

The MRP/CFTR proteins are in family C of the ABC superfamily (ABCC). This family includes a total of nine human MRP genes (MRP1 to MRP9), the gene defective in cystic fibrosis (CFTR), and the two sulfonylurea receptor genes (SUR1, SUR2). The MRP proteins share a topology that is common to the ABC superfamily where there are two membrane spanning domains (MSD1 and MSD2), each containing six transmembrane α-helices (Fig. 2) (Bakos et al., 1996, Higgins, 1992, Kast and Gros, 1997, Kast and Gros,

The Organic Anion-transporting Polypeptide (Oatp/OATP) family

The OATP family of transporters function independently of ATP and sodium gradients and were originally characterized as uptake transporters, although some may function primarily for efflux from the cell (for review see Hagenbuch and Meier, 2003). Two members of the OATP family have been shown to accept GSH as a substrate, namely rat Oatp1 (Li et al., 1998) and rat Oatp2 (Li et al., 2000). Li et al. (1998) demonstrated that Oatp1 can accept GSH as a substrate and that Oatp1 is a GSH/solute

Summary

Despite the many difficulties in the study of GSH transport, two mammalian protein families, MRP/CFTR and OATP, have now been identified as putative GSH transporters. These include MRP1, MRP2, MRP4, MRP5, CFTR, rat Oatp1, and rat Oatp2. In addition, three GSH transport proteins, Hgtp1, Ycf1p, and Bpt1p, have been identified in yeast. GSH also has regulatory affects on transporters as indicated both by the co-transport of GSH and certain substrates on MRP1, MRP2, and MRP4, the ability of GSH to

Acknowledgments

This work was supported in part by the National Institute of Health Grants ES06484 and DK48823, NIEHS Center Grant ES01247, and Toxicology Training Program Grant ES07026.

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