ATP-DEPENDENT GSH AND GLUTATHIONE S-CONJUGATE TRANSPORT IN SKATE LIVER: ROLE OF AN Mrp FUNCTIONAL HOMOLOGUE James F. Rebbeor1 , Gregory C. Connolly1 , John H. Henson2 , James L. Boyer3 , and Ned Ballatori1 1 Dept. of Environmental Medicine, Univ. of Rochester School Medicine, Rochester, NY 14642 2 Department of Biology, Dickinson College, Carlisle, PA 17013 3 Liver Center, Department of Medicine, Yale Univ. School of Medicine, New Haven, CT 06520 Reduced glutathione (GSH) is a tripeptide that is involved in critical cellular functions, including detoxification of reactive metabolic intermediates and foreign chemicals, and maintenance of the thiol redox status. GSH is synthesized in the cell cytosol, but is degraded in the extracellular space by the ectoproteins γ -glutamyl transpeptidase and dipeptidase. Transport of GSH across the cell membrane is therefore required for normal cellular turnover; however, the transport systems involved have not been identified. Recent studies indicate that cellular GSH efflux is mediated in part by Oatp1, the hepatic basolateral organic solute transporter, by Mrp1, an ATP-dependent plasma membrane transport protein, and by Mrp2, the liver canalicular ATP-dependent organic anion transporter (Ballatori and Rebbeor, Seminars in Liver Dis. 18:377-387, 1998). However, the evidence for the roles of Mrp1 and Mrp2 in GSH efflux is indirect. Studies that have attempted to demonstrate ATP-dependent GSH transport in isolated plasma membrane vesicles have provided negative results. In contrast to these findings in mammalian systems, our studies in membrane vesicles isolated from the yeast S. cerevisiae demonstrated the presence of a low-affinity ATP-dependent GSH transport process that was mediated by Ycf1p, the yeast orthologue of mammalian Mrp (Rebbeor et al., J. Biol. Chem. 273:33449-33454, 1998). Ycf1p shows significant homology with rat Mrp1 (42.6%) and Mrp2 (41.9%), and these three proteins appear to transport similar compounds. Recent studies by Paulusma and coworkers (Biochem. J. 338:393-401, 1999) provide additional evidence for a role of Mrp1 and Mrp2 in GSH efflux. Using MDCKII cells stably expressing human MRP1 and MRP2 these investigators observed a direct correlation between MRP expression and GSH efflux from the cells; however, they were unable to detect ATPdependent GSH transport in isolated membrane vesicles. The reason for the inability to measure ATP-dependent GSH transport in mammalian membrane vesicles is not known, but may be related to the combined effects of a low catalytic efficiency of GSH transport (i.e., a high Km, but only modest Vmax), and a comparatively high nonspecific permeability of inside-out mammalian plasma membrane vesicles, which tends to dissipate solute gradients generated by membrane pumps. Although vesicles that are in the insideout configuration are required to demonstrate ATP-dependent transport, these vesicles may be particularly susceptible to a high nonspecific permeability owing to the unnatural curvature of the membrane bilayer. Mammalian liver plasma membrane vesicles are only able to maintain transmembrane ion or solute gradients for brief periods of time, on the order of minutes. In contrast to mammalian liver vesicles, our recent studies with skate liver plasma membrane vesicles indicate that these vesicles can maintain comparable gradients for several hours (Ballatori et al., Am. J. Physiol. 278:G57-63, 2000), indicating a lower nonspecific permeability. Thus, the present study utilized plasma membrane vesicles isolated from liver of the little skate (Raja erinacea) to examine the hypothesis that the ATP-dependent multidrug resistance-associated proteins can transport GSH. As in mammalian livers, skate livers secrete GSH, glutathione Sconjugates, and other organic anions into bile in relatively high concentrations (Boyer et al., Am. J. Physiol. 230:974-981, 1976; Simmons et al., Biochem. Pharmacol. 42:2221-2228, 1991), indicating the presence of a functional homologue of the Mrp2 transporter.
Skate liver plasma membrane vesicles were isolated by a modification of methods developed by Sellinger et al. (Toxicol. Appl. Pharmacol. 107:369-376, 1991) and Song et al. (J. Cell Biol. 41:124-132, 1969). Vesicles were stored at -80o C in transport buffer containing 10 mM Hepes, pH 7.4, 20 mM KCl, 0.1 mM CaCl2 , and 250 mM sucrose. Transport was measured as uptake of radiolabeled substrate into vesicles collected by rapid filtration on Millipore 0.45 µm filter under vacuum. Skate hepatocytes were isolated as described by Smith et al. (J. Exp. Zool. 241:291-296, 1987), using a two step collagenase perfusion protocol. For immunocytochemical localization of Mrp2, skate hepatocyte clusters were first allowed to adhere onto glass cover slips coated with 1 mg/ml high molecular weight poly-L-lysine. The cells were fixed for 30 min in minus 20o C acetone, rehydrated in phosphate buffered saline and then blocked in phosphate buffered saline plus 1% bovine serum albumin and 0.2% Triton X-100. Cells were then incubated in 1:200 dilution of the EAG-15 anti-Mrp2 primary antibody followed by a 1:400 dilution of goat anti-rabbit IgG conjugated with Alexa 488 fluorophore (Molecular Probes, Eugene, OR). Stained cells were viewed on an Olympus Fluoview laser scanning confocal microscope using a 40X (1.15 NA) water immersion objective lens. Skate liver plasma membranes were subjected to SDS-PAGE electrophoresis and the separated proteins were immunoblotted with two different antibodies raised against rat liver canalicular Mrp2 (EAG-15 and MIII-5). Rat liver canalicular plasma membranes containing the Mrp2 antigen, seen as an immunoreactive band at about 190 kDa, were included as a control. A protein band with a molecular mass of between 170-180 kDa was also detected with both the EAG15 and the MIII-5 antibodies in skate liver plasma membranes, indicating the presence of an Mrp2like protein in skate liver membranes. No signal was detected in membranes isolated from skate muscle or heart. Staining of isolated skate hepatocyte clusters with anti-Mrp2 revealed an apical/pericanalicular staining pattern. Interestingly, this pattern was often discontinuous, suggesting an apparent differential accumulation of the transporter in different domains of the apical membrane. To functionally characterize this putative Mrp2 analogue, we tested for the presence of ATPdependent transport of [3 H]S-dinitrophenyl-glutathione (DNP-SG), a prototypic substrate of Mrp2, in skate liver membrane vesicles. DNP-SG is secreted into skate bile in high concentrations (Simmons et al., Biochem. Pharmacol. 42:2221-2228, 1991), indicating the presence of an active canalicular transport mechanism for this organic anion. Uptake of 10 µM [ 3 H]DNP-SG in skate liver plasma membrane vesicles was measured at 200 C for timed intervals up to 4 hours, with 5 mM MgATP and without ATP. Uptake was mediated by both ATP-dependent and ATP-independent mechanisms in this mixed population of skate liver plasma membranes (i.e., canalicular and sinusoidal membrane domains). The ATP-dependent component constituted about 20-30% of the total uptake. ATP-dependent DNP-SG uptake was of relatively high affinity (Km = 32 ± 9 µM), and was cis inhibited by known substrates of Mrp2 and by GSH, but was minimally affected by substrates of the bile salt transporter (Bsep/Spgp), or substrates of the multidrug resistance (MDR) proteins. Interestingly, ATP-dependent transport of [3 H]S-ethylglutathione and [3 H]GSH was also detected in skate liver plasma membrane vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (Km = 12 ± 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter, but was not effected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for transport of GSH by an ATP-dependent, Mrp-like transporter, and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins. These findings also indicate that skate liver plasma membrane vesicles may be a useful model system for studying this and other ATPdependent hepatic transporters. (Supported by ES03828, ES01247, DK34989, DK25636, DK48823, and by NSF BIR9531348 and ESI9452682).
RESPONSIBLE AUTHOR Ned Ballatori Tel 716-275-0262 Fax 716-256-2591 E-mail
[email protected] SPECIES
Little skate, Raja erinacea
KEY WORDS
Organic anion transporter Mrp2 ATP-dependent
LAB ASSOCIATES (Ned Ballatori, Maria Runnegar, and James L. Boyer): Gregory C. Connolly, Univ. Rochester School of Medicine Benjamin E. Lenth, Wesleyan University Isabel A. Heine, Riverside High School, Durham, NC
1999-2000 PUBLICATIONS: Ballatori, N., D.N. Hager, S. Nundy, D.S. Miller, and J.L. Boyer. Carrier-mediated uptake of lucifer yellow in skate and rat hepatocytes: a fluid-phase marker revisited. Am. J. Physiol. 277:G896-G904, 1999. Runnegar, M., D.J. Seward, N. Ballatori, J.M. Crawford, and J.L. Boyer. Hepatic toxicity and persistance of ser/thr protein phosphatase inhibition by microcystin in the little skate Raja erinacea. Toxicol. Appl. Pharmacol. 161:40-49, 1999. Ballatori, N., J.F. Rebbeor, G.C. Connolly, D.J. Seward, B.E. Lenth, J.H. Henson, P. Sundaram, and J.L. Boyer. Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump. Am. J. Physiol. 278:G57-G63, 2000.
