title: Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 Cells.
abstract: Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 muM [3H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (Ki=2.5 muM, 4-biphenylacetic acid (Ki=5.3 muM), and flurbiprofen (Ki=5.2 muM, whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (Ki=0.08 muM), ethacrynic acid (Ki=0.52 muM, and 1-chloro-2,4-dinitrobenzene (Ki=0.77 muM). Bilirubin ditaurate was a potent inhibitor of both transport components (Ki=1.5 and 0.17 muM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 muM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave Km values of 170 and 250 muM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: M
authors.initials: M
authors.lastname: Saxena
authors.forename: G B
authors.initials: GB
authors.lastname: Henderson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017136:Ion Transport / Q000502:physiology; D007700:Kinetics; D007942:Leukemia, Experimental / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice
chemical_list: D000838:Anions; D000255:Adenosine Triphosphate; D008727:Methotrexate