Gary B. Henderson

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 Cells.

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.

Separation and inhibitor specificity of a second unidirectional efflux route for methotrexate in L1210 cells.

L1210 cells mediate the unidirectional and energy-dependent efflux of methotrexate. Efflux occurs primarily via a system which has a high sensitivity to prostaglandin A1, vincristine, reserpine, verapamil, and bromosulfophthalein, but evidence has also been obtained for a second efflux component with a lower response to these inhibitors. Pretreatment of L1210 cells with low concentrations of vincristine reduces methotrexate efflux by three fold and uncovers a second efflux component with an inhibitor specificity which is distinctly different from the primary efflux route. Vincristine treatment increased by 8-20-fold the concentration required for half-maximal efflux inhibition by prostaglandin A1, reserpine, bromosulfophthalein, and verapamil but had no effect on inhibition by probenecid, quinidine, or carbonylcyanide m-chlorophenylhydrazone. A selective block in the primary efflux system and retention of the second component was also achieved in cells exposed to low concentrations of prostaglandin A1 or bromosulfophthalein. These results support prior conclusions that L1210 cells contain both a primary and secondary unidirectional efflux route for methotrexate. The second system has been difficult to detect and quantitate since it comprises only 25% of total unidirectional efflux and shows a relatively low response to various efflux inhibitors.

Altered expression of unidirectional extrusion routes for methotrexate and cholate in an efflux variant of L1210 cells

The specificity and function of two unidirectional anion-efflux pumps in mouse L1210 cells were evaluated using a variant cell line selected for growth in the presence of cholate and bromosulfophthalein. Transport analysis revealed that cholate efflux in the variant L1210/C7 cell line had declined 8-fold, due to the loss of a bromosulfophthalein-sensitive efflux system, the major extrusion route for cholate in parental cells. Efflux measurements showed further that a bromosulfophthalein-sensitive efflux system for methotrexate was also absent in L1210/C7 cells. Total unidirectional efflux of methotrexate, however, was similar in the variant and parental cells, since the loss in the bromosulfophthalein-sensitive system was compensated by a rise in a second probenecid-sensitive route. The latter was identified from inhibitor studies to be the same system which acts as a minor efflux route for methotrexate in parental cells. These results support the hypothesis that L1210 cells contain a bromosulfophthalein-sensitive efflux system which mediates the unidirectional extrusion of either methotrexate or cholate, and a second probenecid-sensitive route which differs from the bromosulfophthalein-sensitive system in inhibitor specificity and also in its ability to transport methotrexate but not cholate.