| Abstract: |
Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominately (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced V(max) for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent K(m) = 0.40 ± 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3/- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATPγS), could also stimulate efflux, but to a lesser extent. Also, ATPγS and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent K(m) = 46 ± 7 μM), but extremely high capacity (106 ± 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP- dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophtalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells. |
| Keywords: |
leukemia; nonhuman; methotrexate; animal cell; mouse; animal; mice; ph; tumor cells, cultured; enzyme activity; mice, inbred c57bl; cell membrane; adenosine triphosphate; verapamil; energy metabolism; biological transport; hydrogen-ion concentration; cell membrane transport; minimum inhibitory concentration; reversed phase high performance liquid chromatography; effusion; temperature dependence; quinidine; mice, inbred dba; leukemia l1210; reserpine; priority journal; article; cholic acid; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; adenosinetriphosphatase; vanadate sodium; osmoregulation; bromsulfophthalein
|
