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 predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic
buffer conditions, in these inside-out membrane vesicles was temperature and
ATP dependent (apparent Km = 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) (
ATP gamma S), could also stimulate efflux, but to a lesser extent. Also,
ATP gamma S 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 Km = 46 +/- 7 microM), 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
bromosulfophthalein-sensitive,
ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.