Identification of the bromosulfophthalein-sensitive efflux route for methotrexate as the site of action of vincristine in the vincristine-dependent enhancement of methotrexate uptake in L1210 cells.
The mechanism by which vincristine enhances the uptake of methotrexate in leukemic L1210 mouse cells has been investigated. Methotrexate uptake after 30 min at 37 degrees C increased 44% relative to untreated controls in cells suspended in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/bicarbonate-buffered saline medium containing 20 microM vincristine. This stimulation was half-maximal at a vincristine concentration of 4 microM. An enhancement of methotrexate uptake by vincristine was also observed in the presence of glucose but a reversal could be achieved by prior treatment of the cells with taxol, a drug which prevents microtubule disassembly by vincristine. When the effect of vincristine was determined on the individual influx and efflux components for methotrexate, the increased uptake of methotrexate correlated with the inhibition of the unidirectional efflux route for methotrexate that is sensitive to bromosulfophthalein. Inhibition of this route was half-maximal at 3 microM vincristine and it exceeded 90% at high concentrations of the inhibitor. Transport via the bidirectional exchange carrier for methotrexate was not affected by vincristine, while the unidirectional efflux route sensitive to probenecid was inhibited by vincristine but only at concentrations 10-fold higher than required to inhibit the bromosulfophthalein-sensitive route. Vincristine did not increase methotrexate uptake in CCRF-CEM lymphoblasts, a human cell line which contains much lower levels of bromosulfophthalein-sensitive efflux route for methotrexate. Concentrations of vincristine which inhibited the bromosulfophthalein-sensitive efflux route of L1210 cells by 80-90% had little or no effect on the intracellular pH or on intracellular levels of ATP, GTP, cyclic AMP, K+, or oxidized glutathione. A significant increase was observed in the cellular uptake of tetraphenylphosphonium ions, suggesting that vincristine causes a hyperpolarization of the plasma membrane.
Henderson
G B
GB
Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037.
Tsuji
J M
JM
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects; D007939:Leukemia L1210 / Q000378:metabolism*; D008727:Methotrexate / Q000493:pharmacokinetics*; D009861:Onium Compounds / Q000493:pharmacokinetics; D009943:Organophosphorus Compounds / Q000493:pharmacokinetics; D013448:Sulfobromophthalein / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000494:pharmacology*
D009861:Onium Compounds; D009943:Organophosphorus Compounds; D013448:Sulfobromophthalein; D014750:Vincristine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; C013289:tetraphenylphosphonium; D008727:Methotrexate