A continuous fluorescence assay for the study of P-glycoprotein-mediated drug efflux using inside-out membrane vesicles

Abstract

A fluorimetric procedure for assaying the transport activity of P-glycoprotein (P-gp) using a membrane vesicle model has been developed. In this assay methylene blue is incorporated into inside-out vesicles prepared from human acute lymphoblastic leukemic cells resistant to 100 ng z ml21 vinblastine (VBL100) and their sensitive controls. The fluorescence of a fluorescent derivative of colchicine (fluorescein¿colchicine) is quenched as the probe is transported across the vesicle membrane. The fluorescein¿colchicine transport was found to be dependent on the presence of P-glycoprotein, required ATP, and was inhibited by vanadate and the reversal agent, verapamil, in a dose-dependent manner. Furthermore, the transport was competed against by the P-gp substrates, vinblastine and methotrexate. The transport of fluorescein¿colchicine by P-gp was found to be cooperative (n 5 1.23).