Use of membrane vesicles to investigate drug interactions with transporterproteins, P-glycoprotein and multidrug resistance-associated protein

Background: The ATP-dependent drug transporter proteins, P-glycoprotein (Pe p) and the multidrug resistance-associated protein (MRP) are known to be in volved in drug efflux that reduces drug accumulation and so renders tumor c ells resistant to the cytotoxic effects of a number of anticancer agents. T he ways in which these transporters bring about drug expulsion are not full y explained and may involve intracellular factors as well. Thus detailed ev idence may be difficult to obtain from studies on intact cells. Material an d methods: Inside-out plasma membrane vesicles prepared from multidrug-resi stant cells expressing high amounts of Pgp or of MRP provide a simpler syst em for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approac h for determining whether potential inhibitors have their effects on transp ort at the cell membrane or by more indirect means. Conclusions:We show tha t information gained from analysis of the ATP-dependence, time course and o smotic sensitivity of accumulation is helpful in distinguishing between tra nsport and changes in binding. We have also used the technique to demonstra te the effects of the resistance modifier, XR-9051 on Pgp-mediated transpor t and to explore interactions of MK571, indomethacin and ethacrynic acid wi th MRP.