Use of membrane vesicles to investigate drug interactions with transporter proteins, P-glycoprotein and multidrug resistance-associated protein.
BACKGROUND
The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells.


METHODS
Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system 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 approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means.


CONCLUSIONS
We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.
Wheeler
R
R
Department of Pharmacology, University of Cambridge, UK.
Neo
S Y
SY
Chew
J
J
Hladky
S B
SB
Barrand
M A
MA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D015703:Antigens, CD / Q000502:physiology*; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004347:Drug Interactions; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D020024:Leukotriene Antagonists / Q000378:metabolism; D008562:Membrane Glycoproteins; D011422:Propionates / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D060245:Tetraspanin 29; D014407:Tumor Cells, Cultured / Q000378:metabolism*; D014750:Vincristine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D000972:Antineoplastic Agents, Phytogenic; C554869:CD9 protein, human; D020024:Leukotriene Antagonists; D008562:Membrane Glycoproteins; D011422:Propionates; D011804:Quinolines; D060245:Tetraspanin 29; C030190:S-(2,4-dinitrophenyl)glutathione; D014750:Vincristine; C059141:verlukast; D000255:Adenosine Triphosphate; D005978:Glutathione