Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.
It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.
Tamai
I
I
Department of Medicine, University of Chicago, Illinois 60637.
Safa
A R
AR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003524:Cyclosporins / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism*; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D014747:Vinblastine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D003524:Cyclosporins; D004095:Dihydropyridines; D008562:Membrane Glycoproteins; D014747:Vinblastine; C041130:azidopine; C065699:SDZ 33-243