Dexniguldipine-HCl is a potent allosteric inhibitor of [ 3H]vinblastine binding to P-glycoprotein of CCRF ADR 5000 cells

Cell membranes were prepared from the multidrug resistant, P-glycoprotein expressing human lymphoblastoid cell line CCRF-ADR 5000. The P-glycoprotein of these membranes possessed high affinity binding sites for [ 3H]vinblastine, with a K d of 8 ± 2 nM and B max of 17 ± 8 pmol/mg of protein. The binding of [ 3H]vinblastine to P-glycoprotein was not ATP-dependent, and was inhibited by cytotoxic drugs with the following potency order; vincristine > doxorubicin > etoposide. The 1,4-dihydropyridine and multidrug resistance reversing agent, dexniguldipine-HCl, inhibited binding with a K i value of 37 nM. The multidrug resistance reversing agent cyclosporin A, and the cytotoxics doxorubicin and etoposide did not alter the kinetics of [ 3H]vinblastine dissociation from P-glycoprotein; however, the 1,4-dihydropyridines dexniguldipine-HCl and nicardipine accelerated dissociation of [ 3H]vinblastine. These data suggest that P-glycoprotein possesses at least two allosterically coupled drug acceptor sites; receptor site 1 which binds vinblastine, doxorubucin, etoposide and cyclosporin A, and receptor site 2 which binds dexniguldipine-HCl and other 1,4-dihydropyridines.