Stereoselective transport of hydrophilic quaternary drugs by human MDR1 and rat Mdr1b P-glycoproteins

Abstract

1. The present study was performed to evaluate and compare the ability of human MDR1-, and rat Mdr1b- and Mdr2-P-glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing insect cells. 2. As model substrates we used the N-methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N-methylquinidine and N-methylquinine. Vincristine, an established MDR1 substrate, was used as a reference. 3. We observed ATP-dependent uptake of all drugs studied into MDR1- and Mdr1b-expressing vesicles. Mdr2 was not able to transport these compounds. MDR1- and Mdr1b-mediated transport was saturable, and could be inhibited by various drugs, including PSC-833. 4. For both MDR1 and Mdr1b the V(max)/K(m) ratios (or clearance) of N-methylquinidine were greater than those determined for N-methylquinine. This stereoselective difference was also evident from differential inhibitory studies with the two isomers. 5. Comparison of normalized clearance indicated that human MDR1 was more effective in transporting the tested substrates than rat Mdr1b. 6. In conclusion, our results demonstrate that MDR1 and Mdr1b, but not Mdr2, are able to transport the monoquaternary model drugs; both MDR1 and Mdr1b display stereospecificity for these cations; and indicate human MDR1 is more efficient in transporting these cations than its rat orthologue Mdr1b.

Figure 1

Immunoblot analysis of MDR1, Mdr1b, and Mdr2 in membrane vesicle preparations. Plasma membrane-enriched vesicles were isolated from A2780/AD cells, non-infected Sf21 insect cells, and Sf21 cells infected with recombinant baculoviruses encoding human MDR1, rat Mdr1b, or rat Mdr2. Proteins (10 μg for A2780/AD vesicles, 2.5 μg for Sf vesicles) were separated by SDS-PAGE and transferred to a nitrocellulose filter. Immunoblotting analysis was performed using the primary antibody C219, a monoclonal antibody recognising all Pgps (Georges et al., 1990). Bound antibody was visualized as described in Methods. Lane 1: A2780/AD membrane vesicles, lane 2: Sf-MDR1 membrane vesicles, lane 3: Sf21 (non-infected) membrane vesicles, lane 4: Sf-Mdr1b membrane vesicles, lane 5: Sf-Mdr2 membrane vesicles. Molecular masses of the protein standards are indicated in kilodaltons.

Figure 2

Time profiles for ATP-dependent uptake of N-methylquinidine and N-methylquinine into membrane vesicle preparations isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing, or non-infected insect cells. At the time points indicated, transport of radiolabelled substrates into Sf-MDR1 (closed circles), Sf-Mdr1b (open triangles), Sf-Mdr2 (closed squares), or Sf21 (open diamonds) membrane vesicles (25 – 30 μg of protein) was determined as described in Methods. Uptake was determined for [³H]-N-methylquinidine (A), and [³H]-N-methylquinine (B), each at a final concentration of 200 nM. The uptake experiments presented in A and B were performed under identical conditions, i.e. on the same day using the same membrane vesicle preparations and reaction buffers. ATP-dependent uptake was calculated by subtracting values obtained in the presence of 4 mM AMP – PCP from those obtained in the presence of 4 mM ATP. Data points represent the mean±standard deviation of triplicate determinations of a typical experiment.

Figure 3

Osmolarity dependence of N-methylquinidine transport into membrane vesicle preparations isolated from MDR1- or Mdr1b-overexpressing insect cells. Sf-MDR1 (closed circles) or Sf-Mdr1b (open triangles) membrane vesicles (25 μg of protein) were incubated for 25 s at 37°C sec in the presence of 8 μM [³H]-N-methylquinidine, and different concentrations of sucrose (0.25, 0.33, 0.5 and 0.8 M). Transport of [³H]-N-methylquinidine was determined as described in Methods. ATP-dependent uptake was calculated by subtracting values obtained in the presence of 4 mM AMP – PCP from those obtained in the presence of 4 mM ATP. Data points represent the mean±standard deviation of triplicate determinations.

Figure 4

Uptake kinetics of N-methylquinidine, N-methylquinine, and vincristine by membrane vesicle preparations isolated from MDR1- or Mdr1b-overexpressing insect cells. Sf-MDR1 or Sf-Mdr1b membrane vesicles (25 μg of protein) were incubated in the presence of increasing concentrations of [³H]-labelled substrates. Initial uptake rates for N-methylquinidine, N-methylquinine, and vincristine were determined after an uptake period of 25, 50 and 15 s, respectively. Data presented are converted into uptake per min, and are corrected for Pgp-content of vesicle preparations. (A) Uptake of N-methylquinidine (closed circles) and N-methylquinine (open triangles) into Sf-MDR1 vesicles; (B) uptake of N-methylquinidine (closed circles) and N-methylquinine (open triangles) into Sf-Mdr1b vesicles; (C) uptake of vincristine into Sf-MDR1 (closed circles) and Sf-Mdr1b (open triangles) vesicles. Data points were analysed by non-linear curve fitting to an equation describing Michaelis – Menten kinetics using SigmaPlot. ATP-dependent uptake was calculated by subtracting values obtained in the presence of 4 mM AMP – PCP from those obtained in the presence of 4 mM ATP. Data points represent the normalized average uptake±standard error of the substrates into vesicles of three different membrane preparations (two for vincristine); the uptake into each vesicle preparation was determined three times and normalized for Pgp content. All individual data points were included in the non-linear curve fitting.