Abstract
The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows that Kapp for inhibition should increase linearly with the amount of pumps present in the membrane for a reverser that inhibits pumping from the cytoplasmic face. In contrast, if the reverser acts by blocking transport from the outer face, i.e., preemptively, Kapp should be independent of the number of pumps present. The experimental data suggest that verapamil blocks pumping at the extracellular face of the membrane, whereas the other three blockers act on pumping from the cytoplasmic phase. The maximum degree of inhibition was the same for all four blockers; thus, they do not act in parallel but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin at the cytoplasmic face and transfers it to the verapamil-binding site, from where daunorubicin is emptied at the extracellular surface.
Footnotes
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During the course of this work, T.L. was a Weimann Associate Research Professor and recipient of research grants from the Novo Nordisk Foundation, and “Eva and Henry Frænkels Mindefond”.
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DOI: 10.1124/jpet.103.056960.
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ABBREVIATIONS: P-gp, P-glycoprotein; EATC, Ehrlich ascites tumor cell; AM, acetoxymethyl ester; Kapp, apparent half-saturation concentration (e.g., IC50); I, inhibitor concentration; ki, the pump rate for pumping from the inside; ki,0, the uninhibited pump rate for pumping from the inside; Ki, intrinsic affinity for P-gp at the inner leaflet of the cell membrane; ko, the pump rate for (preemptive) pumping from the outer face of the membrane; ko,0, the uninhibited pump rate for pumping from the outer face of the membrane; Ko, intrinsic affinity for P-gp at the outer face of the membrane (preemptive pumping); p, the leak rate of a compound that crosses the membrane by passive diffusion; Si, intracellular substrate concentration; So, extracellular substrate concentration; GF120918, elacridar; XR9576, tariquidar.
- Received July 11, 2003.
- Accepted August 21, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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