Pumping of drugs by P-glycoprotein: a two-step process?
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Pumping of drugs by P-glycoprotein : a two-step process? / Litman, Thomas; Skovsgaard, Torben; Stein, Wilfred D.
In: Journal of Pharmacology and Experimental Therapeutics, Vol. 307, No. 3, 12.2003, p. 846-53.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Pumping of drugs by P-glycoprotein
T2 - a two-step process?
AU - Litman, Thomas
AU - Skovsgaard, Torben
AU - Stein, Wilfred D
PY - 2003/12
Y1 - 2003/12
N2 - 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.
AB - 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.
KW - Algorithms
KW - Animals
KW - Anti-Bacterial Agents
KW - Antineoplastic Agents, Phytogenic
KW - Blotting, Western
KW - Calcium Channel Blockers
KW - Carcinoma, Ehrlich Tumor
KW - Cell Line, Tumor
KW - Cyclosporine
KW - Daunorubicin
KW - Fluoresceins
KW - Humans
KW - Immunosuppressive Agents
KW - Kinetics
KW - Models, Biological
KW - P-Glycoprotein
KW - Pharmaceutical Preparations
KW - Quinolines
KW - Verapamil
KW - Vinblastine
U2 - 10.1124/jpet.103.056960
DO - 10.1124/jpet.103.056960
M3 - Journal article
C2 - 14534356
VL - 307
SP - 846
EP - 853
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
SN - 0022-3565
IS - 3
ER -
ID: 119646557