Litman Thomas, Skovsgaard Torben, Stein Wilfred D
Bioinformatics Centre, University of Copenhagen, Universitetsparken 15, Bldg. 10, DK-2100 Copenhagen, Denmark.
J Pharmacol Exp Ther. 2003 Dec;307(3):846-53. doi: 10.1124/jpet.103.056960. Epub 2003 Oct 8.
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.
通过研究维拉帕米、环孢素A、XR9576（他林洛尔）和长春碱这四种药物抑制柔红霉素和钙黄绿素-AM从四株具有不同耐药水平和P-糖蛋白（P-gp）含量的艾氏腹水癌细胞系流出的能力，测定了这四种药物对P-糖蛋白（P-gp）的表观抑制常数Kapp。对于作为转运底物的柔红霉素，维拉帕米的Kapp与[P-gp]无关，但长春碱、环孢素A和XR9576的Kapp随[P-gp]严格线性增加。对药物泵出及其逆转动力学的理论分析表明，对于从细胞质面抑制泵出的逆转剂，抑制的Kapp应随膜中泵的数量线性增加。相反，如果逆转剂通过阻断外表面的转运起作用，即抢先阻断，则Kapp应与存在的泵数量无关。实验数据表明，维拉帕米在膜的细胞外表面阻断泵出，而其他三种阻断剂作用于细胞质相的泵出。所有四种阻断剂的最大抑制程度相同；因此，它们不是并行作用，而是串行作用，即从细胞质相泵出的药物在离开细胞时必须通过抢先途径。我们的结果与Sauna-Ambudkar提出的P-糖蛋白泵出的两步模型一致。我们认为，长春碱/环孢素A/XR9576结合位点在细胞质面接受柔红霉素并将其转移至维拉帕米结合位点，柔红霉素从该位点在细胞外表面被排空。
