Active transport of fluorescent P-glycoprotein substrates: Evaluation as markers and interaction with inhibitors

With P-glycoprotein (P-gp) continuing to have prominence among the ABC tran sporters for its ability to remove various xenobiotics from many cell types , accurate and robust methods for estimating the exposure of drug, carcinog en, toxicant, pesticide, and even some endobiotics to tissues and cells aff ected by P-gp are valuable. The inhibition of P-gp active transport of mole cules, therefore, has often been quantified by concentration dependence of inhibitor effect on fluorescent substrate marker efflux mediated by this en zyme, with much evidence indicating two asymmetric yet interdependent subst rate binding sites on P-gp. A uniqueness in the pair of binding sites could result in distinct effects of an inhibitor on the transport of certain sub strates, thus leading to differences in fluorescent substrate responsivenes s or sensitivity. Seven different fluorescent substrates of P-gp were quant itatively tested for their responsiveness to inhibition by a wide range of P-gp substrates/inhibitors. Interesting differences were observed in the IC 50 values caused by each of the inhibitors employed, in part exemplified by DNR and LDS being generally more sensitive to inhibition effects than any other fluorescent marker. However, no clear trend emerged to designate any fluorochrome marker as the most or least responsive to inhibition. Furtherm ore, LDS is more sensitive to some P-gp inhibitors than the substrate marke r DNR, generally the most responsive. These results support the assertion o f two unequal substrate binding sites that are allosterically dependent on each other. Therefore, an inhibitor that favors binding to the site opposit e from that favored by a particular marker may have significant transduced effects through the protein between the two binding sites. Nevertheless, al though either DNR or LDS is generally the fluorescent substrate most respon sive to inhibition, there may be other substrates yet even more sensitive. (C) 2001 Elsevier Science.