A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C 4 into membrane vesicles

Abstract

This study investigated a high-throughput assay to measure multidrug resistance-associated protein (MRP1)-mediated uptake into membrane vesicles. Typically, a rapid filtration technique using a 12-filter vacuum manifold is used. We report here the development of a 96-well microtiter dish assay. MRP1-transfected HeLa cells (HeLa-T5) were used for the membrane vesicle preparations. The uptake of 50 nM [ 3H]leukotriene C 4 (LTC 4) was measured in a 96-well microtiter dish with rapid filtration onto a Perkin Elmer unifilter GF/B plate using a Perkin Elmer Filtermate 196. Counting of the isotype was conducted with a Perkin Elmer Top Count NXT. Uptake was adenosine 5 ′-triphosphate-dependent and linear over a 120-s time course. Uptake was inhibited by the leukotriene D 4 antagonist, MK 571, with a k i of 0.67 μM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-β-glucuronide was 35-fold greater than inhibition by estradiol-3-β-glucuronide. The kinetic parameters for LTC 4 uptake were determined to be a K m of 157 nM with a V max of 344 pmol/min/mg protein. The properties of MRP1-mediated transport of LTC 4 are consistent with those previously reported. The microtiter dish assay is a more expedient method for measuring transport into membrane vesicles and will have applications to other transporters.