A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C4 into membrane vesicles.
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 50nM [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 microM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-beta-glucuronide was 35-fold greater than inhibition by estradiol-3-beta-glucuronide. The kinetic parameters for LTC(4) uptake were determined to be a K(m) of 157nM with a V(max) of 344pmol/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.
Tabas
Linda B
LB
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285-0424, USA. Tabas_Linda_B@Lilly.com
Dantzig
Anne H
AH
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D001667:Binding, Competitive; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000493:pharmacokinetics*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D014162:Transfection; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D014316:Tritium; D014750:Vincristine / Q000378:metabolism
D000255:Adenosine Triphosphate; D000906:Antibodies; D004958:Estradiol; D005978:Glutathione; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011422:Propionates; D011804:Quinolines; D014316:Tritium; D014750:Vincristine; C025482:estradiol-3-glucuronide; C025483:estradiol-17 beta-glucuronide; C059141:verlukast; C421109:multidrug resistance-associated protein 1