A High-Throughput Assay for Measurement of Multidrug Resistance Protein-Mediated Transport of Leukotriene C4 Into Membrane Vesicles

Anal Biochem. 2002 Nov 1;310(1):61-6. doi: 10.1016/s0003-2697(02)00282-8.

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 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.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Antibodies / chemistry
  • Antibodies / immunology
  • Antibody Specificity
  • Binding, Competitive
  • Biological Transport, Active
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Dose-Response Relationship, Drug
  • Estradiol / analogs & derivatives*
  • Estradiol / pharmacology
  • Glutathione / metabolism
  • Glutathione / pharmacology
  • HeLa Cells
  • Humans
  • Kinetics
  • Leukotriene C4 / antagonists & inhibitors
  • Leukotriene C4 / chemistry
  • Leukotriene C4 / pharmacokinetics*
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / immunology
  • Multidrug Resistance-Associated Proteins / metabolism*
  • Multidrug Resistance-Associated Proteins / pharmacology
  • Propionates / pharmacology
  • Quinolines / pharmacology
  • Transfection
  • Transport Vesicles / drug effects
  • Transport Vesicles / metabolism
  • Tritium
  • Vincristine / metabolism

Substances

  • Antibodies
  • Multidrug Resistance-Associated Proteins
  • Propionates
  • Quinolines
  • Tritium
  • estradiol-3-glucuronide
  • estradiol-17 beta-glucuronide
  • Leukotriene C4
  • Estradiol
  • Vincristine
  • verlukast
  • Adenosine Triphosphate
  • Glutathione
  • multidrug resistance-associated protein 1