Elsevier

Analytical Biochemistry

Volume 310, Issue 1, 1 November 2002, Pages 61-66
Analytical Biochemistry

A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C4 into membrane vesicles

https://doi.org/10.1016/S0003-2697(02)00282-8Get rights and content

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 C4 (LTC4) 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 D4 antagonist, MK 571, with a ki 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 LTC4 uptake were determined to be a Km of 157 nM with a Vmax of 344 pmol/min/mg protein. The properties of MRP1-mediated transport of LTC4 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.

Section snippets

Materials

[3H]LTC4 (110 Ci/mmol) was purchased from DuPont NEN (Boston, MA). LTC4, ATP, AMP-PCP, GSH, creatine phosphate, and creatine kinase were purchased from Sigma (St. Louis, MO). MgCl2 was purchased from E M Science (Cherry Hill, NJ). RPMI 1640 medium was purchased from Life Technologies (Grand Island, NY). Iron-supplemented bovine calf serum (BCS) was purchased from Hyclone Laboratories (Logan, UT). The Pierce BCA Protein Assay Reagent was purchased from Pierce (Rockford, IL).

Cell culture

HeLa-T5 and HeLa-C1

Time course for LTC4 uptake

LTC4 uptake into membrane vesicles prepared from the MRP1-transfected HeLa-T5 cells and the HeLa-C1 vector control was measured over a 120-s time course. Transport was measured in the presence of an ATP-regenerating system with either 4 mM ATP or the nonhydrolyzable analog AMP-PCP (Fig. 1). The initial rate of 50 nM uptake was 8.08 pmol/min/mg protein in HeLa-T5 vesicles, 5-fold higher than that of the AMP-PCP control (1.55 pmol/min/mg protein). By contrast, ATP-dependent uptake in vector-control

Discussion

For the purpose of validating the microtiter dish method reported here, studies were done to determine kinetic constants and examine the effects of inhibitors on MRP1-mediated uptake of LTC4 into membrane vesicles prepared from HeLa-T5-transfected cells. The data generated employing the microtiter dish technology were in good agreement with previously reported studies that used conventional methods.

Kinetic determinations have been reported for LTC4 transport in many MRP1-expressing cell lines.

References (24)

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