A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C4 into membrane vesicles
Section snippets
Materials
(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.
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2006, Journal of Biological ChemistryCitation Excerpt :Cells were examined using a Leica TCS SP2 MS multiphoton system confocal microscope (Leica Microsystems, Heidelberg, Germany) (22). MRP1-mediated Transport of 3H-labeled Substrates by Membrane Vesicles—ATP-dependent uptake of 3H-labeled substrates by membrane vesicles was measured using a modified rapid filtration method (31) that was adapted to a 96-well microtiter plate format (32). LTC4 transport assays were performed at 23 °C in a 50-μl reaction containing 1.8 μg of membrane vesicle protein, 50 nm [3H]LTC4 (20 nCi/reaction), 10 mm MgCl2, 4 mm ATP or 4 mm AMP, 250 mm sucrose, and 50 mm Tris-HCl, pH 7.5 (transport buffer), with an ATP regenerating system consisting of 100 μg ml–1 creatine kinase and 10 mm creatine phosphate.