EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 545

vesicles have been suggested to be a useful tool for pharmaceutical drug screening.
Here we describe a method for preparation of vesicles from HEK293 cells with the
expression vectors harboring transporter cDNAs. 51
HEK293 cells expressing transporters are grown routinely in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum, penicillin, and streptomycin in a
humidified incubator at 37 ◦ C under an atmosphere of 5% CO 2 in air. The transporter-
expressing cell clone is cultured on 30 plastic dishes (150 mm in diameter) containing
appropriate selection drugs, and when the cells reach confluence, they are harvested
by scraping with a rubber policeman and washed twice by centrifugation in PBS. The
cells are suspended in 25 mL of buffer 1 (10 mM NaCl, 1.5 mM MgCl 2 , 0.02 mM
phenylmethylsulfonyl fluoride, 10 mM Tris-HCl, pH 7.4) and placed on ice for 30
minutes. The cells are incubated in a cell disruption bomb (Parr 4635) and equilibrated
with nitrogen gas at 700 psi with gentle stirring for 20 minutes. Then the pressure is
released, ethylenediaminetetraacetic acid is added to a final concentration of 1 mM,
and the content is homogenized with a Teflon-glass homogenizer (20 strokes) at 4 ◦ C.
The cell homogenate is collected and centrifuged for 5 minutes at 1000 rpm and 4 ◦ C.
The supernatant is layered on buffer 2 (35% sucrose, 10 mM Tris-HCl, pH 7.4) in a
centrifuge tube and centrifuged for 90 minutes at 18,000 g and 4 ◦ C. The white-colored
fluffy layer at the boundary of buffer 2 is collected, suspended in buffer 3 (150 mM
mannitol, 75 mM potassium gluconate, 10 mM HEPES-Tris, pH 7.4), and centrifuged
at 100,000 g for 2 hours at 4 ◦ C. The resulting pellet is again suspended in buffer 3 and
centrifuged at 100,000 g for 3 hours at 4 ◦ C. The final pellet is suspended in buffer 3
and stored at 80 ◦ C until used for transport experiments. The content of protein in

each preparation can be measured by the method of Bradford. 52 , 53
We employed membrane vesicles prepared from HEK293 expressing OCTN2 to
examine the Na + dependence of OCTN2. Overshoot uptake of L-[ 3 H]carnitine by
membrane vesicles from OCTN2-expressing cells was observed in the presence of
extravesicular Na + , whereas it was not observed in the absence of extravesicular Na + ,
indicating that OCTN2 mediates the Na + -coupled transport of L-carnitine. 52

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18.5.2. Membrane Vesicles from Tissues
By using membrane vesicles from tissues, we can investigate carrier-mediated drug
transport in organs. Here we describe the preparation of renal BBMVs. The kidneys
are rapidly removed under anesthesia and chilled in ice-cold isolation buffer. The
cortex is separated from the medulla with scissors, minced weighed, suspended in 10
volumes of homogenization buffer (10 mM mannitol, 2 mM Tris-H 2 SO 4 , pH 7.4), and
homogenized. A sample of homogenate is collected for protein and enzyme assays.
After addition of 10 mM MgCl 2 , the homogenate is stirred on ice for 15 minutes
and centrifuged at 1900 g for 12 minutes. The resulting supernatants are centrifuged
at 20,000 g for 12 minutes. The pellet is resuspended in 40 mL of homogenization
buffer with a Dounce glass pestle homogenizer, and the MgCl 2 precipitation step is
repeated. The suspension is centrifuged at 1900 g for 12 minutes and the supernatant
is further centrifuged at 20,000 g for 12 minutes. This pellet is resuspended in 20 mL