EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 543

We have employed X. laevis oocytes for functional characterization of the organic
cation transporter human OCTN1, using tetraethylammonium (TEA) as a model
organic cation. The uptake of [ 14 C]TEA into OCTN1 cRNA-injected oocytes was
significantly higher than that into water-injected oocytes, and the uptake of [ 14 C]TEA
increased linearly until 90 minutes, and was saturable. Calculation of the kinetic
parameters gave apparent K m and V max values of 0.195 1.45

pmol/60 minutes per oocyte, respectively. In addition, we examined the effects of pH
and membrane potential on the uptake of [ 14 C]TEA by human OCTN1 by altering the
transport buffer pH or replacing Na + with K + in the transport buffer. The results of
these experiments suggested pH dependence and membrane potential independence
of human OCTN1. 42

± 0.033 mM and 18.5 ±

18.4.5. Techniques to Study Cellular Uptake or Transport

Dish Method The dish method is suitable for transport study in adherent cells. Cul-
tured cells are cultivated in an appropriate medium in a humidified incubator at 37 ◦ C
under an atmosphere of 5% CO 2 in air. In the transport measurement, the cells cul-
tured on plastic dishes are incubated with transport medium (Hanks' balanced salt
solution: 136.7 mM NaCl, 5.36 mM KCl, 0.952 mM CaCl 2 , 0.812 mM MgSO 4 ,
0.441 mM KH 2 PO 4 , 0.385 mM Na 2 HPO 4 ,25mMD-glucose, 10 mM HEPES, pH
7.4) containing a radiolabeled test compound to initiate the uptake study. After an
appropriate time, the dishes are washed three times with ice-cold Hanks' balanced
salt solution and solubilized 1 M NaOH; then the radioactivity is measured in a liquid
scintillation counter after neutralization with HCl and addition of liquid scintillation
fluid. 23

Silicon Layer Method Cultured cells are cultivated in an appropriate medium in
a humidified incubator at 37 ◦ C under an atmosphere of 5% CO 2 in air. The cells
are harvested with a rubber policeman, washed twice, and suspended in the transport
buffer. After preincubation of cells for 5 minutes at 37 ◦ C, the cell suspension is mixed
with transport medium containing the radiolabeled test compound to initiate the uptake
study. At appropriate times, 200- L aliquots of the mixture are placed in centrifuge

tubes containing 50 L) of silicon oil and

liquid paraffin mixture (density = 1.03). Then the samples are centrifuged, and the
resulting cell pellets are solubilized in 3 M KOH and neutralized with 30

L of 3 M KOH, covered with a layer (100

Lof5M
HCl. The associated radioactivity is measured in a liquid scintillation counter. 49 , 50
In the case of trans- stimulated uptake experiments, the cells are preloaded with
unlabeled compounds for 30 minutes, and subsequent uptake of radiolabeled com-
pounds by the cells is measured. In addition, in the case of trans -stimulated efflux
experiments, the cells are preloaded with radiolabeled compounds for 30 minutes at
37 ◦ C. Then the cells are spun down, suspended in ice-cold transport medium, and
washed twice at 0 ◦ C. The cells are again suspended in the transport medium contain-
ing a test compound, and efflux is initiated at 37 ◦ C. At appropriate times, 100-mL
aliquots of the mixture are sampled. 49