EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 540

The uptake of [ 14 C]M17055 by rat renal cortical slices showed a time-dependent
increase. In addition, we have examined the effects of various compounds on the
uptake of [ 14 C]M17055 by rat renal cortical slices, and the results suggested that the
excretion of M17055 is at least partially mediated by the organic anion transporter
OAT1. An Eadie-Hofstee plot of data obtained at various substrate concentrations
provides the K m value. 30

18.4. PRIMARY CELL CULTURES AND ESTABLISHED
MODEL CELL LINES

Primary cell cultures and established model cells may be the simplest systems that
reflect the physiological conditions and can provide clues to the involvement of trans-
porters in drug absorption, distribution, and excretion. In particular, primary cultured
cells can permit us to evaluate carrier-mediated transport and to identify directly the
molecules responsible. The introduction of established model cell lines has made it
possible to conduct experiments routinely.

18.4.1. Isolated Hepatocytes

Isolated hepatocytes and enterocytes are often used as primary cultured cells. Here
we focus on rat hepatocytes. Rat hepatocytes are isolated by means of the collage-
nase perfusion procedure. 31 After isolation they are suspended at 4 ◦ C in albumin-free
Krebs-Henseleit buffer. In the preparation of isolated primary cells it is important to
check cell viability using the trypan blue (0.4% w/v) exclusion test; we use only hep-
atocytes with more than 95% viability. 32 After preincubation of isolated hepatocytes
for 5 minutes at 37 ◦ C, the cells and the transport buffer containing a radiolabeled
drug are incubated separately at 37 ◦ C for 5 minutes and then mixed to initiate uptake.
At designated times, 200-

L aliquots are taken and analyzed using the silicon layer
method described later. Although primary cultured cells may require maturation, suit-
able combinations of cells and analytic methods permit us to access the effects on
drug uptake, of ATP depressants, low temperature, and various compounds, as well
as to determine the concentration dependences of uptake. 33 , 34 In addition, human
hepatocytes are available from some organizations in the United States and China,
and are useful to examine drug behavior in humans. 23

18.4.2. Fibroblasts

Primary cultured cells can be an excellent tool in comparative studies with knock-
out or knockdown animals. 35 We have studied the loss of transport capability for
cationic compounds in the jvs mouse using primary cultured fibroblasts. Transport
measurements were performed by the dish method. The uptake of [ 14 C]TEA by fi-
broblasts from jvs mice was significantly lower than that by cells from normal mice,
as was also the case for [ 3 H]carnitine. Furthermore, the efflux transport of [ 14 C]TEA
was measured after preincubation of fibroblasts with [ 14 C]TEA, to demonstrate the
bidirectional transport of TEA by Octn2. 23