EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 547

classification of transporters, TEA is characteristically used as a cationic compound;
uridine and thymidine as nucleosides; and probenecid, estradiol-17-glucuronide, bro-
mosulfophthalein (BSP), taurocholate, p -aminohippuric acid (PAH), indomethacin,
and salicylate as anionic compounds. The experimental procedures for inhibition
studies are similar to those used in transport assays with animals, tissues, cells, and
oocytes.
We performed an inhibition study with rat renal cortical slices to characterize
the transport mechanism of a novel diuretic drug, M17055. The effects of vari-
ous compounds on [ 14 C]M17055 uptake were measured at 37 ◦ C. The concentration
of [ 14 C]M17055 was 1 mM, and inhibitors used were 1 mM probenecid, 500 mM
p -aminohippuric acid (PAH), 200 mM benzylpenicillin (PCG,) and 1 mM estradiol-
17-glucuronide, 1 mM estrone 3-sulfate (E3S), 1 mM taurocholic acid (TCA), 1
mM tetraethylammonium (TEA), and 1 mM cimetidine. Probenecid, a well-known
inhibitor of organic anion transport systems, decreased [ 14 C]M17055 uptake sig-
nificantly. E3S also showed remarkable inhibition, whereas TCA was less effec-
tive. PAH inhibited [ 14 C]M17055 uptake, whereas PCG, which selectively inhibits
OAT3 while having little effect on OAT1, did not. In addition, a transport study with
OAT1-expressing HEK293 cells showed higher [ 14 C]M17055 uptake activity than
did cells transfected with vector alone. PAH and probenecid inhibited the uptake of
[ 14 C]M17055, whereas PCG had few effects. Overall, the results indicated that OAT1
contributes at least in part to the renal distribution of M17055. 30
Thus, comparison of results obtained from tissues or primary cells with the known
properties of transporters can allow identification of the responsible or major trans-
porters involved in transport of particular drugs. When there are several transporters
with similar substrate specificities, it will be useful to perform the inhibition study
with tissues and cells devised by knockdown or knockout approaches. For example, in
a comparative knockdown study using transporter-expressing cells and human model
cell lines, the responsible molecule can be suggested by the loss of substrate transport
activity or sensitivity to inhibitors.
18.6.2. Elucidation of Regulatory Mechanisms
The results of the human genome project indicate that approximately 50 ABC trans-
porter genes and approximately 360 SLC transporter genes may exist in humans. So
the question arises: “How is the overall expression pattern of transporters controlled?”
To answer this it is necessary to clarify the mechanisms regulating the function and
expression of individual transporters. In this section we describe methods for studying
transcriptional and posttranscriptional regulation which can provide us with informa-
tion about induction and functional modifications of transporters by administration
of drugs.
Transcriptional Regulation
Reporter Gene Assay In initial transcriptional regulation studies, reporter gene as-
says are often performed to determine the minimal promoter and the region responsible