MULTIDRUG RESISTANCE PROTEINS OF THE ABCC SUBFAMILY - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 287

expressing combinations of uptake transporters and efflux pumps have been estab-
lished to study vectorial transport under more defined conditions. 143 , 247 , 248

11.6.1. Establishment and Characterization of Double-Transfected Cells

Several double-transfected MDCK cell lines have been established that express an
uptake transporter in the basolateral membrane and an ATP-dependent efflux pump in
the apical membrane. 106 , 143 , 247 In the particular case of studies on hepatobiliary elim-
ination, members of the organic anion-transporting polypeptide (OATP) family, com-
prising OATP1B1, 251 − 253 OATP1B3, 254 and OATP2B1 255 , 256 as well as the sodium-
dependent bile salt transporter NTCP, 12 the organic cation transporter OCT1, 257 and
the organic anion transporter OAT2, 257 are of interest because they contribute to
the uptake of endogenous substances and drugs across the sinusoidal (basolateral)
membrane of hepatocytes. Although several members of the ABCC subfamily are
expressed in hepatocytes, only ABCC2 contributes to the efflux across the canalicu-
lar (apical) hepatocyte membrane. However, other ATP-dependent efflux pumps are
also localized in the apical membrane of the hepatocyte and mediate transport of
endogenous and xenobiotic substances, including ABCB1 (MDR1 P-glycoprotein),
ABCG2 (BCRP), and ABCB11 (BSEP, bile salt export pump).
The first double-transfected cells for vectorial transport were MDCK cells,
which expressed recombinant human OATP1B3 in the basolateral membrane and
ABCC2 in the apical membrane. 143 Subsequently, double-transfected MDCK cells
expressing OATP1B1 and ABCC2 or OATP2B1 and ABCC2 were established and
characterized 106 , 247 (Figure 11.2 c ). Additional transporter combinations in transfected
cells have been established: NTCP and ABCB11, 258 , 259 OATP1B1 and ABCB1, or

OATP1B1 and ABCG2. 260

11.6.2. Functional Characterization of Double-Transfected Cells:
Vectorial Transport

For functional characterization, double-transfected cells are grown polarized on
semipermeable filter supports, and transcellular transport is measured either from
the basolateral to the apical side or from the apical to the basolateral side. In the case
of cells expressing ABCC2 and an uptake transporter, a vectorial transport resulting
from the combined uptake and efflux is obtained only from the basolateral to the
apical side, not in the opposite direction. This has been shown for BSP, a prototypic
amphiphilic organic anion and a substrate for OATP1B1, OATP1B3, OATP2B1, as
well as ABCC2, 106 , 143 and also for DHEAS, E 2 17

G, pravastatin, LTC 4 , CCK-8, and
E 1 3S. 106 , 107 , 143 , 247 All these compounds are substrates for the efflux pump ABCC2
and for at least one of the three OATPs expressed in human hepatocytes. When cells
express an uptake transporter exclusively, the vectorial transport may be negligible
and only intracellular substrate accumulation may be detected. 143
Vectorial transport of organic anions by double-transfected cells also permits the
analysis of transport inhibitors. 143 A selective inhibition of the efflux pump should
result in a decreased vectorial transport from the basolateral to the apical side and

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