P. Bravo et al., EFFICIENT IN-VITRO VECTORIAL TRANSPORT OF A FLUORESCENT CONJUGATED BILE-ACID ANALOG BY POLARIZED HEPATIC HYBRID WIF-B AND WIF-B9 CELLS, Hepatology, 27(2), 1998, pp. 576-583
Efficient transport of bile acids, a typical characteristic of hepatoc
ytes, is partially lost in most hepatoma cell lines and ire normal hep
atocytes after some days in culture. We have tested whether the polari
zed rat hepatoma-human fibroblast hybrid WIF (hybrids between W138 and
Fao cells) cells previously obtained by our group were able to perfor
m vectorial transport of the fluorescent bile acid derivative cholylgl
ycylamidofluorescein (CGamF) towards the bile canaliculi (BC). Four if
frenl WIF clones were analyzed. All were well polarized, as shown by t
he formation of spherical and even tubular BC-like structures and by t
he restricted localization at the BC, visualized by immunofluorescence
, of the apical membrane marker HA4, a possible bile acid carrier. WIF
-B and its subclone WIF-B9 were found to accumulate CGamF in 65% to 75
% of their BC. This transport was time, temperature, and partly sodium
dependent and was inhibited by coincubation with the parental natural
bile salt cholylglycine. Dinitrophenyl glutathione, a substrate of th
e canalicular multispecific organic anion transporter, did not inhibit
CGamF canalicular secretion, whereas it greatly impaired the canalicu
lar secretion of a non-bile acid organic anion, fluorescein, generated
intracellularly from fluorescein diacetate. Confocal microscopy confi
rmed the presence of CGamF in the cytoplasm, supporting a transcellula
r route from medium to BC. In contrast, two other polarized clones exh
ibited a poor ability (WIF 12-6) or no ability (WIF12-1 TG delta) to v
ectorially transport CGamF. In conclusion, WIF-B and WIF-B9 exhibit no
t only structural but also functional polarity, at least as far as vec
torial organic anion transport is concerned.