EFFICIENT IN-VITRO VECTORIAL TRANSPORT OF A FLUORESCENT CONJUGATED BILE-ACID ANALOG BY POLARIZED HEPATIC HYBRID WIF-B AND WIF-B9 CELLS

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.