The rat liver in vivo transfers bile salts, proteins, and dyes from blood into bile. It is the purpose of this communication to demonstrate the maintenance of this transcellular transport in cultured adult rat hepatocytes. Two minutes after adding fluorescein (20 ,ug/ml) to the culture medium, maximal cellular fluorescence was observed through the fluorescence microscope. Subsequently, intercellular clefts showed a steadily increasing fluorescence with a maximum between 5 and 20 min, resulting in a brightly fluorescent network of intercellular gaps. The following observations are taken as evidence that these findings reflect cellular uptake and canalicular secretion of the dye. First, the same sequence of observations was made upon addition of fluorescein diacetate (a nonfluorescent precursor offluorescein), proving that the compound had been taken up and metabolized in the cells to fluorescein before secretion into intercellular clefts. Second, preincubation of the monolayers with the cholestatic bile salt taurolithocholate (100 j.mol/liter) suppressed almost completely intercellular but not cellular fluorescence. It is concluded that hepatocytes in culture show a functional polarity permitting the transcellular transport of substances bound for biliary secretion.The hepatocyte is an epithelial cell whose morphology and physiology show a marked polarity. Its cell membrane has two distinct domains: the sinusoidal membrane, which mediates solute exchange with blood, and the canalicular membrane, which faces the bile canaliculus and is involved in bile formation (1, 2). Functional polarity is most clearly demonstrated by transcellular transport of bile salts, proteins, and various organic compounds from the blood compartment to bile. This transcellular transfer is mediated by two transport systems-one sinusoidal and one canalicular-operating in sequence (3). Isolated hepatocytes obtained by dissociation ofthe liver after perfusion with collagenase (4) have been described as reaggregating to organotypic structures in monolayer culture (5-8). These cells in culture maintain the capability to take up bile salts by the sinusoidal transport system (9). Moreover, their ultrastructure displays numerous elements that show the anatomical features of bile canaliculi (8,10,11). At present, it is open to debate whether these ultrastructural elements of the cultured hepatocyte are still operative and perform secretory work.Recent reports offunctional polarization of epithelial cells in culture by demonstration ofasymmetric virus budding (12) and transcellular net movement of ions (13,14) in MDCK cells led us to study whether this applies to biliary transport in cultured adult rat hepatocytes. For this purpose, we have investigated uptake and secretion offluorescent dyes that are subject to biliary elimination in vivo (15) with fluorescence microscopy.MATERIALS AND METHODS Materials. Fluorescein was obtained from Drobena GmbH, (Berlin), fluorescein diacetate was from Sigma, taurolithocholate was from Calbiochem, and c...
