Transcellular transport of fluorescein in hepatocyte monolayers: evidence for functional polarity of cells in culture.

Abstract

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...

A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig

“…Other investigators found that in primary parenchymal hepatocytes cultures, small biliary canaliculi often developed within the monolayers (Furukawa et al, 1987;Barth and Schwartz, 1982). Likewise, small canaliculi of the same size and character developed in the monolayers of the PICM-19 cells (Fig.…”

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…36,37 The accumulation or release of fluorescein after hydrolysis of FDA is dependent on the cell membrane permeability and integrity. [37][38][39][40] Hepatocyte polarity in culture has been observed by examining the secretion of fluorescein from hepatocytes through gap junctions into intercellular clefts. 38,41 When paracellular junction integrity is compromised, intercellular gap junction communication is disrupted, and fluorescein is able to passively diffuse out of the cells through the gap junctions.…”

“…[37][38][39][40] Hepatocyte polarity in culture has been observed by examining the secretion of fluorescein from hepatocytes through gap junctions into intercellular clefts. 38,41 When paracellular junction integrity is compromised, intercellular gap junction communication is disrupted, and fluorescein is able to passively diffuse out of the cells through the gap junctions. The fluorescein is not retained intercellularly and consequently, the fluorescein can be washed out of cells with compromised paracellular junctions by successive washings with PBS.…”

Laser−Micropipet Combination for Single-Cell Analysis

“…Once the fluorescent, free acid is formed; it is no longer membrane permeant and remains trapped within the cell, although cells do transport the dye out over time via anionic transporters. 41 For the single-cell experiments, cells grown in DMEM in a cell chamber were washed once in buffer A. Solutions of fluorescein (20 nM) and/or Oregon Green (500 nM) diacetates were made in buffer A with glucose (10 mM) immediately prior to use and added to the cell chamber after removing the wash buffer.…”