Abstract. We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (>70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of 3,-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4°C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4°C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

Earlier studies have shown that native methionyl-tRNA synthetase from Escherichia coli K12 is composed of four, probably identical, subunits having a molecular weight of 43000 each.Incubation of the purified enzyme with trypsin results in irreversible conversion into an enzymatically active modified form which, by a number of criteria, including molecular size, catalytic and antigenic properties, electrophoretic and chromatographic behaviour, appears to be undistinguishable from the altered methionyl-tRNA synthetase previously observed after incubation a t 37" C of a crude extract. Furthermore, a similar modification of methionyl-tRNA synthetase resulted upon incubation of the purified enzyme with several other proteolytic enzymes, including papain, subtilisin and pronase.The structural and catalytic properties of the homogenous trypsin-modified methionyl-tRNA synthetase were examined in detail. It has a molecular weight of 64000 established by equilibrium ultracentrifugation, and dissociates in the presence of 8 M urea to yield two, apparently identical subunits of molecular weight 32000. Furthermore, it is shown that proteolysis of the native enzyme by trypsin is accompanied by release of enzymatically inactive fragments corresponding to approximately 20 Olio of the original protein. The simplest interpretation of these results is that proteolysis selectively removes a portion of each original subunit, corresponding to a quarter of its size, with consequent dissociation of the original tetramer into dimers, each now composed of modified subunits.No significant differences in the Michaelis constants for any of the substrates or inhibitors tested were found between native and modified enzyme. Nor was conversion accompanied by any detectable change in the specificity towards the amino acid. The molecular activity (expressed as moles of substrate converted/mole subunit) of the homogenous trypsin-modified enzyme was found to be 20°/, lower than that of native methionyl-tRNA synthetase, for both reactions catalyzed by the enzyme. An interesting difference between the two forms of the enzyme appeared in their magnesium ion requirement for the aminoacylation of tRNAs. Thus, under otherwise identical assay conditions, the trypsin-modified enzyme required significantly less magnesium ions for the aminoacylation of both tRNAfdet and tRNAEt a t optimal rates. Another remarkable feature of the trypsin-modified enzyme is its enhanced stability against heat inactivation, compared to the native enzyme.A structural model is proposed for methionyl-tRNA synthetase which attempts to account for its great susceptibility to limited proteolysis, and for the maintenance of its catalytic properties despite the resulting radical changes in its molecular structure.

Cytosolic Ca 2؉ (Ca i 2؉ ) regulates secretion of bicarbonate and other ions in the cholangiocyte. In other cell types, this second messenger acts through Ca 2؉ waves, Ca 2؉ oscillations, and other subcellular Ca 2؉ signaling patterns, but little is known about the subcellular organization of Ca 2؉ signaling in cholangiocytes. Therefore, we examined Ca 2؉ signaling and the subcellular distribution of Ca 2؉ release channels in cholangiocytes and in a model cholangiocyte cell line. The expression and subcellular distribution of inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) isoforms and the ryanodine receptor (RyR) were determined in cholangiocytes from normal rat liver and in the normal rat cholangiocyte (NRC) polarized bile duct cell line. Subcellular Ca 2؉ signaling in cholangiocytes was examined by confocal microscopy. All 3 InsP 3 R isoforms were expressed in cholangiocytes, whereas RyR was not expressed. The type III InsP 3 R was the most heavily expressed isoform at the protein level and was concentrated apically, whereas the type I and type II isoforms were expressed more uniformly. The type III InsP 3 R was expressed even more heavily in NRC cells but was concentrated apically in these cells as well. Adenosine triphosphate (ATP), which increases Ca

Metal overload plays an important role in several diseases or intoxications, like in Wilson's disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.