Bile canalicular barrier function and expression of tight-junctional molecules in rat hepatocytes during common bile duct ligation

Tight junctions of hepatocytes form the intercellular barrier between the blood circulation and bile flow. We focused on early stages of common bile duct ligation to observe changes in tight junctions without the irreversible changes seen after lengthy ligation. Common bile ducts of 12-week-old male rats were ligated for 6 h because, at this time point, no histological changes were observed. Serum bilirubin and bile acid levels began to increase 3 h after ligation and were restored to the control level immediately after surgical removal of the ligation. To examine the barrier of hapatocytes, horseradish peroxidase was injected via the femoral vein, and bile was collected for the first 10 min. A four-fold elevation of the secretion and concentration was observed in the bile of ligated rats compared with that of control animals. We next examined lanthanum permeability by perfusion fixation of the liver. At 6 h after ligation, both dilation of the bile canaliculi and partial loss of microvilli were commonly observed. There were dense deposits of lanthanum in almost all bile canaliculi of ligated rats. In control animals, neither dilation of the bile canaliculi nor loss of microvilli was detected, and only 44% of bile canaliculi exhibited deposits. An apparent increase of occludin mRNA expression was detected in livers after 6 h ligation, whereas the expression of claudin-1, -2, and -3 was not influenced by ligation. These results indicate that regulation of occludin gene expression is different from that of claudin-1, -2, and -3. The early phase of bile stasis employed in this study is thought to be an indispensable approach for understanding the precise regulation of tight junctions.     

Loss and reappearance of gap junctions in regenerating liver

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.     

Differentiation of the plasma membrane of hepatic cells in monolayer cultures

Hepatocytes from rats were isolated by treatment with trypsin and cultured. Plasma membranes at different culture stages were observed by electron microscopy. The activities of 5' nucleotidase and adenosinetriphosphatase on the plasma membranes were examined. The cell coat was also studied by use of the concanavalin A-peroxidase technique. The surfaces of single cells, covered with microvilli, are the site of adenosinetriphosphatase activity only and are devoid of 5'-nucleotidase activity. After a few h of culture, the cells are grouped together in tight clusters or long trails and are separated by an intercellular space of 250 A, partially permeable to lanthanum nitrate. The juxtaposed plasma membranes on which 5'-nucleotidase and adenosinetriphosphatase activities occur also delimit spaces similar to bile canaliculi. The formation of junction complexes and their permeability to lanthanum nitrate was also studied. No enzymatic activity is observed at the junctions. The numerous tight junctions, impervious to the tracer, are always accompanied by a profusion of microfilaments. Mature desmosomes are rare, and are present only in the form of "maculae adhaerentes diminutae." The gap junctions, nearly always permeable to the tracer, form rapidly and assume a variety of shapes (trail, bulge and ring-like), the significance of which is open to discussion. The use of concanavalin A permits localization of the free sugar sites on the surface of the cells, in the pinocytotic vesicles and in the internal space of the gap junctions.     

Microtubule disruption interferes with the structural and functional integrity of the apical pole in primary cultures of rat hepatocytes.

The effect of microtubule disruption on the development and maintenance of cell polarity was studied in rat hepatocytes cultured as primary monolayers in the presence of colchicine or nocodazole. Addition of colchicine immediately after plating did not inhibit the generation of bile canaliculi (the apical pole) after 1 day of culture, as judged by electron microscopic examination, and did not allow penetration of Ruthenium Red through the tight junctions. However, the bile canaliculi developed in the presence of colchicine or nocodazole were not fully normal since they were not able to concentrate fluorescein diacetate in their lumina, and did not enrich with proteins of the apical plasma membrane domain, as control cells did. When the drugs were added after 1 or 2 days of culture, the new bile canaliculi appeared to be unaffected when examined by electron microscopy, but many of them did not concentrate fluorescein and were not enriched with apical membrane proteins within 4 to 24 h after drug addition. Whenever the drugs were added, the proteins that would normally concentrate on the membrane of the bile canaliculi accumulated intracellularly in endocytic vesicles after 2 to 4 h of drug treatment, and in vacuoles resembling lysosomes when the drugs were maintained for 24 h or more. These results show that microtubule disruption does not inhibit the structural reconstitution of bile canaliculi, but impairs their normal function and the transport of proteins of the apical plasma membrane domain.     

How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi

Few in vitro models expressing complex hepatocyte polarity are available. We used the unpolarized rat Fao cell line to isolate the polarized WIF-B line. These complex rat-human hybrid cells form functional simple bile canaliculi. To obtain Fao-derived polarized models with a simpler chromosome content and developed bile canaliculi, we employed two approaches. Partial success was achieved with monochromosomal hybrids. As shown by the immunolocalization of apical, basolateral, and tight-junctional proteins, monochromosomal hybrid 11-3 cells were polarized. They formed simple functional bile canaliculi and transiently expressed the typical polarity of simple epithelial cells. One subclone blocked in this polarity state was isolated. A more robust approach was provided by spheroid culture, a three-dimensional system that strengthens cell-cell contacts. Transient spheroid culture induced irreversible polarization of Fao cells. This induction occurred in most spheroids (approximately 1% of the cells). From populations enriched in stably polarized cells, we generated new polarized cell models, designated Can. Can 3-1 cells formed simple functional bile canaliculi when plated at high density. Regardless of plating density, Can 9 and Can 10 cells formed long tubular branched canaliculi competent for vectorial transport of organic anions and bile acids, and involving several dozen adjacent cells. Thus, we have generated new cell models stably expressing typical hepatocyte polarity. Among these models, Can 9 and Can 10 are the first capable of forming functional, highly developed bile canaliculi similar to those formed in vivo. This work was supported by grants from the Association pour la Recherche sur le Cancer (no.6551), the Institut Curie (PIC Signalisation Cellulaire, no. 914) and the Institut National de la Santé et de la Recherche Médicale (contract PRISME 98-09).     

Tight junctions in the rat parotid gland

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.     

Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

In vivo assembly of tight junctions in fetal rat liver

Examination of glutaraldehyde-fixed, freeze-fractured livers from 14-15-day rat fetuses provided the basis for the following observations. Membrane particles align in otherwise poorly particulated areas of the presumptive pericanalicular plasma membrane (A face), frequently forming a discontinuous "honey-comb" network joining small particle islands. Even at this early stage, contiguous B-fracture faces contain furrows, rather than rows of pits, distinguishing the linear particle aggregates on the A face as developing tight junctions rather than gap junctions. Short segments of these linear arrays merge with smooth ridges clearly identifiable as segments of discontinuous tight junctions. With the continuing confluence of particulate and smooth ridge segments, mature tight junctions become fully appreciable. We conclude that tight junctions form de novo by the alignment and fusion of separate particles into beaded ridges which, in turn, become confluent and are transformed into continuous smooth ones. At 21 days of fetal life, most of the images of assembly have disappeared, and the liver reveals well-formed bile canaliculi sealed by mature tight junctions.     

Formation of a barrier by brain microvessel endothelial cells in culture

Endothelial cells (EC) isolated from bovine brain microvessels produce a continuous monolayer when grown in primary culture. The EC are joined together by tight junctions and contain few pinocytotic vesicles. Horseradish peroxidase (HRP) is unable to penetrate this in vitro barrier system. Exposure of the cells to 1.6 M arabinose produces a reversible separation of the tight junctions with penetration of HRP across the monolayer in a pattern similar to that observed in animals after infusion of hyperosmotic solutions into the carotid artery. The behavior of brain microvascular cells in culture suggest that they retain properties important to the formation of the blood-brain barrier.     

Assembly and functional analysis of tight junction in a colon adenocarcinoma cell line: effect of glucose depletion.

We describe morphologic and biochemical changes in the colonic epithelial HCT-116 cell line following depletion of glucose from the culture medium. Cultured cells under permissive differentiation conditions (inosine-supplemented glucose-free medium) exhibited, after confluence, an enterocytic differentiation, in contrast to cells grown under standard culture conditions, where they remain in an undifferentiated state. The differentiated phenotype was characterized by the presence of a monolayer of polarized cells displaying an apical tight junction, and by the presence of alkaline phosphatase, a well known brush border marker. We demonstrated that the formed tight junctions were functional using the following criteria: a) labeling of the junctions with antibodies recognizing the tight juntion proteins occludin and ZO-1, as observed by immunofluorescence and immunoblotting analysis; b) characteristic organization of the tight junction strands, as observed in freeze-fracture replicas; c) increase ofthe transepithelial resistance across the monolayer; d) not permeation of the ruthenium red stain across the tight junction, and e) presence of the hyperphosphorylated form of occludin.     

The unique polarity phenotype of hepatocytes

Hepatocytes, the main epithelial cell type of the liver, function like all epithelial cells to mediate the vectorial flow of macromolecules into and out of the organ they encompass. They do so by establishing polarized surface domains and by restricting paracellular flow via their tight junctions and cell–cell adhesion. Yet, the cell and tissue organization of hepatocytes differs profoundly from that of most other epithelia, including those of the digestive and urinary tracts, the lung or the breast. The latter form monolayered tissues in which the apical domains of individual cells align around a central continuous luminal cavity that constitutes the tubules and acini characteristic of these organs. Hepatocytes, by contrast, form capillary-sized lumina with multiple neighbors resulting in a branched, tree-like bile canaliculi network that spreads across the liver parenchyme. I will discuss some of the key molecular features that distinguish the hepatocyte polarity phenotype from that of monopolar, columnar epithelia.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

A protein associated with the mouse and rat hepatocyte junctional complex

Summary In this study we have examined a protein associated with bile canaliculi of mouse and rat hepatocytes that is detected by monoclonal antibody BG9.1. The protein is seen by indirect-immunofluorescence microscopy as 2 discrete parallel lines at the lateral borders of adjacent hepatocytes. This pattern is present during development in the day 13 fetal mouse liver. Electron microscopy with immuno-gold labeling indicated that the protein is associated with the cytoplasmic surface of junctional complexes located on either side of bile canaliculi. BG9.1 reacts with a protein of 192000 apparent molecular weight on immunoblots of plasma membrane isolated from mouse and rat hepatocytes. It has been reported that unlike most cellular components, tight junctions are not soluble in sodium deoxycholate. Extraction of isolated hepatic plasma membrane sheets with deoxycholate and other reagents did not eliminate the pattern seen by indirect-immunofluorescence microscopy and enhanced the intensity of reactions on immunoblots. BG9.1 also binds to the junctional-complex region in other epithelial cell types. These results indicate that BG9.1 detects a deoxycholate-insoluble protein associated with junctional complexes and suggests that the protein is a component of tight junctions.     

Ultrastructural characteristics of intercellular contacts and bile canaliculi in neonatal rat hepatocytes in primary culture

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal heptocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocyts the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The difference in the development of cellular polality could be caused by the proliferating activity of neonatal hepatocytes.     

Development of bile canaliculi between chicken embryo liver cells in vivo and in vitro.

The development of an organized network of bile canaliculi is essential for the normal functioning of the liver. We have characterized bile canaliculus development in situ from Days 3-19 and in vitro in cultured hepatocyte monolayers using electron microscopical and immunofluorescent staining with antibodies that specifically recognize antigens of the bile canaliculus. Although the liver first forms as a discrete epithelial bud of endodermal tissue at stage 12-14 (45-53 h after laying), canaliculi were first detected by our antibodies at low levels in 4-day embryos and at high levels in stage 27 (5 days after laying) and later embryos. During Days 4, 5, and 6 the canaliculi near the periphery of the rudiment do not stain while canaliculi in central areas, closer to the gut, are strongly stained. During this transition period the ultrastructure of the canaliculi in the peripheral regions is also less developed than the central canaliculi where the antigens appear. By 7 days post laying, canaliculi throughout the entire liver rudiment express the marker antigens equally and have the ultrastructural characteristics of mature, functional canaliculi. Cells prepared from liver of embryos of 11 days incubation and grown in monolayer culture reformed discernible canalicular specializations, as determined by immunofluorescent staining and electron microscopy, but only transiently (for 1 to 3 days after plating). Not all of the antigens were expressed or polarized in these cultures. The capacity of the embryonic parenchymal cells to develop and maintain polarity appears to depend on factors possibly including age-dependent changes in the cells themselves, interactions with other cell types or extracellular matrix, or the shape of the cells.     

The reaggregation of adult rat liver cells maintained in vitro

The reaggregation of dissociated adult rat liver cells maintained in vitro for up to 96 h is described. Cultures were examined by dark-ground fluorescence and electron microscopy. Spaces resembling bile canaliculi were formed between reaggregated hepatocytes. Desmosomes and ‘tight’ junctions were formed between hepatocytes but ‘gap’ junctions were not detected. In older cultures structural damage was observed in many hepatocytes and some of them ingested cell debris by phagocytosis. Structures resembling bile ducts and sinusoids were also formed but complex association between all three types of cell aggregate was not observed.     

Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

The antigen of bile canaliculi of the mouse hepatocyte: identification and ultrastructural localization

Summary The AgB10 antigen of bile canaliculi of the mouse hepatocyte was identified using monoclonal antibodies. The M_r value of 116000 for AgB10 was measured by immunoblotting. The tissue localization of AgB10 was studied by light and electron microscopy using the immunoperoxidase technique. AgB10 was predominantly present on the microvillus membrane of bile canaliculi, the brush border of intestinal mucosa and apical surfaces of the epithelial cells in some other organs. A small amount of AgB10 was detected on the basolateral domain of the hepatocytes. AgB10 was specific for hepatocytes and was not found in the other cell types of the liver. In primary hepatocyte culture, AgB10 was localized on the surface of cells during the first 24 h, predominantly at the sites of cell-cell and cell-substratum contacts. After 48 h of culture AgB10 gradually disappeared from contracting cell surfaces and became concentrated only in the reconstituted bile canaliculi.