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Studies of the mechanisms of bile formation have been facilitated by the development of in-vitro models that enable the transport functions of hepatocytes and bile duct epithelial cells to be analyzed in isolation. Isolated hepatocyte couplets represent a primary bile secretory unit that retain secretory polarity. Isolated bile duct units are intact polarized segments of interlobular bile duct segments. Both preparations can be maintained in short term culture, respond appropriately to choleretic agonists and facilitate a variety of functional applications. [ABSTRACT FROM AUTHOR]

Morphological alterations in the pattern of liver cell gap junctions were examined in phalloidin-treated rats to assess the role of gap junctions in experimental intrahepatic cholestasis. Double-labelled fluorescent staining of gap junctions and F-actin were performed using a monoclonal antibody against rat hepatocyte connexin 32 and rhodamine-phalloidin. Immunoelectron microscopy, using the anti-connexin 32 antibody, freeze-fracture replica electron microscopy, and conventional electron microscopy were also performed. In phalloidin-treated rat livers, the specific immunofluorescent staining of connexin 32 was markedly decreased in the pericentral area after 1 day of phalloidin treatment and, after 5 days of phalloidin treatment, there was a decrease in connexin 32 staining in the entire hepatic lobule. On the other hand, F-actin staining at the cell periphery and at the bile canaliculi was markedly increased in the pericentral area of the hepatic lobule after 1 day of phalloidin treatment and in the entire lobule after 5 days of treatment. Immunoelectron micropscopy showed that both sides of the cytoplasmic domains of gap junctions were stained with anti-connexin 32 antibody in controls, whereas, in cholestatic rats, only one side of the cytoplasmic domain of some gap junctions was stained with anti-connexin 32 antibody after 1 or 3 days of phalloidin treatment. No gap junctions were observed after 5 days of phalloidin treatment either by freeze-fracture replica electron microscopy or by conventional electron microscopy. These results indicate that with phalloidin treatment, hepatocyte gap junctions decrease, first in the pericentral area, and finally throughout the entire lobule. [ABSTRACT FROM AUTHOR]

The immunoelectron microscopic localization of actin in hepatocytes was studied in normal, common bile duct-ligated, and phalloidin treated rats. We used the low temperature embedding procedure with Lowicryl K4M and protein A-gold technique was applied to demonstrate the localization of actin. The tissues embeded in Lowicryl K4M at low temperature provided good preservation of ultrastructure and antigenicity of actin. In control rats bile canaliculi were clearly visualized and most of the gold particles were observed on the bile canalicular microvilli, at the pericanalicular ectoplasm, at the cell border, and on the sinusoidal microvilli. In common bile duct-ligated rats and phalloidin treated rats, pericanalicular ectoplasm was thickened and many gold particles were seen there. [ABSTRACT FROM AUTHOR]

The cytoskeletons of hepatocytes and biliary epithelial cells in bile duct ligated rate livers were investigated by transmission and scanning electron microscopy. The three dimensional organization of the intermediate filaments (IFs) of hepatocytes and biliary epithelial cells was clearly demonstrated by scanning electron microscopy. Cell borders and dilated bile canaliculi were well preserved after perfusion with detergent solution. A very dense filamentous network of IFs was seen throughout the cytoplasm, especially around the dilated bile canaliculi and at the cell borders. IFs in biliary epithelial cells were more numerous compared with hepatocytes. Morphometric analysis showed that the IFs of hepatocytes significantly (p>.001) increased in amount in bile duct ligated rats. The IFs of biliary epithelial cells showed no significant changes in bile duct ligated rats compared to controls. These results suggest that the increase in IFs in hepatocytes results from the adaptation of the hepatocytes to the stress imposed by bile duct ligation. It may be that the resulting intracanalicular pressure and back diffusion of bile induces a metaplastic change in hepatocytes so that they acquire more IFs to function like the bile duct epithelium to conduct bile flow. [ABSTRACT FROM AUTHOR]

We observed pericanalicular webs (PCW) of liver cells in cases with familial non-hemolytic hyperbilirubinemia using electron microscopy. The area and width of PCW were determined by morphometric methods as a way of quantitating this feature. The mean PCW width was 0.175±0.003 μm (mean ± SE) in Dubin-Johnson syndrome and 0.184±0.005 μ,m in Rotor's syndrome. In both of these syndromes PCW width was significantly larger than that in Gilbert's syndrome (0.124±0.003 /μm) (p<0.01). The mean PCW area was 0.585±0.017 /xm in Dubin-Johnson syndrome and 0.582±0.030/i,m in Rotor's syndrome. Values in these two syndromes were significantly larger than that in Gilbert's syndrome (0.382±0.014ju,m) (p<0.01). Widths and areas of PCW in these three syndromes were not significantly different between central, intermediate, and peripheral zones of the hepatic lobules. There was a positive correlation between serum direct bilirubin levels and widths or areas of PCW in these syndromes. These results suggested that disturbances of bile flow caused by the dysfunction of pericanalicular microfilaments are partly involved in the pathogenesis of Dubin-Johnson syndrome and Rotor's syndrome. [ABSTRACT FROM AUTHOR]

Pathological and pathophysiological study on the intestinal lymphatics was carried out clinically and experimentally. Jejunal biopsy study revealed marked dilatation of intestinal lymphatics in liver cirrhosis with ascites and BehÇet's disease as well as protein-losing enteropathy. Lymphangiographic study showed also abnormal findings in above-mentioned diseases. In cases of McKee dogs in which mechanism of blockade of lymphatic flow was already clarified, McKee dogs showed marked malabsorption of fat. When lymphatic flow was blocked mechanically at the thoracic duct, fat accumulation was seen in the absorptive cells as well as in dilated intestinal lymphatics. In administration of colchicine to rats, fat accumulation was seen in the matrix of absorptive cells as well as in the endoplasmic reticulums and Golgi apparatus. However, amorphous substances instead of chylomicrons were seen in dilated intestinal lymphatics. These studies suggested that a tract of fat transportation from intestinal cells to the thoracic duct should be considered as a functional unit in fat absorption. [ABSTRACT FROM AUTHOR]

Microfilaments in epithelial cells are important for the structural and functional integrity of tight junctions. In the present study, we examined the relationship between microfilaments and tight junctions in hepatocytes of rat liver following common bile duct ligation (CBDL) for up to 2 weeks. Actin filaments and tight junctions were studied by fluorescence microscopy using 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph) and an anti-ZO-1 antibody, respectively. Double-stained sections were examined with confocal laser scanning microscopy (CLSM). Electron microscopy was applied for the assessment of structural alterations in microfilaments and in tight junctions with detergent-extraction and freeze-fracture preparations. Our results showed that F-actin was present at the entire plasma membrane of hepatocytes in control liver, whereas CBDL increased the patocytes in control liver, whereas CBDL increased the amount of F-actin mainly at the bile canalicular and lateral plasma membranes. Simultaneously, the immunofluorescence of ZO-1 underwent striking changes, i.e., from a uniform to an irregular staining pattern with various fluorescence intensities. CLSM demonstrated a colocalization of ZO-1 and F-actin in control liver and its deterioration in CBDL liver. Electron microscopy showed marked alterations of microfilaments and tight junctions due to CBDL. It is concluded that actin filaments are intimately associated with tight junctions in normal hepatocytes. CBDL impairs this association by progressively diminishing the structural interaction between F-actin and ZO-1, which may in turn lead to functional disturbances of tight junctions. [ABSTRACT FROM AUTHOR]

In short-term cultures of rat hepatocytes, bile canaliculi enclosed between unseparated cell couplets are able to perform periodical contractions resulting in expulsion of bile. Pericanalicular cytoskeletal proteins are involved in canalicular contractility: F-actin, myosin and tropomyosin are associated around bile canaliculi, as revealed by staining with tetramethylrhodaminyl-phalloidin and by immunofluorescence. Bile canalicular contractility is distributed by cholestatic agents that are known to interfere with actin polymerization; e.g., phalloidin and also cytochalasin B inhibit canalicular contractility and cause pericanalicular vacuolization and formation of blebs. Whereas the association of the cytoskeletal proteins is not affected by treatment with cytochalasin B, treatment with phalloidin results in dissociation of F-actin and myosin, indicating that binding of phalloidin to F-actin impairs its molecular interaction with myosin. [ABSTRACT FROM AUTHOR]

Time lapse cinematographic analysis revealed that the frequency of canalicular contractions in primary monolayer-cultured hepatocytes (couples and triplets) isolated from a rat liver was increased by endothelin-1 (ET-1)-treatment as compared with that of a control group. By transmission electron microscopy, vesicles were more frequently observed to be opened to the canalicular lumens during the contracting stage of the bile canaliculi in the ET-1-treated couplets and triplets than in the control group. [ABSTRACT FROM AUTHOR]

Time lapse cinematographic analysis revealed that the frequency of canalicular contractions in primary monolayer-cultured hepatocytes (couplets or triplets) isolated from rat livers increased in the presence of monolayer-sinusoidal endothelial cells by the N-L monomethyl arginine (L-NMMA)-treatment as compared to that in a control. Transmission electron microscopy revealed an enhanced vesicular transport to the canalicular lumens at the contracted bile canaliculi in the L-NMMA-treated couplets or triplets. [ABSTRACT FROM AUTHOR]

Effects of phalloidin on the early (5–60 min) processes of wound repair were examined in the ethanol-injured mouse gastric mucosa. Within 5 min after ethanol was washed out from the stomach, survived epithelial cells changed their shape and became flat extending both lamellipodia and filopodia. The denuded basal lamina was re-covered by these stretched cells within 30 min. When a low dose of phalloidin (5 μg) was applied to the wounded stomach, it took 60 min or more to cover the exposed basal lamina. Epithelial cells at the wound edge of the phalloidin-treated stomach often contained unusually thick layer of microfilaments in their subapical region. This observation suggests that phalloidin induces active microfilament assembly mainly in the apical region of epithelial cell processes. [ABSTRACT FROM PUBLISHER]

Composition and interactions of cell types in rainbow trout ( Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120× g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver. [ABSTRACT FROM AUTHOR]

To elucidate early changes and the mechanism of ischaemia-reperfusion liver injury, we investigated three-dimensional microstructural changes of cellular actin filaments in rat livers using confocal laser scanning microscopy. The liver tissues of a reperfusion group were examined 12 h after removal of a vascular clamp. Fixed tissues were stained with fluorescein-labelled phalloidin to obtain stereoscopic images of the actin filaments and these were compared with histological findings. The images of bile canaliculi showed that multiple abnormal minute diverticula arose from the canalicular membranes and fused with one another, resulting in irregular dilation of the bile canaliculi. These changes were observed after 15 min of ischaemia and reperfusion in which no significant necrosis was seen. The frequency and degree of these changes were strictly dependent on the periods of ischaemia (15-60 min). We called these bile canacilular lesions "varicoid changes". The liver of an ischaemia group taken after persistent clamping without reperfusion did not show these changes. Our findings suggest that the varicoid change in the bile canaliculi is probably due to alterations in the actin polymerization-depolymerization cycle and is a pathognomonic change of ischaemia-reperfusion liver injury. [ABSTRACT FROM AUTHOR]

Studies which water compartment in the lung contributes to the Hahn T2f and T2s components. T2 decay curves for peripheral lung tissue; Restriction of the protons of water; Determination of intra and extravascular water; Histological examination results.

Interactions between homing receptors on circulating leucocytes and endothelial addressins regulate tissue-specific cellular extravasation. Although integrin α4β7 appears to be the main receptor for gut-homing T lymphocytes, less is known about molecules mediating mucosal B cell homing. Expression of integrin α4β7 on B lymphocytes, B cell blasts, and plasma cells in human gut-associated lymphoid tissue (GALT; the Peyer's patches and appendix) and lamina propria was studied by multi-colour immunofluorescence applied on cryosections. Isolated mononuclear cells from the same tissue compartments were examined by flow cytometry and compared with peripheral blood B cells. Integrin α4β7 was expressed by IgA+ B cell blasts and plasma ceils (CD38high) in the lamina propria, B cell blasts in GALT, and sIgD+ B lymphocytes in peripheral blood. In contrast, GALT sIgD+ B lymphocytes were negative or only weakly positive for β4β7. These results suggested that B lymphocytes down-regulate α4β7 upon extravasation in GALT but up-regulate this integrin after antigen-priming. Thus, α4β7 may be a homing receptor also for B cell blasts extravasating in the gut lamina propria, where this integrin is maintained on plasma cells, perhaps as a local retention factor. [ABSTRACT FROM AUTHOR]

It is conceivable that brush border enzyme activities of the intestinal mucosa will change when bacterial toxins are exposed to the intestinal microvillous membranes. The effect of cholera toxin on the activity of intestinal alkaline phosphatase in rats was therefore determined in the intestinal mucosa by the histochemical method as well as in intestinal lymph by using lymph fistulated-rats. Activity of intestinal alkaline phosphatase in the intestinal mucosa and lymphatics changed biphasically after the oral administration of cholera toxin to rats. For the first three hours after the administration of cholera toxin it was depressed; it then increased and at eight hours reached a maximum. These changes in the activity of intestinal alkaline phosphatase were prevented by the administration of chlorpromazine, a known inhibitor of adenylate cyclase activity. [ABSTRACT FROM PUBLISHER]