Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.     

Changes in the epithelium of the proximal part of the nephron tubule after bile loss by the body

As demonstrate the experiments performed in 45 male rats of Wistar line, a complete bile loss by the organism results in 24-72 h in certain distrophic changes of epithelium in the proximal parts of the nephron canaliculi. The brush border microvilli undergo destruction, the cellular membranes disorganize, cytoplasm of epitheliocytes vacuolizes up to their complete rupture. The PAS-positive reaction of the basal membrane in the canaliculi increases. In cytoplasm of epitheliocytes in the straight portions of the canaliculi of the distal parts of the nephron glycogene is deposited. Alkaline phosphatase, succinate dehydrogenase, cytochromoxydase activity decreases, and that of acidic phosphatase increases. The changes advance with increasing time from the beginning of bile loss.     

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.     

A morphometric study on growth of bile canaliculi after partial hepatectomy

19-33 h after partial hepatectomy there is a time-dependent increase of the bile canalicular luminal volume, of the total length of bile canaliculi, and of their non-microvillous, smooth surface, when measured per unit volume of lobular parenchyma. On average the luminal volume fraction is increased by a factor of 2.8, the length per volume by a factor of 1.4, and the density of smooth surface by a factor of 2.0, when compared with sham-operated controls. On the other hand, the volume and surface density of bile canalicular microvilli are only slightly increased after partial hepatectomy. These findings are interpreted as indicating disproportional growth of bile canaliculi which is due predominantly to the formation of new, at first non-microvillous, membrane.     

Fine structure of the liver in the larval lamprey, Petromyzon marinus L.; hepatocytes and sinusoids

The ultrastructure of hepatocytes, bile canaliculi, and hepatic sinusoids of the larval lamprey, Petromyzon marinus, was examined using thin-sectioned and freeze-fractured tissues. The liver is a "tubular gland" with hepatocytes arranged in a tubular fashion around large bile canaliculi. Hepatocytes are roughly conical in shape, with their tapered apices facing a bile canalicular lumen. They possess extensive rough and smooth endoplasmic reticulum, a well-developed Golgi complex, abundant mitochondria, and varying numbers of large secondary lysosomes. Both secondary lysosomes and the Golgi complex are concentrated in the apical or peribiliary cytoplasm, indicating a possible role in bile secretion. The apical surfaces of the hepatocytes bear numerous elongate microvilli and occasional cilia, which project into the bile canaliculi. The hepatocytes are joined, apically, by junctional complexes composed of zonulae occludentes and adhaerentes. In freeze-fracture, the zonulae occludentes are of variable apicobasal depth and consist of honeycomb-like meshworks of fibrils. Spaces of variable width frequently appear in the P-face grooves, indicating that the zonulae occludentes are "leaky." Numerous communicating (gap) junctions join the hepatocytes laterally. Varying numbers of lateral microvilli project into the intercellular spaces and, basally, the plasma membrane is deeply infolded, resulting in the formation of apparently interdigitating basal processes resting upon a thin basal lamina. Sinusoids are composed of both a heavily-fenestrated, continuous endothelium, and phagocytic reticulo-endothelial (Kupffer) cells. Depsite the difference in arrangement of their hepatocytes, the mammalian and lamprey livers show similar ultrastructural features.     

Curcumin Prevents Bile Canalicular Alterations in the Liver of Hamsters Infected with Opisthorchis viverrini

Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.     

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. IV. Disintegration of bile canaliculi and disturbance of tight junction formation caused by vinblastine

Treatment of cultured hepatocytes with vinblastine or colchicine caused striking perturbations of the structural organization of the biliary pole and of the junctional complexes. During the early hours of cultivation the reassociation of the bile canaliculi was impaired by the drug, whereas at later times in culture preformed canaliculi were disintegrated to small vesicular remnants lacking microvilli. Vinblastine did not impair tight junction formation per se. However, under the influence of the drug, tight junctional strands associated in an atypic manner perpendicular to the upper surface of the hepatocytes, whereas those strands lining the canaliculi were decomposed to smaller entities and dislocated within the lateral membrane. Concomitantly to the structural disintegration of the biliary pole an accumulation of vesicles in the pericanalicular cytoplasm was noted. As indicated by numerous filipin-induced lesions, they were characterized by a high content of membrane cholesterol. The apical pole and the contiguous membrane on the other hand contained only very few filipin-cholesterol lesions. These findings suggest that antimicrotubular drugs impair the fusion of pericanalicular vesicles with the luminal membranes of the canaliculi, thus interrupting the delivery of membraneous material to the apical pole. In addition, microtubules seem to play an important role in the coordinated development and the structural fixation of the biliary pole of cultured hepatocytes.     

Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations

Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.     

Analysis of hepatocyte plasma membrane domains during rat development using monoclonal antibodies

The plasma membrane of adult rat hepatocyte consists of three domains, which have been identified by the monoclonal antibodies A39 and A59 as markers of the sinusoidal domain, B1 of the lateral, and B10 of the canalicular domains (Eur J Cell Biol 39:122, 1985). These monoclonal antibodies were used to study, by indirect immunocytochemistry, formation of the hepatocyte plasma membrane domains during development, from day 15 of gestation to day 35 post partum. The antigens defined by A39, B1, and B10 were detected, from day 15, over the major part of the hepatocyte plasma membrane except for the membranes of newly formed bile canaliculi, which were not labeled by B1 and only poorly labeled, if at all, by A39 and B10. As soon as fetuses were 16 days old, B1 labeled predominantly the lateral domain, as in the adult. Labeling with B10 progressively intensified on the membranes of bile canaliculi, but localization was not exclusively canalicular until day 21 post partum. A39 intensely labeled the canalicular membranes at 19-21 days of gestation, while at 35 days post partum it exhibited the predominantly sinusoidal labeling observed in adult hepatocytes. The antigen defined by A59 was not detected before birth and was found exclusively on the sinusoidal domain, as in the adult. These results show that the patterns of antigen distribution on different plasma membrane domains establish themselves at different rates. The marked differences observed between fetal or neonatal and adult hepatocytes might be responsible for immaturity of liver functions in the neonate.     

Ultracytochemical study of ATP-ase activity in mucosal parietal cells and in the cells of human adenocarcinoma of the stomach]

Ultracytochemical investigation of ATP-ase activity was carried out in parietal cells of the mucosa and in cancer cells of human stomach carcinoma possessing a similar ultrastructure. In parietal cells the reaction product of ATP-ase was observed on the membranes of microvilli of intracellular canaliculi, on the membranes delineating the lateral intercellular space, on the basal plasmolemma and in the nucleoli. The reaction product was absent on the membranes of tubuvesicles and on the apical surface of the plasmalemma. In cancer cells the reaction product was found on the membranes of the microvilli of the intracellular canaliculi, basal plasmolemma and in the nucleoli. Comparative examination of ATP-ase activity in these cells implies that at least the part of the mechanism of hydrochloric acid secretion which is involved in the transfer of H+ and Cl- is retained in cancer cells. A steady decrease in hydrochlorid acid secretion observed in the stomach mucosa in cancer as well as in the tumour itself seems to be associated with other mechanisms.     

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.     

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

The AgB10 antigen of bile canaliculi of the mouse hepatocyte was identified using monoclonal antibodies. The Mr 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 contacting cell surfaces and became concentrated only in the reconstituted bile canaliculi.     

Senescence marker protein-30 (SMP30) induces formation of microvilli and bile canaliculi in Hep G2 cells

Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with aging. To elucidate the physiological functions of SMP30, we transfected human SMP30 cDNA into the human hepatoma cell line, Hep G2. These Hep G2/SMP30 transfectants, which stably expressed large amounts of SMP30, proliferated at a slower rate and synthesized less DNA than mock transfectants (Hep G2/pcDNA3 controls). Thus, enhanced expression of SMP30 retarded the growth of Hep G2/SMP30 cells. Ultrastructural studies by scanning electron microscopy revealed numerous microvilli covering the surfaces of Hep G2/SMP30 cells, whereas few microvilli appeared on control cells. Subsequently, transmission electron microscopy revealed that groups of Hep G2/SMP30 cells exhibited bile canaliculi and possessed specialized adhesion contacts, such as tight junctions and desmosomes, at interplasmic membranes. However, in controls, units of only two cells were seen, and these lacked specialized adhesion junctions. Moesin and ZO-1 are known to be concentrated in microvilli and at tight junctions, respectively. Double-immunostaining was performed to examine whether moesin and ZO-1 were expressed in bile canaliculi with microvilli at the apical regions of Hep G2/SMP30 cells. The intensity of moesin and ZO-1 staining in the contact regions of each cell was markedly higher in Hep G2/SMP30 than in control cells. Moreover, moesin stained more interior areas, which corresponded to the microvilli of bile canaliculi. Clearly, bile canaliculi with microvilli formed at the apical ends of Hep G2/SMP30 cells. These results indicate that SMP30 has an important physiological function as a participant in cell-to-cell interactions and imply that the down-regulation of SMP30 during the aging process contributes to the deterioration of cellular interactivity.Akihito Ishigami and Toshiko Fujita contributed equally to this work.This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (to S.H., A.I., and N.M.), and a grant from the Health and Labour Sciences Research Grants for Comprehensive Research on Aging and Health and Research on Dementia and Fracture supported by the Ministry of Health, Labour, and Welfare, Japan (to A.I and N.M.), and a Grant-in-Aid for Smoking Research Foundation, Japan (to N.M.).     

Aggregation of macrophages in the tips of intestinal villi in guinea pigs: their possible role in the phagocytosis of effete epithelial cells

The immunoreactivity of a monoclonal antibody against cell suspensions from guinea pig adrenal glands was examined at light- and electron-microscopic levels. In addition to the cell surface membrane of adrenocortical cells, the antibody labeled specific sites in the pancreas, liver and testis, but did not label any of the other tissues examined. In the pancreas, microvilli-like processes and the cell surface membrane of centroacinar cells were immunoreactive to the antibody. The microvilli of interlobular duct cells and pancreatic duct cells were also immunoreactive. In the liver, bile canalicular microvilli of hepatocytes were exclusively labeled. Membrane structures of cell organelles, mainly mitochondria, in testicular Leydig cells were also labeled. Immunoblot analysis showed that the monoclonal antibody bound to two common bands at molecular weights of approximately 62 kDa and 110 kDa in the pancreas, liver, testis, and adrenal gland. The two bands reacted with the digoxigenin-conjugated lectin, Sambucus nigra agglutinin (SNA), which recognizes sialic acid linked (2–6) to galactose. Reaction patterns of SNA in the pancreas, liver and testis were similar to those of the monoclonal antibody; pancreatic centroacinar cells and interlobular duct cells, hepatocyte bile canaliculi and testicular Leydig cells were densely stained with SNA. Thus, the monoclonal antibody recognizes two common membrane glycoproteins containing sialic acids in the pancreas, liver, testis and adrenal cortex.     

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.     

Intracellular lumen in cultured hepatocytes of rats. Study of an original cytoplasmic infrastructure

A few hours after plating, isolated rat hepatocytes reassociate into clusters and differentiate intercellular cavities bordered by junctional complexes. These structures show a great resemblance to bile canaliculi seen in vivo. Intracellular lumens surrounded by microvilli are observed in the cytoplasm of some cultured hepatocytes. The formation of these structures, which contain an osmiophilic substance, probably results from modifications in the functioning of the secretory apparatus. It can be speculated that these intracellular lumens may contribute to the formation of new canaliculi differentiated between reassociated cells.     

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.     

Ultrastructural review of Choleoeimeria spp., a coccidium infecting the gall-bladder epithelium of reptiles

Choleoeimeria Paperna and Landsberg, 1989 is a reptile coccidium with unique features. Its endogenous development occurs in the cells of the bile epithelium. Its host cell while becoming hypertrophic emerges above the epithelial surface. The following species studied by electron microscopy: C. alloagamae Paperna, 2007 from Agama sp. West Africa; C. allogehyrae Paperna, 2007 from Gehyra australis and C. heteronotis Paperna, 2007 from Heteronotia binoei, from Australia, and C. pachydactyli Paperna and Landsberg, 1989 from Pachydactylus capensis from South Africa. The fine structure of the respective endogenous stages is fairly uniform. The host-cell hypertrophy coincides with a drastic depletion of the microvilli, their junction zone with the underlying cell extends into numerous long and fine membranal out-folds. The PV of all infected cells is filled with typical round granular particles. Young meronts undergo binary fission. The differentiating microgamont develops an expanded multilobed body. Macrogamont's organelles include type 1 and type 2 wall forming bodies, canaliculi and granular bodies, suspected to be the precursors of the sporozoites refractile bodies. The oocyst wall forms from 4 wall-membranes consolidating over the zygote plasmalemma.     

Surface changes at fertilization: integration of sea urchin (Arbacia punctulata) sperm and oocyte plasma membranes

To examine the integration and fate of the sperm plasma membrane following its incorporation into the oocyte plasma membrane, we have fertilized sea urchin (Arbacia punctulata) gametes reciprocally labeled with cationized ferritin. When unlabeled oocytes were inseminated with labeled sperm, cationized ferritin acceptors moved laterally from the sperm plasma membrane into the fertilization cone and surrounding microvilli, mixing with components of the oocyte plasmalemma. Labeled oocytes inseminated with unlabeled sperm produced extremely large fertilization cones, completely devoid of cationized ferritin, while the remainder of the oocyte surface remained heavily labeled. Surface area measurements indicated that if all the sperm plasmalemma were utilized to delimit a fertilization cone it would provide less than 10% of the total surface membrane. Evidence is presented indicating that a principal source of membrane to the expanding fertilization cone of inseminated oocytes is from microvilli, i.e., microvilli are retracted to accommodate fertilization cone formation. Membrane delimiting the fertilization cone has a much lower affinity for agents (cationized ferritin and concanavalin A) that stain negatively charged and carbohydrate moieties compared to other regions of the oocyte surface. These ultrastructural observations indicate that significant rearrangements occur in the oocyte and sperm plasma membranes following gamete fusion which give rise to asymmetries in membrane topography; components of both membranes are redistributed within the bilayer adjacent to and delimiting the fertilization cone.     

Electron microscopic cytochemical characterization of bile canaliculi and bile ducts in vitro.

Electron microscopic cytochemical localization of Mg++-activated adenosine triphosphatase (Mg++-ATPase) and 5-nucleotidase (AMPase) was investigated in bile canaliculus-rich and bile duct-containing fractions isolated from rat liver. Comparative cyochemical studies between prefixed and non-prefixed fractions revealed that the activity of both enzymes could be detected in the fractions under appropriate experimental conditions. However, the cytochemical activity of AMPase was much more sensitive to glutaraldehyde than that of Mg++-ATPase. Mg++-ATPase and AMPase reaction products were localized primarily on bile canalicular microvilli, that is, along the outer (luminal) surface of canalicular plasma membranes, but they were never observed on bile ductal microvilli. AMPase was also detectable on lateral hepatic plasma membranes. Mg++-ATPase demonstrated by the cytochemical technique described is a reliable enzyme marker for isolated bile canalicular membranes. At high magnification, Mg++-ATPase reaction product was also observed on the microfilaments surrounding isolated bile canaliculi. The possibility that the reaction product on the pericanalicular microfilaments may result from the hydrolysis of ATP byan actomyosin ATPase-like enzyme associated with these filaments is briefly discussed.