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1.
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. 相似文献
2.
H Robenek J Rassat V Grosser H Themann 《Virchows Archiv. B, Cell pathology including molecular pathology》1982,40(2):201-215
Over a period of 20 weeks estradiol valerate (1.5 mg/kg body weight/week) was administered subcutaneously to male Wistar rats from which the livers were examined at four week intervals employing a freeze-fracture technique and colloidal lanthanum tracer studies. In connection with intrahepatic cholestasis, distinct alterations in the tight junctions were observed, consisting of disorganization, rarification and proliferation. Disruption of the tight junctions was not seen and colloidal lanthanum did not penetrate into the bile canalicular lumen. Holding the view that the term "leakiness" of tight junctions should be defined with reference to the tracer employed, we conclude that in the liver one tight junctional strand is sufficient to prevent the escape of larger bile constituents such as bile acids and that a back diffusion of bile acids over the tight junctional barrier does not play a role in the pathogenesis of the estrogen-induced cholestasis. Interruptions of tight junctions, as described by other authors, are interpreted as a secondary mechanical effect. On the other hand, we consider an increased permeability of the tight junctions to water and small solute molecules as probable; possibly this increased permeability is brought about by alterations in the microfilaments. A model for the pathogenesis of the estrogen-induced intrahepatic cholestasis is proposed. 相似文献
3.
A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis 总被引:1,自引:1,他引:0
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C J Connell 《The Journal of cell biology》1978,76(1):57-75
What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction. 相似文献
4.
S. Sakisaka H. Koga K. Sasatomi Y. Mimura T. Kawaguchi K. Tanikawa 《The Yale journal of biology and medicine》1997,70(4):403-408
Previous studies suggested endotoxin, derived from the intestine through the portal blood to the liver, was predominantly metabolized by Kupffer cells. In the present study, fluorescent-labeled endotoxin injected into the rat portal vein was demonstrated not only in Kupffer cells but also in hepatocytes. Furthermore a great amount of labeled endotoxin was recovered in bile. In the livers of patients with primary biliary cirrhosis (PBC), immunohistochemistry demonstrated significant retention of endotoxin in the biliary epithelial cells, and treatment with ursodeoxycholic acid significantly reduced the retention in those cells. The study for detection of apoptosis demonstrated increased rates of apoptosis in hepatocytes and biliary epithelial cells in PBC liver, and the rate of apoptosis in biliary epithelial cells was significantly reduced after treatment with ursodeoxycholic acid. Immunohistochemistry in PBC liver demonstrated significant reduction of fluorescence intensity for a 7H6 antigen in biliary epithelial cells, indicating the increased paracellular permeability of bile ducts, because cellular immunolocalization of that antigen has been shown to be inversely correlated with the paracellular permeability of the tight junction. These results suggest that, in biliary epithelial cells, retention of endotoxin, increased apoptosis, and increased permeability of tight junctions may be involved in the pathogenesis of PBC. 相似文献
5.
The hagfish liver was histochemically examined with special attention to biliary structures around the portal veins. Hepatocytes were organized into tubular structures surrounded by sinusoids. Biliary ductule structures, which resemble the ductal plates transiently appearing in mammalian liver development, were observed around the portal veins, but they did not appear around central veins. Thus, the hagfish liver demonstrates the same basic structure as the mammalian liver; that is, a vascular system from portal to central veins via sinusoids, and portal triad structures consisting of portal veins, hepatic arteries, and intrahepatic bile ducts. The epithelial cells of the ductal platelike structures strongly expressed cytokeratin, had some lectin-binding sites, and were delineated by the basal lamina, which was reactive for periodic acid-Schiff (PAS) staining and Iectin histochemistry. The lumina of the ductal plate-like structures were comparatively small and heterogeneous in diameter around the portal veins, suggesting that the biliary structures may not be efficient for bile secretion. The epithelial cells of the gall bladder had a simple columnar shape and were a PAS-positive cytoplasm. Those of bile ducts near the hilus, including extrahepatic and hepatic ducts, were simple columnar or cuboidal cells, and had large lumina. The cytoplasm in these cells was PAS-positive. These phenotypes with the expression of lectin-binding sites were clearly different from those of the ductal plate-like structures in the liver proper, suggesting that the extrahepatic and intrahepatic biliary structures may have different developmental origins. 相似文献
6.
Bile canalicular barrier function and expression of tight-junctional molecules in rat hepatocytes during common bile duct ligation 总被引:3,自引:0,他引:3
Takakuwa Y Kokai Y Sasaki K Chiba H Tobioka H Mori M Sawada N 《Cell and tissue research》2002,307(2):181-189
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. 相似文献
7.
A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly by electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues. 相似文献
8.
A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly be electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues. 相似文献
9.
Summary The time of establishment of the blood-testis barrier in the rabbit was studied by electron microscopy using lanthanum nitrate. This electron-dense tracer was present in the intercellular spaces in all regions of the seminiferous cords in 7 to 9-week-old animals. In 10 and 11-week-old rabbits, the penetration of lanthanum nitrate was restricted to the basal region of the seminiferous cords. Closer examination revealed the presence of numerous tight junctions between adjacent Sertoli cells. The morphological appearance of these junctions was similar to those described previously in other mammals. Entry of the tracer substance was restricted at these junctions. Pachytene germ cells, which reside beyond the junctions, were never surrounded by the tracer. Based on our observations it was concluded that the blood-testis barrier in the rabbit is formed between the 9th and 10th postnatal week, and that it is functionally effective by the 10th week. 相似文献
10.
The epithelia of a number of glands and cavitary organs of the rat and guinea pig have been surveyed, and in all cases investigated, a characteristic tripartite junctional complex has been found between adjacent cells. Although the complex differs in precise arrangement from one organ to another, it has been regularly encountered in the mucosal epithelia of the stomach, intestine, gall bladder, uterus, and oviduct; in the glandular epithelia of the liver, pancreas, parotid, stomach, and thyroid; in the epithelia of pancreatic, hepatic, and salivary ducts; and finally, between the epithelial cells of the nephron (proximal and distal convolution, collecting ducts). The elements of the complex, identified as zonula occludens (tight junction), zonula adhaerens (intermediary junction), and macula adhaerens (desmosome), occupy a juxtaluminal position and succeed each other in the order given in an apical-basal direction. The zonula occludens (tight junction) is characterized by fusion of the adjacent cell membranes resulting in obliteration of the intercellular space over variable distances. Within the obliterated zone, the dense outer leaflets of the adjoining cell membranes converge to form a single intermediate line. A diffuse band of dense cytoplasmic material is often associated with this junction, but its development varies from one epithelium to another. The zonula adhaerens (intermediate junction) is characterized by the presence of an intercellular space (~200 A) occupied by homogeneous, apparently amorphous material of low density; by strict parallelism of the adjoining cell membranes over distances of 0.2 to 0.5 µ; and by conspicuous bands of dense material located in the subjacent cytoplasmic matrix. The desmosome or macula adhaerens is also characterized by the presence of an intercellular space (~240 A) which, in this case, contains a central disc of dense material; by discrete cytoplasmic plaques disposed parallel to the inner leaflet of each cell membrane; and by the presence of bundles of cytoplasmic fibrils converging on the plaques. The zonula occludens appears to form a continuous belt-like attachment, whereas the desmosome is a discontinuous, button-like structure. The zomula adhaerens is continuous in most epithelia but discontinuous in some. Observations made during experimental hemoglobinuria in rats showed that the hemoglobin, which undergoes enough concentration in the nephron lumina to act as an electron-opaque mass tracer, does not penetrate the intercellular spaces beyond the zonula occludens. Similar observations were made in pancreatic acini and ducts where discharged zymogen served as a mass tracer. Hence the tight junction is impervious to concentrated protein solutions and appears to function as a diffusion barrier or "seal." The desmosome and probably also the zonula adhaerens may represent intercellular attachment devices. 相似文献
11.
V Franceschini F Ciani M Lazzari P Del Grande G Minelli 《Basic and applied histochemistry》1988,32(3):327-332
The permeability of the newt cerebral capillaries to lanthanum ion has been studied after perfusion with mannitol solutions of increasing molarity. In the control specimens lanthanum deposits were limited to the luminal side of the capillaries and tracer did not spread to the pericapillary spaces due to the tight junctions. Treatment with hypertonic solutions of mannitol (0.25M, 0.5M, 1M) caused opening of the blood brain barrier with a progressive increase in lanthanum between the endothelial cell edges, in the basal lamina and in the extracellular spaces of the nervous parenchyma in relation to the molarity of the mannitol solution. The spread of lanthanum is probably due to opening of the tight junctions between the endothelial cells, since pinocytotic vesicles labelled with tracer were not evident. 相似文献
12.
The intercellular junctions of guinea-pig placental capillaries: a possible structural basis for endothelial solute permeability 总被引:1,自引:0,他引:1
The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars. 相似文献
13.
The value of the dynamic 99mTc-mebrofenin liver scintigraphy in the diagnosis of extrahepatic bile ducts patency has been assessed. The study included 176 patients in whom an occlusion of bile ducts of any degree was excluded in 111 cases, and bile ducts patency disorders were diagnosed in 65 patients. Time of the appearance of radiolabelled mebrofenin was measured in the selected segments of bile ducts. Time of appearance of radioactivity in the intestines was also noted. Another diagnostic criterion was persistent stasis of radioactivity in the choledochus. Basing on these criteria, patients were classified as "sick" or "healthy". It was possible to exclude the disease in case of negative result with the probability level of 91%. Positive result of scintigraphy points to the impaired patency diagnosis with 96% probability. Technique is simple, free of complications and contraindications. It is also of high predictive value. These features make the procedure particularly useful in the diagnosis of bile ducts patency. 相似文献
14.
The cell junctions of the notochord of Xenopus laevis tadpoles were examined with the electron microscope using thin sections, lanthanum tracer experiments, and freeze-fracture replicas. Both the peripheral and vacuolated cells of the notochord are connected by numerous spot desmosomes characterized by an intercellular desmogloea and intermediate filaments on the cytoplasmic sides. The peripheral cells also display numerous hemidesmosomes facing the underlying basal lamina. Staining with rhodamine-phalloidin for F-actin yielded negative results and suggested that adhaerens-type junctions are absent. Tracer experiments with lanthanum and freeze-fracture replicas clearly revealed the presence of gap junctions between both cell types but no indications of tight junctions were found and no intercellular barrier existed for tracer infiltration of the notochord. 相似文献
15.
PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES 总被引:3,自引:1,他引:2
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William B. Neaves 《The Journal of cell biology》1973,59(3):559-572
The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous. 相似文献
16.
Cheung ID Bagnat M Ma TP Datta A Evason K Moore JC Lawson ND Mostov KE Moens CB Stainier DY 《Developmental biology》2012,361(1):68-78
The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis. 相似文献
17.
THE USE OF BEEF LIVER CATALASE AS A PROTEIN TRACER FOR ELECTRON MICROSCOPY 总被引:18,自引:11,他引:7
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Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells. 相似文献
18.
The reaggregation of adult rat liver cells maintained in vitro 总被引:2,自引:0,他引:2
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. 相似文献
19.
Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation 总被引:5,自引:3,他引:2
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《The Journal of cell biology》1984,98(5):1865-1877
The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland. Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min. Secretion was induced in some animals by intraperitoneal injection of isoproterenol. In resting glands, the tracers microperoxidase , cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts. In glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase , cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands. Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells. The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution. Freeze- fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight greater than or equal to 1,900; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight less than or equal to 34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights. Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution. 相似文献
20.
Dynamic changes in protein components of the tight junction during liver regeneration 总被引:4,自引:0,他引:4
Takaki Y Hirai S Manabe N Izumi Y Hirose T Nakaya M Suzuki A Mizuno K Akimoto K Tsukita S Shuin T Ohno S 《Cell and tissue research》2001,305(3):399-409
The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2-3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2-3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2-3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes. 相似文献