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PubMed1982

Tight junction contact events and temporary gap junctions in the sciatic nerve fibres of the chicken during Wallerian degeneration and subsequent regeneration.

Tight and gap junctions are described on the basis of freeze-fractures in normal chicken sciatic nerves as well as during Wallerian degeneration and subsequent regeneration. 1. Small calibre nerve fibres display a fairly continuous tight junction contact zone in the membranes of the mesaxons, paranodal loops and Schmidt-Lanterman incisures. Large fibres with more than 40 lamellae have only focal tight junction contacts in the mesaxonal membranes. 2. With the onset of Wallerian degeneration (days 2-4 post-crush, distal stump) myelinic tight junctions become arranged as maculae composed of one circular or several polygonally oriented strands that are criss-crossed by other tight junctional strands. These maculae are subsequently found in the membranes of cytoplasmic vacuoles of the Schwann cells, indicating an endocytotic mode of uptake. Tight junctions are not found between the 5th and 6th day after crush. 3. During the proliferation phase of the Schwann cells and the arrangement of these cells into Büngner cell bands (2 to 8 days post-crush) gap junctions appear between the Schwann cells of the bands. These junctions then disappear with the onset of remyelination (8 days post-crush). 4. With the onset of remyelination (from the 8th day onwards) short focal tight junctions appear in the membranes of the outer mesaxons. Shortly thereafter, when the sheaths possess 4 to 8 lamellae, tight junctions also appear in the membranes of the inner mesaxons, the paranodal loops and the cytoplasmic inclusions. The characteristic differences of tight junction elaboration in small versus large nerve fibres are re-established after three months of regeneration. The elaborated tight junctions in small and early remyelinating fibres point to a specific function; in small fibres (versus large fibres) the tight junctions might effect a separation of the intramyelinic extracellular space as a single compartment. The tight junction contacts in early remyelinating fibres support the hypothesis that myelin growth occurs within the myelin spiral and not by a free rotation and elongation of the Schwann cell tongues. It is assumed that the gap junctions between the Schwann cells contribute to the co-ordination of the Schwann cell band formation, which is involved in the guidance of sprouting axons.

Animals↗
PubMed1987

Protamine alters structure and conductance of Necturus gallbladder tight junctions without major electrical effects on the apical cell membrane.

Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.

Animals↗
PubMed2000

Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme.

The aim of the study was to characterize the interendothelial junctions in tumor microvessels of five cases of human glioblastoma multiforme. In addition to morphological analysis, tumors were screened for the expression of junctional proteins, such as occludin, claudin-1, ZO-1 and catenins. The expression of the tight junction protein claudin-1 was lost in the majority of tumor microvessels, whereas claudin-5 and occludin were significantly down-regulated only in hyperplastic vessels. As shown by freeze-fracture analysis, under the conditions of tumor growth tight junction particles of endothelial cells were almost exclusively associated with the exocytoplasmic fracture face, providing evidence for a switch of the particles from the protoplasmic to the external leaflet of the endothelial membrane. These results suggest a relationship between claudin-1 suppression and the alteration of tight junction morphology, which is likely to correlate with the increase of endothelial permeability. Underlining the undifferentiated state of tumor microvessels, plakoglobin, a crucial protein for mature endothelial junctions, was not detectable in most microvessels, whereas beta-catenin was abundantly labeled. In this context, it is of particular interest that the majority of microvascular pericytes were negative for alpha-smooth muscle actin, which is a marker of differentiated pericytes, although pericytes were frequently found in electron micrographs. In conclusion, the data suggest that the increase in microvascular permeability in human gliomas, contributing to the clinically severe symptoms of brain edema, is a result of a dysregulation of junctional proteins.

Blood Vessels↗
PubMed1995

Chorda-evoked opening of tight junctions in rat submandibular salivary acini demonstrated by microperoxidase.

The permeability of these junctions from the interstitium to the lumen was examined by using an ultrastructural tracer, microperoxidase, in conjunction with electron microscopy. In the resting gland, the reaction product of microperoxidase was seen in the interstitial and intercellular spaces, but not within acinar lumina; thus the tight junction was impermeable to microperoxidase (junction closed). Intraductal injection of hypertonic sucrose solution (1000 mOsm; 30 microliters) caused a sustained elevation of the luminal pressure, indicating osmotic water flow into the lumen due to the presence of a hypertonic solution. In this gland no opening of the tight junctions was observed. In the chorda-stimulated gland, microperoxidase entered the lumen through the tight junctions, that is, they became permeable to microperoxidase (junction open). These findings suggest that chorda stimulation opens the acinar tight junctions and that the paracellular secretory pathway may be involved in the secretion of small molecules and water from the submandibular acini.

Animals↗
PubMed1982

The structure of tight junctions between uterine luminal epithelial cells at different stages of pregnancy in the rat.

The ultrastructure of tight junctions between uterine luminal epithelial cells of the pregnant rat was studied by the freeze-fracture technique. On day 5 of pregnancy, the day of implantation, the region of tight junctions extended three times as far down the lateral cell membrane as on day 1 of pregnancy, and the strands of the complex interlinked more frequently. These observations suggest that tight junctions on day 5 may be more efficient in preserving the contents of the uterine lumen from dilution or escape than at earlier times of pregnancy.

Animals↗
PubMed2006

Differential expression of genes encoding tight junction proteins in colorectal cancer: frequent dysregulation of claudin-1, -8 and -12.

BACKGROUND AND AIMS: As integral membrane proteins, claudins form tight junctions together with occludin. Several claudins were shown to be up-regulated in various cancer types. We performed an expression analysis of genes encoding tight junction proteins to display differential gene expression on RNA and protein level and to identify and validate potential targets for colorectal cancer (CRC) therapy. PATIENTS AND METHODS: Amplified and biotinylated cRNA from 30 microdissected CRC specimen and corresponding normal tissues was hybridized to Affymetrix U133set GeneChips. Quantification of differential protein expression of claudin-1, -8 and -12 between normal and corresponding tumour tissues was performed by Western blot analyses. Paraffin-embedded CRC tissue samples, colon cancer cell lines and normal tissue microarray were analysed for protein expression of claudin-1 by immunohistochemistry (IHC). RESULTS: Claudin-1 (CLDN1) and -12 (CLDN12) are frequently overexpressed in CRC, whereas claudin-8 (CLDN8) shows down-regulation in tumour tissue on RNA level. Quantification of proteins confirmed the overexpression of claudin-1 in tumour tissues, whereas changes of claudin-8 and -12 were not significantly detectable on protein level. IHC confirmed the markedly elevated expression level of claudin-1 in the majority of CRC, showing membranous and intracellular vesicular staining. CONCLUSIONS: Differential expression of genes encoding claudins in CRC suggests that these tight junction proteins may be associated to and involved in tumorigenesis. CLDN1 is frequently up-regulated in large proportion of CRC and may represent potential target molecule for blocking studies in CRC.

Aged↗
PubMed1995

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile.

BACKGROUND: Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected small-molecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. METHODS: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze-substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. RESULTS: A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. CONCLUSIONS: The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall.

Air Sacs↗
PubMed1988

Functional coupling of tight junctions and microfilaments in T84 monolayers.

The actin-binding agent cytochalasin D (CD) in intact intestinal epithelium appears to elicit segmentation and contraction of a perijunctional ring of actomyosin and, consequently, to diminish tight junction resistance. We determined if an intestinal epithelial model composed of T84 cells also displayed such a perijunctional cytoskeletal specialization and, if so, whether exposure to CD also affected the tight junction barrier. We find T84 cells display a prominent perijunctional microfilament ring that is actin rich. CD elicits large decreases in transepithelial resistance due specifically to perturbed tight junction permeability as determined with dual Na+-mannitol flux analysis. Transepithelial mannitol and insulin fluxes also increase after CD exposure, but these molecules remain differentially restricted in accordance with their sizes, indicating that gross disruption of the monolayer has not occurred. Structurally, CD elicits segmentation and condensation of the perijunctional ring into actin-rich plaques. These features have similarity to those seen in native intestinal epithelia. This system may represent a simple model for studies of cytoskeletal-tight junction relationships.

Actin Cytoskeleton↗
PubMed1994

Effect of once daily milking and concurrent somatotropin on mammary tight junction permeability and yield of cows.

Six pairs of monozygous Friesian twin cows during late lactation were used to assess the effect of once daily milking and bST treatment on yields and tight junction permeability of mammary epithelial cells. During the first 7 d, all cows were milked twice daily; on d 8 through d 21, all cows were milked once daily, but one cow of each twin pair was treated daily with 20 mg of bST on d 13 through 21; and, finally, during d 22 through 28, all cows were again milked twice daily. Once daily milking, a common management practice in New Zealand, resulted in a small (7%) but significant decrease in milk yield. Treatment with bST increased milk yield by 19%, thereby exceeding the milk yield loss from once daily milking. The integrity of mammary tight junctions was assessed indirectly by measuring concentrations of plasma lactose and milk BSA. Once daily milking temporarily disrupted tight junction integrity, based on a 4- to 5-fold increase in plasma lactose and a 42 to 55% increase in the concentrations of milk BSA. In the present study, bST did not affect the permeability of mammary tight junctions.

Animals↗
PubMed1980

Interlamellar tight junctions of central myelin. I. Developmental mechanisms during myelogenesis.

The process of myelination in the central nervous system (CNS) of the rat (optic nerve) was studied with the freeze-fracturing technique and ultrathin sectioning to obtain information on the developmental mechanisms of interlamellar tight junctions. Using a tilting cartridge for analysis of thin sections, it could be demonstrated that during the initial phase of wrapping a tight junction formation develops between the joining tips of the oligodendrocytic process. In tannic acid-stained samples these junctions appear as typical quintuple-layered membrane fusions, while in potassium permanganate-stained material membrane thickenings between the apposing glial tips are prevalent. The latter configuration represents the characteristic feature of the so-called radial component of central myelin. Using the freeze-fracturing technique, a biphasic mode of the myelinic tight junction assembly was detected. It is suggested that tight junctions represent a prerequisite of the myelination process.

Animals↗
PubMed2000

Short communication: effects of isolation stress on mammary tight junctions in lactating dairy cows.

Eighteen cows had been selected for their responsiveness to psychological stress during the first lactation and were classified as having low (n = 10) or high (n = 8) cortisol concentrations in response to isolation-induced stress. In the present study these cows, now in their second lactation, were used to determine the effect of social isolation stress on the permeability of mammary tight junctions. During the experiment, each cow was isolated from the rest of the herd for 55 h. After the 1st h of isolation, each cow received a bolus infusion of endotoxin in one hind quarter in order to challenge tight junctions. Blood samples were taken throughout to measure lactose, which was used as an indicator of tight-junction leakiness. After 1 h of isolation, stress caused an increase in tight junction permeability in both groups, which was further enhanced by the endotoxin treatment. Although the permeability did not differ significantly between the two groups, it was consistently higher in the high-cortisol group, which was also the most stress-responsive group. Thus, psychological stress may adversely affect milk quality by allowing serum components to leak into milk.

Animals↗
PubMed2004

Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells.

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.

Cell Line↗
PubMed1991

Three-dimensional model of tight junction fibrils based on freeze-fracture images.

To study the three-dimensional structure of tight junction fibrils, the epithelia of the jejunum and epididymis of adult mice were examined by the freeze-fracture technique in unfixed and in aldehyde-fixed specimens. The fibrils have a stronger affinity for the protoplasmic (P) face of the lipid bilayer in fixed material, and for the external (E) face in unfixed and rapidly frozen material. Therefore we can observe the fibrils both from the outside and inside of the cell. Fibrils appearing on the P-face are smoothly contoured ridges and rows of hemispherical particles, while those appearing on the E-face are exclusively rows of hemispherical particles. Based on these observations, we wish to propose a new fibril model for the tight junction. There are two distinctive types of junctional elements. One type is composed of a smooth and continuous strand in the external view of the cell, but is studded with hemispherical bulgings in its internal view. This type will be referred to as the "continuous type". The other type is bead-like, and will be referred to as the "particle type". The relative proportion of these two types of elements appearing within a tight junction network differs among tissues.

Animals↗
PubMed2005

S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF.

Pulmonary pathologies including adult respiratory distress syndrome are characterized by disruption of pulmonary integrity and edema compromising respiratory function. Sphingosine 1-phosphate (S1P) is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, with production up-regulated by the proinflammatory cytokines IL-1 and TNF. S1P administration via the airways but not via the vasculature induces lung leakage. Using receptor-null mice, we show that S1P, acting on S1P3 receptor expressed on both type I and type II alveolar epithelial cells but not vascular endothelium, induces pulmonary edema by acute tight junction opening. WT but not S1P3-null mice showed disruption of pulmonary epithelial tight junctions and the appearance of paracellular gaps between epithelial cells by electron microscopy within 1 h of airways exposure to S1P. We further show by fluorescence microscopy that S1P induced rapid loss of ZO-1 reactivity, an essential component of the cytoplasmic plaque associated with tight junctions, as well as of the tetraspannin Claudin-18, an integral membrane organizer of tight junctions. S1P shows synergistic activity with the proinflammatory cytokine TNF, showing both pulmonary edema and mortality at subthreshold S1P doses. Specifically, preexposure of mice to subthreshold doses of TNF, which alone induced no lung edema, exacerbated S1P-induced edema and impaired survival. S1P, acting through S1P3, regulates epithelial integrity and acts additively with TNF in compromising respiratory barrier function. Because S1P3-null mice are resistant to S1P-induced pulmonary leakage, either alone or in the presence of TNF, S1P3 antagonism may be useful in protecting epithelial integrity in pulmonary disease.

Animals↗
PubMed1994

Microtubules are oriented with their minus-ends directed apically before tight junction formation in rat Sertoli cells.

We have examined the polarity of microtubules in Sertoli cells of the seminiferous epithelium during testicular development to test the hypothesis that microtubules change their polarity during tight junction formation. Microtubules in a number of polarized epithelial cells, including Sertoli cells, are oriented with their minus-ends directed toward the apical surface of the cell. Indirect evidence from cultured epithelial cell models suggests that this orientation may be achieved during the relocation of centrioles, reorganization of microtubules, and repositioning of the Golgi that occurs during tight junction formation. Using the microtubule hook decoration technique, we have determined the polarity of Sertoli cell microtubules at 5 and 15 days postnatally, prior to the establishment of the tight junctional complex of the blood testis barrier at 19 days. Our results indicate that, although centrioles and the Golgi apparatus migrate from an infranuclear location at 5 days to a supranuclear location by 15 days, the minus-ends of microtubules are already directed toward the apical surface of the cell by 5 days of age. These data indicate that the establishment of the apically directed minus-end orientation of microtubules of mature Sertoli cells precedes rearrangement of the centrioles and Golgi and is not temporally related to the formation of the tight junctional complex of the blood testis barrier.

Animals↗
PubMed2006

A modified technique for the impregnation of lanthanum tracer to study the integrity of tight junctions on cells grown on a permeable substrate.

Ionic lanthanum is commonly used to trace permeability pathways across epithelia and endothelia in biological electron microscopy. A method for obtaining a uniformly dense precipitate of lanthanum is described. The method, which is a modification of the technique described by Shaklai and Tavassoli (1977) was suitable for fixation of cell cultures grown on permeable filter inserts and was successfully applied to study opening of tight junctions by hypertonic solutions in the airway epithelial cell line 16HBE14o(-). The preparation method formed the basis for a semiquantitative morphological determination in which the tight junctions were subdivided as "intact," "weakened," and "open." By using this modified technique, it could be demonstrated that opening of tight junctions in airway epithelial cells increased, with increasing osmolarity with electrolytes having a stronger effect than nonelectrolytes. A significant linear relationship was found between the osmolarity of the medium and the open state of the tight junctions (as determined by the semiquantitative morphological technique) or the transepithelial electrical resistance.

Cell Culture Techniques↗
PubMed1999

A systematic analysis of 40 random genes in cultured vascular smooth muscle subtypes reveals a heterogeneity of gene expression and identifies the tight junction gene zonula occludens 2 as a marker of epithelioid "pup" smooth muscle cells and a participant in carotid neointimal formation.

An accumulation of evidence suggests that vascular smooth muscle is composed of cell subpopulations with distinct patterns of gene expression. Much of this evidence has come from serendipitous discoveries of genes marking phenotypically distinct aortic cultures derived from 12-day-old and 3-month-old rats. To identify more systematic differences, we isolated 40 genes at random from libraries of these 2 cultures and examined message expression patterns. To determine consistency of differential expression, we measured mRNA levels in 4 sets of cultures in 6 phenotypically distinct aortic cell clones and in balloon injured rat carotid arteries to determine the relevance of these differences in vitro to in vivo biology. The following 5 consistently differentially expressed genes were identified in vitro: zonula occludens 2 (ZO-2); peroxisome proliferator-activated receptor delta (PPARdelta); secreted protein, acidic and rich in cysteine (SPARC); alpha1(I)collagen; and A2, an uncharacterized gene. We examined these 5 clones during carotid artery injury and an inconsistently differentially expressed clone Krox-24 because, as an early response transcription factor, it could be involved in the injury response. PPARdelta, A2, and Krox-24 mRNAs were upregulated during the day after injury. ZO-2 and alpha1(I)collagen messages were modulated for up to a month, whereas SPARC message showed no consistent change. An analysis of ZO-2 and other tight junction genes indicates that tight junctions may play a role in smooth muscle biology. These data suggest that a systematic analysis of these libraries is likely to identify a very large number of differentially expressed genes. ZO-2 is particularly intriguing both because of this tight junction gene's pattern of prolonged over-expression after injury and because of its potential role in determining the distinctive epithelioid phenotype of smooth muscle cells identified in rat and other species.

Age Factors↗
PubMed1993

Altered expression and localization of the tight junction protein ZO-1 after common bile duct ligation.

Hepatocyte tight junctions form the intercellular barrier between bile and blood. Cholestasis due to common bile duct ligation (CBDL) results in structural changes in the tight junction (TJ) and an overt paracellular leak, although the molecular basis for these alterations is undefined. Using the epithelial isoform of the TJ protein ZO-1 (ZO-1 alpha +) as a marker for molecular changes in hepatocyte TJs, we investigated the effects of CBDL on ZO-1 alpha + immunofluorescence (IF) localization and on ZO-1 alpha + mRNA and protein expression over 2 wk of CBDL. ZO-1 alpha + IF staining was altered after 2 days of CBDL and appeared to accumulate in pericanalicular regions after 7 and 9 days. Quantitative immunoblotting and ribonuclease protection revealed a marked increase in hepatic ZO-1 alpha + protein expression and ZO-1 alpha + mRNA levels, respectively. In contrast to changes in ZO-1 alpha + IF, which occurred throughout the lobule, and changes in mRNA and protein expression, which were maximal after 9 days of ligation, the maximal hepatocyte proliferation occurred within 2 days after CBDL and was confined to periportal regions. CBDL results in altered hepatic localization and increased expression of the TJ protein ZO-1 alpha + and appears to represent a specific response by hepatocytes to pathological junction injury independent of cell proliferation.

Animals↗