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PubMed1997

Transepithelial paracellular leakiness induced by chronic phorbol ester exposure correlates with polyp-like foci and redistribution of protein kinase C-alpha.

Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp-like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.

Animals↗

PubMed1993

Seasonal changes of the blood-testis barrier in viscacha (Lagostomus maximus maximus): a freeze-fracture and lanthanum tracer study.

Adult male viscachas (Lagostomus maximus maximus) were gathered from their natural habitat during the period of complete spermatogenesis (June) and during the month of maximum testicular regression (August). The testes were processed by conventional electron microscopic technique using lanthanum nitrate (electron-dense intercellular tracer) to define the intercellular spaces below the inter-Sertoli tight junctions and by freeze-fracture techniques. During complete spermatogenesis the tracer surrounds spermatogonia, preleptotene, and leptotene spermatocytes and stops at the level of the inter-Sertoli tight junctions below all germ cells displaying synaptonemal complexes (zygotene-pachytene spermatocytes) and germ cells in more advanced stages of differentiation. Conversely, during testicular regression the tracer percolates all intercellular spaces between Sertoli cells and the remaining germ cells (spermatogonia and few preleptotene and leptotene spermatocytes). During complete spermatogenesis, freeze-fracture replicas exhibit numerous inter-Sertoli tight junction strands parallel to each other and to the basal lamina. During spermatogenesis decay, the inter-Sertoli tight junctions are found to be short, tortuous, frequently interrupted, and often associated with extented membranous areas of gap junctions.

Animals↗

PubMed2003

Interleukin-1beta and barrier function of retinal pigment epithelial cells (ARPE-19): aberrant expression of junctional complex molecules.

PURPOSE: To examine the effects of interleukin (IL)-1beta on the resistance and permeability of the tight junctions of cultured retinal pigment epithelial cells. METHODS: A human RPE cell line (ARPE-19) cultured on microporous filter-supports was used. IL-1beta and monoclonal anti-IL-1beta antibody-treated IL-1beta (mAbIL-1beta) were added to the standard culture medium. Transepithelial resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein, horseradish peroxidase, and inulin was measured. The expression of the occludin and claudin was determined by real-time polymerase chain reaction (PCR), immunohistochemistry, and Western blot analysis. RESULTS: A significantly greater decrease of TER occurred in IL-1beta-supplemented medium than in standard medium plus mAbIL-1beta after several days of stimulation. A significantly greater increase of sodium fluorescein, horseradish peroxidase, and inulin permeability occurred in IL-1beta-supplemented medium than in standard medium. The expression of the occludin gene and some types of claudin genes was observed. The expression of occludin was downregulated and that of claudin-1 upregulated more in IL-1beta-supplemented medium than in standard medium by real-time PCR, immunohistochemistry, and Western blot analysis. CONCLUSIONS: The tight junctions of ARPE-19 cells are altered by IL-1beta supplementation either directly or through other factors activated by IL-1beta. The downregulation of occludin and upregulation of claudin-1 may have participated in the dysfunction of the RPE tight junctions in these in vitro experiments.

Biological Transport↗

PubMedDate not supplied

Taurine protects rat bronchioles from acute ozone exposure: a freeze fracture and electron microscopic study.

Dietary taurine has been shown to protect rat and hamster lung epithelia from acute oxidant injury. One of the earliest morphologic criteria of oxidant injury is the alteration of tight junctions of the peripheral lung airways. In the present study, we have used this criteria to evaluate whether taurine was capable of protecting rat lungs from ozone exposure. Rats were treated for 10 days with 50% taurine in their drinking water, prior to exposure to 2 ppm of ozone for 3 hours. The lungs from rats pretreated with taurine and exposed to ozone were compared to untreated rats exposed to ozone and air-exposed controls. At 2, 6, 12, 24, 48, and 72 hours after exposure to air or ozone, rats were anesthetized and the lungs perfusion-fixed through the right side of the heart with a solution of glutaraldehyde and paraformaldehyde. Light microscopy revealed the typical, mild inflammatory cell infiltrate beginning at 6 hours after ozone exposure in bronchioles, alveolar ducts, and surrounding alveoli which was absent in the lungs of animals treated with taurine. Electron microscopic analysis of thin sections indicated alterations in tight junctions which was confirmed by tracer studies using ruthenium red and lanthanum. Alterations in airway epithelium tight junctions were seen 2 and 6 hours after ozone treatment and only in the 2-hour tissues from animals pretreated with taurine prior to ozone exposure. Freeze-fracture replicas from all exposure groups by electron microscopy revealed that only the 2- and 6-hour groups showed alterations in tight junctions. The alterations were characterized by decreased number of fibrils and breaks in the fibrils. Rats treated with taurine and exposed to ozone exhibited these alterations focally at 2 hours exposure and no changes were noted at 6 hours post ozone exposure. These data confirmed previous findings that injury induced by ozone is transient and that taurine protects the bronchioles from this form of oxidant injury.

Animals↗

PubMed2001

Mucosal barrier and immune mediators.

The intestinal mucosa functions is an immunologic organ that plays a major role in the development of oral tolerance and host-defense mechanisms. Antigens must cross the intestinal epithelium in a controlled manner to interact with dendritic antigen-presenting cells, because bacteria or their products are a primary risk factor for the development of intestinal inflammation. Therefore, the regulation of the intestinal epithelial cell barrier is central to the development of intestinal immunity and inflammation, but the involved mechanisms are largely unknown. Intestinal barrier function relies on the formation of tight junctions at the apical contact areas of intestinal epithelial cells. Tight junctions have a highly dynamic structure whose permeability, assembly, or disassembly can be regulated by a variety of cellular and metabolic mediators, including cytokines, which have major functions in the immune system. Immune modulators control tight junction dependent intestinal barrier function during development, wound healing, and pathologic processes such as cancer, infection, and chronic inflammation.

Journal Article↗

PubMed2004

Rab13 mediates the continuous endocytic recycling of occludin to the cell surface.

During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells.

Adherens Junctions↗

PubMed1979

Intercellular junctional specializations in human basal cell carcinoma. A freeze-fracture study.

Intercellular junctions of various types were found on the membrane fracture faces of human nodular basal cell carcinoma (BCC) cells. The junctional types represented include desmosomes, tight junctions, and gap junctions. A semiquantitative comparison of undifferentiated and differentiated nodular BCC showed that gap and tight junctions were observed on all exposed membrane fracture interfaces of the differentiated tumors, while only fifty six per cent of the membrane interfaces of the undifferentiated tumor exhibited similar junctional specializations. These membrane specializations may be a partial reflection of differentiation among the different types of BCC and their contribution to the less invasive character of nodular BCC cannot be ruled out.

Basal Cell Carcinoma↗

PubMed1984

A thin-section and freeze-fracture study of the pulp blood vessels in feline and human teeth.

The fine structure of blood vessels with a diameter of less than 50 micrometers, i.e. small arterioles, capillaries and postcapillary venules were studied. The number of pinocytotic vesicles was greater in capillary endothelium than in arterioles and venules. There was regional variation in vesicle frequency in capillary endothelial cells (arteriolar and venule cells not studied). Fenestrations and pinocytotic vesicles were closely associated in some capillary-endothelial membrane areas and segmental differentiation of tight junctions (zonula occludens) in the vascular endothelium. The arteriolar tight junctions were classified as relatively tight, capillary junctions as leaky and venule junctions as very leaky. No nexuses (gap junctions) were found in the endothelium. Only four nexuses were found in the arteriolar smooth-muscle cells. Perivascular fibrocytes were frequently interconnected with nexuses. Nerve fibres in the vicinity of blood vessels contained granular and clear vesicles. The narrowest gap between vesicle-containing nerves and smooth-muscle cells was 0.3 micrometer. No specialized contacts were found between nerve fibres and vascular cells. Solitary cilia with a 9 + 0 pattern were occasional in capillary endothelial cells fibroblasts and odontoblasts. Only one arterio-venous anastomosis was found.

Adolescent↗

PubMed1984

Redistribution of Na-K-ATPase in the dystrophic rat retinal pigment epithelium.

Our previous studies have shown that a breakdown in tight junctions in the dystrophic retinal pigment epithelium (RPE) of Royal College of Surgeons' rats is accompanied by changes in intramembrane structure which suggest a redistribution of intramembrane particles. We have now investigated, using the p-nitrophenyl phosphate technique, the possibility that a specific membrane protein, Na-K-ATPase, is redistributed as tight junctions break down in the dystrophic RPE. In the normal RPE, Na-K-ATPase activity is restricted to the apical membrane. Junctional membranes and membranes around phagosomes are free of enzyme activity, suggesting a segregation of the transport enzyme from the junctional and phagocytic membrane. In the dystrophic RPE, prior to changes in tight junctions, enzyme activity is restricted to the apical membrane. During the initial stages of junctional breakdown, junctional membranes and membranes around cytoplasmic inclusions are also labelled. As the breakdown progresses, Na-K-ATPase activity is often present laterally and basolaterally and is sometimes absent apically. Enzyme activity is seen basally only where RPE cells have detached from Bruch's membrane and are superimposed over each other. These changes suggest that Na-K-ATPase redistributes during junctional breakdown, but that attachments between the RPE and Bruch's membrane may restrict the redistribution. The apparent reduction of enzyme activity apically suggests that active transport across the dystrophic RPE may be reduced as the tight junctions break down.

Animals↗

PubMed1978

The functional and structural border of the neurohemal region of the median eminence.

In stressed rats the tanycytes of the ventrolateral wall of the third ventricle exhibit by light microscopic immunohistochemistry a positive staining for neurohormones which is distinctly limited to the distal perivascular end of the tanycyte process. Since by electron microscopic immuncytochemistry the tanycyte cytoplasm does not show any reaction product, the light microscopic reaction most likely results from a labeling of the intercellular space in the direct vicinity of the subendothelial cleft. Whether this subendothelial space is permeable to neurohormones was tested by injection of HRP1. In the region of the arcuate nucleus 30 min after intravenous application, the marker is affixed to the membranes of the perivascular tanycyte processes in the subendothelial cleft of capillaries possessing non-fenestrated endothelia. Occasionally, HRP penetrates for a short distance between the tanycytes. Then the labeling of the intercellular cleft ends abruptly. Here, several parallel ridges of tight junctions between the perivascular distal tanycyte processes are found by the freeze-etching technique. Since HRP cannot reach the subendothelial clefts of this region by passing through capillary walls due to the presence of a blood-brain barrier, it is suggested that the marker penetrates from the median eminence this far via the subendothelial extracellular space. It is prevented from spreading further by the tight junctions of the perivascular tanycyte endings. The same way may be taken by the neurohormones. Hence, a border area exists adjacent to the dorsolateral aspect of the neurohemal region of the median eminence where the tanycytes isolate the neuropil from the cerebrospinal fluid not only by their apical tight junctions, but also by basal tight junctions from the subendothelial cleft. This communicates with the perivascular space of the portal vessels.

Animals↗

PubMed2000

Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells.

Microinjection of fluorophore-tagged cytoskeletal proteins has been a useful tool in studies of formation of focal adhesions (FA). We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (TJ) of epithelial Madin-Darby bovine kidney cells. We chose alpha-actinin and vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterized. Isolated FITC-labelled chicken alpha-actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade within an hour after injection and the cells were joined to polykaryons having 5 to 13 nuclei. Short fragments of cell membranes containing injected proteins, actin, beta-catenin, cadherin, claudin, occludin and ZO-1 were visible inside the polykaryons indicating that both AJs and TJs were disintegrated as a single complex. Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidly induced the detachment of the cells from the substratum probably due to high affinity of vinculin head to talin. Vinculin tail domain had no apparent effect on the cell morphology. Since small GTPases are involved in the building up of AJs, we injected active and inactive forms of cdc42 and rac proteins together with vinculin to see their effect. Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect. We suggest that excess alpha-actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskeleton which leads to fragmentation of junctional complexes and subsequent cell fusion. The results show that cell-cell adhesion sites are more dynamic and more sensitive than FAs to an imbalance in the amount of free alpha-actinin and intact vinculin.

Actinin↗

PubMed2004

Functional crosstalk between Wnt signaling and Cdx-related transcriptional activation in the regulation of the claudin-2 promoter activity.

Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and beta-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/beta-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression.

Base Sequence↗

PubMed1979

The tight epithelium of the Mongolian gerbil subcommissural organ as revealed by freeze-fracturing.

Examination of neonatal Mongolian gerbil subcommissural organ (SCO) by freeze-fracture electron microscopy reveals that the epithelial layer (the ependyma) possesses distinctive and very complex tight junctions which connect the apical portions of adjacent ependymal cells. The tight junction network of the Mongolian gerbil is more extensive neonatally than in the adult, and is in general, more conspicuous than in other animal species. The consistent finding of such very complex tight junctions at the interface between the C.S.F. and the SCO suggests that the SCO ependyma, at least in neonatal Monglian gerbils, might function as a tight epithelium with an ability to maintain steep solute gradients and high potential differences. The sealing-off of the SCO towards the C.S.F. (the C.S.F.--SCO barrier) and the effective blood-brain barrier in the organ (the blood-SCO barrier) might well be of functional significance, perhaps in relation to mechanisms involved in the control and/or detection of the stability of the internal environment of the brain.

Aging↗

PubMed1997

Effects of cyclosporin A on paracellular and transcellular transport of horseradish peroxidase in perfused rat livers.

The immunosuppressive agent cyclosporin A (CsA) is known to cause cholestasis. Transcellular and paracellular transport of macromolecules contribute, albeit to a minor extent, to bile formation, but little is known about the effects of CsA on these pathways. The aims of this study were to investigate the influence of CsA on tight junction (paracellular) permeability and on transcytotic vesicular pathways labeled with horseradish peroxidase (HRP) in perfused rat liver. Livers from male Sprague-Dawley rats were perfused with Krebs-Henseleit buffer (albumin 1%, RBC 20%, and amino acid mixture). Taurodehydrocholate (1 microM/min) was coinfused into the portal vein; 1 microg/ml of CsA, dissolved in Cremophor-EL (CsA livers), or the vehicle alone (CEL livers), was added to the medium. Tight junction permeability was assessed by administering HRP (25 mg) as a short pulse to perfused rat livers, operating under single-pass conditions. Under such conditions, HRP output into the bile shows two components: an initial peak at approximately 3-5 min, corresponding to paracellular transfer across tight junctions, and a second peak at approximately 15 min, corresponding to vesicular transport. Furthermore, we assessed the vesicular transport pathway by examining HRP-labeled vesicles in the perisinusoidal (PS) and pericanalicular (PC) areas using ultrastructural morphometric analysis. To analyze HRP in hepatocytes and to study rapid and late transcytotic vesicular pathways, a 1-min pulse of a high dose of HRP (500 and 200 mg, respectively) was given. Two and 18 min after single-pass perfusion the livers were fixed with 2.5% glutaraldehyde-0.8% paraformaldehyde in 0.1 mM cacodylate buffer, pH 7.8. The total pericanalicular area, the HRP-containing structures, were quantified morphometrically in liver samples. At concentrations of 1.2 microg/ml, CsA produced a twofold increase in the paracellular transfer of HRP to bile. The areas under the second peak (transcellular vesicular pathway) of the biliary HRP secretion curve were similar in CEL- and CsA-treated livers. Morphometric analysis confirmed that CsA treatment did not affect the percentage area of HRP-labeled vesicles in either the pericanalicular or in the perisinusoidal area at 2 min (rapid pathway) and 18 min (late pathway). These results indicate that CsA increases tight junctional permeability whereas it does not inhibit rapid or late transcytotic vesicle pathways.

Animals↗

PubMed1976

Redistribution of surface macromolecules in dissociated epithelial cells.

A number of ultrastructural and cytochemical techniques were used to study intact epithelial cells lining the frog urinary bladder: high resolution autoradiography after administration of [3H]glucosamine or [3H]fucose; 125I iodination of external protein; concanavalin A-peroxidase, periodic acid-chromic acid silver methenamine; and colloidal thorium. Results indicate that the material (probably glycoprotein) coating the apical surface differs from that which lines the lateral and basal surfaces. After dissociation and isolation of the epithelial cells, the material previously confined to the apical surface invaded progressively the opened "tight junctions" (about 5 min), then the lateral membranes (about 40 min), and finally the basal membrane (about 80 min): at that time, the whole cell surface was entirely enveloped by the apical material. Since, on the one hand, the reacting material was confined to the apical surface when the tight junctions were closed (in intact epithelial cells) and since, on the other hand, the apical material was sliding down the laterobasal membranes when the tight junctions were opened (in dissociated cells), it may be concluded that tight junctions contribute to maintain the cell surface specialization in epithelia.

Animals↗

PubMed2000

The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1.

A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6. We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.

Animals↗

PubMed2005

Phenotypic changes in colonocytes following acute stress or activation of mast cells in mice: implications for delayed epithelial barrier dysfunction.

BACKGROUND AND AIMS: Stressful life events are known to modulate the development or relapse of disease in both inflammatory bowel disease and irritable bowel disease patients but underlying mechanisms remain unclear. Stress is known to effect mast cells, interferon gamma (IFN-gamma), and myosin light chain phosphorylation to trigger colonic epithelial barrier dysfunction. The aim of this study was to investigate whether acute stress induced or chemical mast cell activation impaired expression and function of epithelial tight junctions, and altered colonocyte differentiation in mice. METHODS: Colonic paracellular permeability was assessed as the in vivo lumen to blood ratio of 51Cr-EDTA in different groups of mice (controls, stressed, mast cell degranulator BrX-537A treated), pretreated or not with the mast cell stabiliser doxantrazole. Involvement of mast cells and IFN-gamma was evaluated in wild-type and IFN-gamma deficient mice. Tight junction alteration was assessed by histology, transmission electron microscopy, and real time reverse transcription-polymerase chain reaction. Colonocyte differentiation was determined by protein kinase C zeta (PKCzeta) immunofluorescence and western blotting, and alkaline phosphatase activity assay. RESULTS: Acute stress induced a three day delayed increase in colonic paracellular permeability which involved mast cell degranulation and overproduction of IFN-gamma. The colonic epithelial barrier was morphologically altered and expression of mRNA encoding tight junction proteins ZO-2 and occludin was decreased. Moreover, three days after acute stress, colonocyte differentiation was reduced, as shown by decreased expression of both PKCzeta isotype and alkaline phosphatase. CONCLUSION: These data highlight new mechanisms whereby an acute stress acts on the gastrointestinal tract by inducing alterations in colonocyte differentiation and decreased expression of mRNA encoding tight junction proteins. Thus phenotypic changes in colonocytes could pave the way for stress related intestinal disorders.

Alkaline Phosphatase↗

PubMed1999

Cellular mechanisms of blood-retinal barrier dysfunction in macular edema.

PURPOSE: To determine the mechanism of blood-retinal barrier (BRB) dysfunction in human and experimental specimens using immunocytochemistry. METHODS: Extravascular albumin was localized in clinical specimens and retinas from transgenic mice that overexpress vascular endothelial growth factor (VEGF) in the photoreceptors. Transgenic mouse retinas were also labeled with Griffonia simplicifolia isolectin-B4 (GSA), a lectin that binds to endothelial cells. RESULTS: The BRB is established by the presence of tight junctions between the retinal vascular endothelial (RVE) cells and the RPE cells and by a paucity of intraendothelial cell vesicles. When BRB breakdown occurs in human ocular disorders such as diabetic retinopathy, retinitis pigmentosa, or cystoid macular edema, staining for extravascular albumin reveals leakage through the tight junctions, an upregulation of intraendothelial vesicles, and permeation of RVE or RPE cells that have undergone degenerative changes. VEGF, in addition to inducing neovascularization (NV), promotes vascular leakage. In VEGF transgenic mice, BRB failure is confined to the outer retina, the area where NV occurs. GSA binds to the luminal and abluminal surfaces of RVE cells in new and established vessels and to intraendothelial vesicles and interendothelial cell junctions in areas of vascular leakage. CONCLUSION: BRB dysfunction may be mediated by leakage through the tight junctions of RVE or RPE cells, by trans-endothelial vesicular transport, or by permeation of RVE or RPE cells that have undergone degenerative changes. GSA may be a useful marker to assist in recognizing open tight junctions and an increase in intraendothelial cell vesicles, which are indicative of BRB failure.

Animals↗