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PubMed1995

Characterization of drug transport through tight-junctional pathway in Caco-2 monolayer: comparison with isolated rat jejunum and colon.

Drug transport through the tight-junctional pathway in Caco-2 monolayer was studied by examining the relationship between its permeability to hydrophilic drugs and membrane conductance. Compared with the rat isolated jejunum or colon, Caco-2 monolayer displayed high electrical resistance and low conductance, as well as low permeability to sulfanilic acid and FITC-dextran (M.W. 4000). However, there was a linear relationship between the drug permeability and partial Cl- ion conductance for Caco-2 monolayer, rat jejunum and colon. Hence, the permeability to those drugs per unit of Cl- conductance is similar in the three membranes, suggesting that the size (radius) of the tight-junctional pathway in the three membranes is similar. In addition, when the electrical resistance of Caco-2 monolayer was reduced to the same level as that of the jejunum or colon by pretreatment with disodium ethylenediaminetetraacetate, its permeability to FITC-dextran became significantly higher than that of other membranes. Accordingly, the high resistance and the low permeability of Caco-2 monolayer compared with rat intestinal membrane may be due to structural differences between the membranes, rather than a difference in the tightness of the junction.

Animals↗
PubMed2002

A ZO1-GFP fusion protein to study the dynamics of tight junctions in living cells.

To examine the dynamics of tight junctions (TJs) in living cells, chimera between the TJ-associated protein ZO-1 and green fluorescent protein (GFP) were constructed. If ZO-1 fused to the C-terminus of GFP (ZO1-CGFP) was stably expressed in MDCK cells, it was fully incorporated into TJs and colocalized with endogenous ZO-1. The GFP tag did not influence cell growth, transepithelial electrical resistance, and paracellular mannitol transport. The morphology of the transfected cells was unchanged. The ZO1-CGFP MDCK cell line thus represents an excellent tool to study TJ dynamics. The influence of the external calcium ion concentration on the formation and dynamics of TJs in living cells was thus explored. Upon opening of the TJs under short-term treatment with EGTA (up to 20 min), the localization of ZO1-CGFP at the membrane persisted. The rim-like pattern around the individual cells appeared fuzzier than in non-treated cells. Long-term calcium depletion resulted in the localization of ZO1-CGFP in the cytoplasm and the nucleus. After restoration of normal Ca(2+) concentrations, cell-cell contacts were restored and the localization of ZO1-CGFP was indistinguishable from the one in control cells kept at normal Ca(2+) concentrations. It remains open how the different localizations of ZO-1 correspond to changes in the signal transduction activity of the molecule.

Animals↗
PubMed1979

Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas.

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.

Animals↗
PubMed1979

Tight junctions in a fluid-transporting epithelium of an insect.

Occluding junctions have been found between the lateral cell borders at the base of the rectum of Periplaneta americana. They appear as punctate membrane appositions in thin sections, and after incubation in physiological solutions containing lanthanum before fixation the inward penetration of tracer is impeded in this same basal area. Moreover, freeze-fracture studies of this region reveal simple linear ridges on fracture face P and grooves on fracture face E, which are similar to the less complex vertebrate tight junctions. The luminal clefts, which permit free inward diffusion of tracers, present no tight junctions, but do have septate junctions. These results support the contention that, contrary to earlier speculation, arthropods do possess tight junctions; these, rather than septate junctions, appear to form the morphological basis of at least some of the permeability barriers observed in invertebrates.

Animals↗
PubMed1992

Implications of a non-lamellar lipid phase for the tight junction stability. Part II: Reversible modulation of transepithelial resistance in high and low resistance MDCK-cells by basic amino acids, Ca2+, protamine and protons.

The transepithelial resistance of confluent epithelial cell monolayers was monitored to investigate the influence of basic amino acids, Ca2+, protamine and protons on tight junction electrical resistance. In an accompanying paper we investigated the effect of these substances on the lamellar/hexagonal II phase transition in reconstituted phospholipid membranes containing phosphatidylserine and phosphatidylethanolamine. We conclude that the permeability of tight junctions may be described by a lipid phase equilibrium where the lamellar phase corresponds to an open state and the hexagonal lipid phase to the closed state of the cell contact. This dynamic lipid model is well suited to describe the morphological as well as functional properties of the tight junctions.

Amino Acids↗
PubMed2006

Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement.

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Adolescent↗
PubMed1992

Rabbit corneal epithelial wound repair: tight junction reformation.

Previous studies have shown that the corneal epithelium will close a limbus to limbus scrape wound in four to five days, but requires 10 days to become firmly attached to the stroma. In order to determine if the restoration of the corneal epithelial barrier follows a similar sequence, we have used freeze-fracture to study tight junction reformation in a rabbit epithelial wound model. Dutch-belted rabbit corneal epithelium was removed with a limbus to limbus scrape wound and sampled from 0 to 30 days post-wounding. A minimum of 3 animals from each time point were processed for electron microscopy and freeze-fracture. Freeze-fracture showed that the cells at the wound margin had a reduction in the number of intramembrane particles on their apical surface. In areas adjacent to the wound edge, fragments of tight junctions were first observed on two days post-wounding specimens. The junctions became progressively more complex in the area behind the wound edge until wound closure at four days when the junctions were also present in the central region of the cornea. The maturation of the junctions continued and at five days after surgery they resembled control junctions. This sequence suggests that the the establishment of morphologically mature tight junctions may be necessary before the corneal epithelium can firmly reattach to the stroma.

Animals↗
PubMed2005

Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Adult↗
PubMed2001

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells.

Zonula occludens protein 1 (ZO-1) is a cytosolic tight junction protein that tethers transmembrane proteins such as occludin, claudin and junctional adhesion molecule to the actin cytoskeleton. The interaction between ZO-1 and claudin or junctional adhesion molecule occurs via the amino-terminal PSD95/Dlg/ZO-1 (PDZ) domains in ZO-1. A yeast two-hybrid screen to search for proteins that interact with the PDZ domains of ZO-1 identified connexin (Cx) 45. Cx45 interacts with the PDZ domains of ZO-1 and ZO-3, but not ZO-2, via a short C-terminal PDZ binding motif (SVWI). In transfected epithelial Madin-Darby canine kidney cells, Cx45 co-localizes with endogenous ZO-1 at or near tight junctions and co-precipitation experiments show that Cx45 and ZO-1 directly interact. Inactivating the C-terminal PDZ-binding motif in Cx45 affects its co-precipitation and co-localization with ZO-1. The growing number of connexins (i.e. Cx43 and Cx45) that can associate with ZO proteins indicate that ZO proteins may play a more general role in organizing gap junctions and/or in recruiting signaling molecules that regulate intercellular communication.

Animals↗
PubMed2003

Role of iNOS in hepatocyte tight junction alteration in mouse model of experimental colitis.

A variety of hepatobiliary abnormalities occur in inflammatory bowel diseases (IBDs). The role of tight junction (TJ) in hepatobiliary complications have been well described. The purpose of this study was to investigate the role of inducible nitric oxide (NOS) in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated iNOS wild-type (WT) mice, DNBS-treated iNOS knock out mice (iNOSKO) mice experienced a significant less rate of the extent and severity of the histological signs of colon injury. Colon levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also significantly reduced in iNOS-KO mice in comparison to wild-type mice. Liver histology from iNOSKO and wild-type mice iNOSWT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and alanine aminotransferase, were significantly reduced in DNBS-iNOSKO mice vs DNBS-iNOSKO mice. Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice, lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional iNOS gene in iNOSKO mice resulted in a significant reduction of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labeling of frozen liver sections from DNBS-iNOSWT mice showed a significant alteration of the immunolocalization for claudin-1 and zonula occludens (ZO)-1. In contrast, a significant reduced alteration in the localization of the immunosignals for claudin-1 and ZO-1 was observed in the liver from iNOSKO mice after DNBS administration. In conclusion, we suggest that the iNOS may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.

Animals↗
PubMed1979

The formation of gap and tight junctions between retinal pigment cells in cell cultures.

The reformation of the junctional complex of retinal pigment cells was studied after trypsin disaggregation and in vitro reaggregation. Control specimens show a zonula occludens (tight junction) with integrated gap junctions and very large macular gap junctions. Isolation after trypsination results in disaggregation of the large gap junctions and fragmentation of the tight junctions with disaggregation of their integrated gap junctions. After two to four days of incubation the restoration of the zonula occludens is complete. After approximately five days of incubation, large gap junctions are found with a patchy arrangement of particles similar to that seen in vivo.

Animals↗
PubMed1997

Extracellular Ca2+ directly regulates tight junctional permeability in the human cervical cell line CaSki.

Lowering extracellular calcium concentration ([Ca2+]o) increases acutely and reversibly the transepithelial electrical conductance (G(TE)) and the epithelial permeability to pyranine (Ppyr) across CaSki cultures. Effects were already observed after lowering calcium from 1.2 to 1.0 mM and were maximal at 0.1 mM. The dose-response curves were sigmoidal (calcium concentration that produces half-maximal effect = 0.3 mM), and the time courses indicated simple exponential trends (time constants of 4-5 min). The effect of calcium was not mediated by mobilization of cytosolic calcium or altering calcium influx, and manganese was found to be a partial agonist to [Ca2+]o. The effects of [Ca2+]o, on permeability were additive to those of hypertonic conditions, indicating that calcium modulates junctional permeability. The experimental data were fitted to theoretical models that relate changes in G(TE) to the probability of assembled/disassembled tight junctions. The results suggest that calcium interacts directly and cooperatively at extracellular sites with junctional elements that are arranged in parallel, and it shifts the probability state of the junctions from "open" to "closed" state. Changes in extracellular calcium may affect the permeability of tight junctions of the cervical epithelium and may play a role in regulating production of cervical mucus.

Calcium↗
PubMed2001

Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C.

Zonula occludens 2 (ZO-2) protein is a tight-junction phos phorylated protein that belongs to the membrane-associated guanylate kinase ('MAGUK') family. Here we study the interaction between ZO-2 and protein kinase C (PKC). We have constructed two ZO-2 fusion proteins of the middle (3PSG) and C-terminal (AP) regions of the molecule and demonstrate that they are phosphorylated by PKC isoenzymes beta, epsilon, lambda and zeta. To understand the physiological significance of the interaction between ZO-2 and PKC, we analysed the phosphorylation state of ZO-2 immunoprecipitated from monolayers with mature tight junctions or from cells that either lack them or have them disassembled through Ca(2+) chelation. We found that in the latter condition the phosphorylation level of ZO-2 is significantly higher and is due to the action of both PKC and cAMP-dependent protein kinase. These results therefore suggest that the phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex.

Animals↗
PubMed1995

Transforming growth factor-alpha abrogates glucocorticoid-stimulated tight junction formation and growth suppression in rat mammary epithelial tumor cells.

The glucocorticoid and transforming growth factor-alpha (TGF-alpha) regulation of growth and cell-cell contact was investigated in the Con8 mammary epithelial tumor cell line derived from a 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma. In Con8 cell monolayers cultured on permeable filter supports, the synthetic glucocorticoid, dexamethasone, coordinately suppressed [3H]thymidine incorporation, stimulated monolayer transepithelial electrical resistance (TER), and decreased the paracellular leakage of [3H]inulin or [14C]mannitol across the monolayer. These processes dose dependently correlated with glucocorticoid receptor occupancy and function. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevented the glucocorticoid growth suppression response and disrupted tight junction formation without affecting glucocorticoid responsiveness. Treatment with hydroxyurea or araC demonstrated that de novo DNA synthesis is not a requirement for the growth factor disruption of tight junctions. Immunofluorescence analysis revealed that the ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells and that TGF-alpha caused-ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, our results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha, can override both effects of glucocorticoids. These results have uncovered a novel functional "cross-talk" between glucocorticoids and TGF-alpha which potentially regulates the proliferation and differentiation of mammary epithelial cells.

Animals↗
PubMed1998

Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood-brain barrier breakdown in vivo.

The tight junctions found between cerebral vascular endothelial cells form the basis of the blood-brain barrier. Breakdown of the blood-brain barrier is a feature of a variety of CNS pathologies that are characterized by extensive leucocyte recruitment, such as multiple sclerosis and stroke. The molecular mechanisms associated with opening of the blood-brain barrier and leucocyte recruitment in vivo are currently poorly understood. We have used an in vivo rat model to investigate the molecular response of the CNS endothelium to neutrophil adhesion and migration. Injection of interleukin-1 beta into the striatum of juvenile brains results in a neutrophil-dependent increase in vessel permeability at 4 h. Only a subset of blood vessels were associated with neutrophil recruitment. These particular vessels displayed an increase in phosphotyrosine staining, loss of the tight junctional proteins, occludin and zonula occludens-1, and apparent redistribution of the adherens junction protein vinculin. Examination of these vessels under the electron microscope indicated that the cell-cell adhesions in such vessels are morphologically different from normal junctions. This study provides the first direct evidence in vivo that leucocyte recruitment can trigger signal transduction cascades leading to junctional disorganization and blood-brain barrier breakdown. Our results have established an endothelial cell molecular profile associated with leucocyte-induced blood-brain barrier breakdown in vivo, and the relevance of different in vitro cell culture models may now be viewed more objectively.

Aging↗
PubMed1997

Canalicular retention as an in vitro assay of tight junctional permeability in isolated hepatocyte couplets: effects of protein kinase modulation and cholestatic agents.

A simple, fast method to evaluate acute changes of tight junctional permeability in isolated hepatocyte couplets is proposed. The method consists of the recording of the number of canalicular vacuoles able to retain the previously accumulated fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF), as visualized by inverted fluorescent microscopy, following acute exposure to the compounds under study. The method was validated by (i) making a systematic documentation of the effect on CLF retention of a variety of hormonal modulators (vasopressin and phorbol esters), as well as several cholestatic (taurolithocholic acid, cyclosporin A, and estradiol 17 beta-glucuronide) and hepatotoxic agents (menadione, A23187, and t-butyl hydroperoxide), all known to affect biliary permeability in intact liver, and (ii) carrying out a comparative analysis of the results obtained with those recorded using rapid canalicular access of horseradish peroxidase (HRP) as an alternative procedure. The compounds tested all decreased canalicular vacuolar retention of CLF in a dose-dependent manner. Vasopressin- and phorbol ester-induced decline in CLF retention were prevented by pretreatment with the protein kinase C inhibitors H-7 and staurosporine, thus confirming a role for this enzyme in canalicular permeability regulation. A significant direct correlation (r = 0.934, p < 0.001) was obtained when the decrease in canalicular retention of CLF was compared with the increment in the canalicular access of HRP. Image analysis revealed that cellular fluorescence was not increased following exposure to these compounds, suggesting a paracellular rather than transcellular route for CLF egress. These results all support canalicular vacuolar retention of CLF as a suitable method to readily evaluate acute changes in tight junctional permeability in isolated hepatocyte couplets induced by physiological modulators or hepatotoxic agents.

Animals↗
PubMed1988

Development, organization, and function of tight junctional complexes in the tracheal epithelium of infant ferrets.

The surface epithelium of newborn ferret airways matures rapidly in the first month of life. Prominent developmental features include a transition from predominantly non-ciliated to ciliated cells, quantitative and qualitative changes in secretion of macromolecules, and a transition from secretory to absorptive patterns of ion transport. Freeze-fracture replicas of ferret tracheal epithelium from 0 to 28 days of age exhibited progressive developmental patterns in tight junctional structure from beaded, unclosed patterns in newborns to more closed patterns at 28 days. Strand number increased while the depth of tight junctional structures and the proportion of strands exhibiting discontinuity decreased postnatally. Total transepithelial conductance, paracellular conductance, and cell size decreased over the first month. Our data suggest that changes in physiological parameters that reflect epithelial tight junction permeability can be attributed, at least in part, to maturation of this intercellular junction during the postnatal period.

Animals↗
PubMed1984

Preservation of the structural integrity of tight junctions during the freeze-fracture of ciliary epithelium.

It has recently been reported that losses of tight junction material could result from the freeze-fracture process. To verify this assumption, we tried to increase the possibility, if any, of losses of junction material, by inducing an important fragmentation of junctional fibrils by bathing ciliary epithelium in a 0.5M sucrose solution before glutaraldehyde fixation and freeze-fracturing at -160 degrees C. In spite of a significant redistribution of junctional material on both fracture faces, careful examination of complementary replicas and measurements of junction elements and interruption lengths showed that no loss of junctional material occurred in this tissue. The influence of physical parameters (i.e. temperature) on the preservation of the structural integrity of the tight junction during fracturing is now a problem to be considered.

Animals↗