scholarly journals Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells

2003 ◽  
Vol 284 (4) ◽  
pp. C1021-C1030 ◽  
Author(s):  
Lawrence W. Leung ◽  
Ruben G. Contreras ◽  
Catalina Flores-Maldonado ◽  
Marcelino Cereijido ◽  
Enrique Rodriguez-Boulan
Keyword(s):  
Tight Junction ◽  
Electrical Resistance ◽  
Freeze Fracture ◽  
Transepithelial Electrical Resistance ◽  
Fold Reduction ◽  
A Cell ◽  
Rat Thyroid ◽  
Sphingolipid Biosynthesis ◽  
Mdck I ◽  
Darby Canine Kidney

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Ω · cm2. When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 Ω · cm2. These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.

Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 53-59 ◽  
Author(s):  
Tracey Duff ◽  
Simon Carter ◽  
Gemma Feldman ◽  
Gordon McEwan ◽  
Walter Pfaller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Ussing Chamber ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.

scholarly journals NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1540-1546 ◽  
Author(s):  
Farideh Tafazoli ◽  
Carl Q. Zeng ◽  
Mary K. Estes ◽  
Karl-Erik Magnusson ◽  
Lennart Svensson
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Electrical Resistance ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Permeability Barrier ◽  
Filamentous Actin ◽  
Specific Effects ◽  
Polarized Epithelia ◽  
Polarized Epithelial Cells

ABSTRACT The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.

scholarly journals Novel Effects of Azithromycin on Tight Junction Proteins in Human Airway Epithelia

2006 ◽  
Vol 50 (5) ◽  
pp. 1805-1812 ◽  
Author(s):  
Valthor Asgrimsson ◽  
Thorarinn Gudjonsson ◽  
Gudmundur Hrafn Gudmundsson ◽  
Olafur Baldursson
Keyword(s):  
Cystic Fibrosis ◽  
Tight Junction ◽  
Electrical Resistance ◽  
Transepithelial Electrical Resistance ◽  
Tight Junction Proteins ◽  
Airway Epithelia ◽  
Diffuse Panbronchiolitis ◽  
Human Airway ◽  
Junction Proteins ◽  
Human Airway Epithelia

ABSTRACT The macrolide antibiotic azithromycin improves lung function and prognosis among patients with cystic fibrosis or diffuse panbronchiolitis, independently of bacterial eradication. Anti-inflammatory effects have been implicated, but data from in vivo studies are scarce, and the link between abnormal electrolyte content in airway surface liquid and bronchial infections remains uncertain. In the present study, we treated human airway epithelia on filter supports with azithromycin and monitored transepithelial electrical resistance. We found that azithromycin increased transepithelial electrical resistance of airway epithelia in a dose-dependent manner. Immunocytochemistry and Western blotting showed that addition of azithromycin changed the locations of proteins in cell cultures and induced processing of the tight junction proteins claudin-1 and claudin-4, occludin, and junctional adhesion molecule-A. These effects were reversible, and no effect was seen when cells were treated with penicillin or erythromycin. The data indicate that azithromycin increases the transepithelial electrical resistance of human airway epithelia by changing the processing of tight junction proteins. The results are novel and may help explain the beneficial effects of azithromycin in patients with cystic fibrosis, diffuse panbronchiolitis, and community-acquired pneumonia.

Tricellulin Modulates Transport of Macromolecules in the Salivary Gland

2019 ◽  
Vol 99 (3) ◽  
pp. 302-310 ◽  
Author(s):  
S.N. Min ◽  
X. Cong ◽  
Y. Zhang ◽  
R.L. Xiang ◽  
Y. Zhou ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Tight Junction ◽  
Tight Junctions ◽  
Electrical Resistance ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Specific Expression ◽  
Fluorescein Isothiocyanate Dextran ◽  
Specific Expression Pattern ◽  
Transport Of Macromolecules

Volume and composition of saliva are crucial for oral and systemic health. How substances, particularly macromolecules, are transported across the salivary gland epithelium has not been established in detail. Tricellulin is a component of tricellular tight junctions that form a central tube to serve as an important route for macromolecule transport. Whether tricellulin is expressed in the submandibular gland (SMG) and involved in salivation has been unknown. Here, by using Western blotting and immunofluorescence, tricellulin was found to be characteristically localized at tricellular contacts of human, rat, and mouse SMGs. Knockdown of tricellulin significantly increased, whereas overexpression of tricellulin decreased, paracellular permeability for 40-kDa but not for 4-kDa fluorescein isothiocyanate–dextran, while transepithelial electrical resistance was unaffected. Conversely, claudin-4 knockdown and overexpression affected transepithelial electrical resistance but not 40-kDa fluorescein isothiocyanate–dextran transport, suggesting that tricellulin regulated transport of macromolecules but not ions, which were mainly regulated by bicellular tight junctions (bTJs). Moreover, tricellulin was dynamically redistributed from tri- to bicellular membranes in cholinergically stimulated SMG tissues and cells. Immunoglobulin-like domain-containing receptor 1 (ILDR1) recruits tricellulin to tricellular contacts. The proportion of macromolecules in the saliva was increased, whereas the amount of stimulated saliva was unchanged in Ildr1-/- mice, which displayed abnormal tricellulin distribution in SMGs. Furthermore, tricellulin interacted with bTJ proteins, such as occludin, claudin-1, claudin-3, claudin-4, and ZO-1, in rat SMG epithelial polarized cell line SMG-C6. Knockdown of tricellulin decreased occludin levels. Thus, we revealed a specific expression pattern of tricellulin in SMG epithelium. Tricellulin not only functioned as a barrier for macromolecules but also modulated the connection of bTJs to the tight junction complex. Alterations in tricellulin expression and distribution could thereby change salivary composition. Our study provided novel insights on salivary gland tight junction organization and function.

scholarly journals Functional significance of the variations in the geometrical organization of tight junction networks.

1976 ◽  
Vol 68 (3) ◽  
pp. 688-704 ◽  
Author(s):  
B E Hull ◽  
L A Staehelin
Keyword(s):  
Tight Junction ◽  
Alimentary Tract ◽  
Freeze Fracture ◽  
Cell Types ◽  
Stress Conditions ◽  
Apical Region ◽  
Absorptive Cells ◽  
Stress Changes ◽  
Before And After ◽  
A Cell

Using freeze-fracture techniques, we have examined the morpholog of tight junction networks found along the length of the alimentary tract of Xenopus laevis before and after metamorphosis. We have developed the hypothesis, based on these observations, that the geometrical organization of the network determined by the stress-induced shape changes normally experienced by the cells linked by the network. Consistent with this theory, tight junctions can be classified into two distinct types of network organization which differ in their response normal and experimentally induced stress conditions: (a) loosely interconnected networks which can stretch or compress extensively under tension, thereby adapting to stress changes in the tissue; and (b) evenly cross-linked networks which retain their basic morphology under normal stress conditions. The absorptive cells of the large intestine as well as the mucous cells of the gastrointestine or stomach are sealed by the first, flexible type of tight junction. The second type of junctional organization, the evenly cross-connected network, is found between absorptive cells of the small intestine and ciliated cells of the esophagus, and reflects in its constant morphology the relative stability of the apical region of both of these cell types. Networks intermediate between these two types arise when a cell which would normally form a lossely interconnected network borders a cell which tends to form a more evenly cross-linked network, as is found in the esophagus where ciliated and goblet cells adjoin. Despite the change in the animal's diet during metamorphosis from herbivorous to carnivorous, the basic gemetrical organization of the networks associated with each tissue of the alimentary tract remains the same.

Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells

1999 ◽  
Vol 277 (6) ◽  
pp. G1138-G1148 ◽  
Author(s):  
Simonetta Ferruzza ◽  
Maria-Laura Scarino ◽  
Giuseppe Rotilio ◽  
Maria Rosa Ciriolo ◽  
Paolo Santaroni ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Electrical Resistance ◽  
Transepithelial Electrical Resistance ◽  
Complete Medium ◽  
Zonula Occludens ◽  
Intracellular Effect ◽  
Tight Junction Permeability ◽  
Zonula Occludens 1 ◽  
Effect Of Copper

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10–100 μM CuCl2(up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to l-histidine [Cu(His)2]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of64CuCl2, both exhibiting a maximum at pH 6.0. Treatment with CuCl2produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 μM Cu(His)2for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.

Protamine increases the permeability of cultured epithelial monolayers

1990 ◽  
Vol 68 (1) ◽  
pp. 220-227 ◽  
Author(s):  
M. W. Peterson ◽  
D. Gruenhaupt
Keyword(s):  
Actin Cytoskeleton ◽  
Electrical Resistance ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Type Ii ◽  
Epithelial Monolayers ◽  
Organ Specific ◽  
Darby Canine Kidney

Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

2008 ◽  
Vol 294 (4) ◽  
pp. G906-G913 ◽  
Author(s):  
Francesco Raimondi ◽  
Pasquale Santoro ◽  
Maria Vittoria Barone ◽  
Serena Pappacoda ◽  
Maria Luisa Barretta ◽  
...  
Keyword(s):  
Tight Junction ◽  
Bile Acids ◽  
Electrical Resistance ◽  
Egf Receptor ◽  
Time Frame ◽  
Paracellular Permeability ◽  
Transepithelial Electrical Resistance ◽  
Confocal Laser ◽  
Gut Permeability

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 μM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 μM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

scholarly journals Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.

1996 ◽  
Vol 134 (4) ◽  
pp. 1031-1049 ◽  
Author(s):  
M S Balda ◽  
J A Whitney ◽  
C Flores ◽  
S González ◽  
M Cereijido ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Diffusion Barrier ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Intercellular Junctions ◽  
Transepithelial Electrical Resistance ◽  
Full Length ◽  
Wild Type ◽  
Small Molecular Weight

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.

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