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At least 1,135 records · Page 63Linked to original sources
PubMed1996

Tight junction dynamics in the frog urinary bladder.

In a previous study in frog skin (Castro et al., J. Memb. Biol. 134:15-29, 1993), it was shown that TJs experimentally disrupted by a selective deposition of BaSO4 could be resealed upon addition of Ca2+ to the apical solution; in the absence of apical Ca2+, the normal Ca2+ activity of the Na2SO4-Ringer's bathing the basolateral side was not able to induce TJ resealing. We now show that apical Ca2+ also activates the TJ sealing mechanism in frog urinary bladders. Three known procedures were utilized to increase TJ permeability, all in the absence of apical Ca2+: (i) exposure to high positive transepithelial clamping potentials; (ii) exposure of the apical surface to hypertonic solutions; and (iii) selective deposition of BaSO4 in the TJs. The resealing of the TJs was promoted by raising the concentration of Ca2+ in the apical solution. This effect of Ca2+ is not impaired by the presence of Ca2+ channel blockers (nifedipine, verapamil, Mn2+ or Cd2+) in the apical solution, indicating that junction resealing does not depend on Ca2+ entering the cells through the apical membrane. TJ resealing that occurs in response to raised apical Ca2+ most likely results from a direct effect of Ca2+, entering the disrupted TJs from the apical solution and reaching the zonula adhaerens Ca2+ receptors (E-cadherins). Protein kinase C (PKC) must play a significant role in the control of TJ assembly in this tight epithelia since the PKC inhibitor (H7) and the activator (diC8) markedly affect TJ recovery after disruption by apical hypertonicity. H7 treated tissues show marked recuperation of conductance even in the absence of apical Ca2+. In contrast, diC8 prevents tissue recuperation which normally occurs after addition of Ca2+ to the apical solution.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2006

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force.

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead. Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

Animals↗
PubMed2006

Bench-to-bedside review: the role of activated protein C in maintaining endothelial tight junction function and its relationship to organ injury.

Activated protein C (APC) has emerged as a novel therapeutic agent for use in selected patients with severe sepsis, even though the mechanism of its benefit is not well established. APC has anticoagulant, anti-inflammatory, antiapoptotic, and profibrinolytic properties, but it is not clear through which of these mechanisms APC exerts its benefit in severe sepsis. Focus has recently turned to the role of APC in maintaining endothelial barrier function, and in vitro and in vivo studies have examined this relationship. This article critically reviews these studies, with a focus on potential mechanisms of action.

Endothelium↗
PubMed2004

Transforming growth factor-beta1 upregulates the tight junction and P-glycoprotein of brain microvascular endothelial cells.

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Ochratoxin A increases permeability through tight junctions by removal of specific claudin isoforms.

On interaction with the intestine, the mycotoxin ochratoxin A is know to cause rapid inflammation, diarrhea, and increased bacterial translocation. All these effects are consistent with a decrease in epithelial barrier function. However, this has not been shown directly. We determined that ochratoxin A is able to reduce the barrier properties of the model intestinal cell line Caco-2. Over 24 h, ochratoxin A reduces the transepithelial electrical resistance of Caco-2 monolayers growing on Transwell filters by approximately 40%. At the same time, the permeability of the monolayer is increased with respect to 4- and 10-kDa FITC dextrans, but not to 20- or 40-kDa dextrans. Immunoblotting and immunofluorescence reveal that the decrease in barrier properties is concomitant with disappearance of claudins 3 and 4, but not claudin 1 from Caco-2 cell membranes. These results suggest that ochratoxin A is able to modulate the barrier function of Caco-2 cells by removal of specific claudin isoforms.

Caco-2 Cells↗
PubMed2004

Hypoxia-induced changes in tight junction permeability of brain capillary endothelial cells are associated with IL-1beta and nitric oxide.

We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.

Animals↗
PubMed1996

Signal transduction and glial cell modulation of cultured brain microvessel endothelial cell tight junctions.

Noncontact coculture of postconfluent bovine brain microvessel endothelial cell (BMEC) monolayers with rat C6 glioma cells results in markedly decreased transmonolayer permeability measured by transendothelial electrical resistance (TER) and solute flux. An elevation in adenosine 3',5'-cyclic monophosphate (cAMP) in response to forskolin correlates with an increase in TER through a threshold event; however, unlike forskolin, the severalfold increase in TER induced by C6 cells is cAMP independent. Activation of protein kinase C (PKC) enhances the C6 cell-induced increase in TER, and PKC inhibition blocks C6 cell induction. Treatment of control or C6 cell-induced BMEC monolayers with pertussis toxin immediately and irreversibly obliterates TER, without an apparent change in guanosine 3',5'-cyclic monophosphate levels or viability, indicating the importance of a G protein-mediated event. A similar effect is observed with transforming growth factor-beta 1, and both responses are polarized, since the loss in TER is significantly faster in BMEC monolayers exposed basolaterally. These results suggest that astroglia modulate the blood-brain barrier through a cAMP-independent PKC-dependent pathway maintained by a G protein-coupled mechanism.

Animals↗
PubMed2003

Association of nonreceptor tyrosine kinase c-yes with tight junction protein occludin by coimmunoprecipitation assay.

Immunoprecipitation is one of the most commonly used techniques to study protein-protein interaction in vivo. There are three major steps involved in an immunoprecipitation procedure: 1) lysis of the cells to make the antigen of interest available; 2) formation of the antibody-antigen complex by adding specific antibody; 3) separation and detection of the immune complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Both polyclonal and monoclonal antibodies can be used to immunoprecipitate the antigen. Polyclonal antibodies usually bind to multiple sites on the antigen and, therefore, are more efficient than monoclonal antibodies. The advantage of using monoclonal antibodies for immunoprecipitations is the specificity of the antibody-antigen interaction because monoclonal antibodies bind to only one epitope of the antigen. If the antigen of interest is tyrosine phosphorylated, and this tyrosine phosphorylation is under investigation, each step has to be handled with extra care to ensure success of the procedure. The buffer used to lyse the cells depends on the nature of the antigen, with more stringent conditions required for integral membrane proteins than for soluble cytosolic proteins. In either case, it is very important to add the protease inhibitors to the lysis buffer in order to prevent degradation of the antigen.

Epithelial Cells↗
PubMed2000

Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate.

The airway epithelium is resistant to infection by gene transfer vectors when infected from the luminal surface. One strategy for enhancing airway epithelial gene transfer is to modify paracellular permeability, thereby permitting the diffusion of vectors to the basolateral surface, where uptake receptors are expressed. We investigated the ability of a medium-chain fatty acid known to enhance drug absorption, sodium caprate (C10), to increase airway paracellular permeability in comparison with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Apical application of C10 decreased transepithelial resistance by > 90% within minutes, whereas EGTA required an hour or more to produce a similar effect. C10 increased mannitol and dextran permeability by sevenfold, as compared with a twofold increase produced by EGTA. A greater enhancement of adenoviral lacZ gene transfer was mediated by C10 (50-fold over controls) than by EGTA (10-fold over controls). This correlated with a significant enhancement of adenoviral CFTR-mediated correction of Cl(-) transport in polarized human airway epithelial (HAE) cells from cystic fibrosis (CF) patients. Confocal microscopy revealed a redistribution of claudin-1 following C10 but not EGTA treatment as a possible mechanism of gene-transfer enhancement by C10. These data suggest that C10 may be a better agent for enhancing gene transfer than is EGTA, and that this effect occurs through disruption of claudin-1.

Decanoic Acids↗
PubMed2005

Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier.

EspG, a secreted effector of enteropathogenic Escherichia coli (EPEC), as well as its homologue Orf3, has been shown to disrupt microtubules (MTs) in fibroblasts and non-polarized epithelial cells. The roles of MTs and the effects of MT disruption in these cell types differ significantly. The aim of this study was to investigate the effects of EspG on polarized, host target intestinal epithelial cells. Immunofluorescent labelling of tubulin showed that EPEC caused progressive fragmentation and loss of the MT network in cells harbouring attached organisms. Immunoblots of proteins extracted from EPEC-infected cells showed a corresponding loss of alpha-tubulin. Type III secretion system (TTSS)-deficient strains had no effect on MT suggesting TTSS dependence. Mutation of espG, but not espF or map, ablated EPEC's effects on MTs for up to 6 h. Ectopic expression of EspG in HeLa cells caused MT disruption. While deletion of espG alone had no effect on the EPEC-induced decrease in transepithelial electrical resistance (TER), mutation of both espG and orf3 significantly delayed the kinetics of this response. Complementation of the double mutant with espG alone restored the kinetics of TER drop to that of wild type. Herein, we describe a previously unrecognized phenotype for the EPEC effectors EspG and Orf3.

Bacterial Adhesion↗
PubMed2007

Cleavage of MAGI-1, a tight junction PDZ protein, by caspases is an important step for cell-cell detachment in apoptosis.

MAGI-1, a member of the MAGUK family of proteins, is shown to be rapidly cleaved during Fas-induced apoptosis in mouse 3T3 A31 cells, and in UV irradiation- and staurosporine-induced apoptosis in HaCaT cells. This generates a 97 kDa N-terminal fragment that dissociates from the cell membrane; a process that is largely prevented in the presence of the caspase inhibitor Z-VAD-fmk. In addition, we show that in vitro translated radiolabelled MAGI-1 is efficiently cleaved into 97 kDa and 68 kDa fragments by caspases-3 and -7 at physiological concentrations and mutating the MAGI-1 Asp(761) to Ala completely abolished the caspase-induced cleavage. Moreover, in HaCaT cells overexpressing the MAGI-1 Asp(761)Ala mutant the disruption of cell-cell contacts was delayed during apoptosis, whereas other caspase-dependent processes such as nuclear condensation were not affected, suggesting that cell detachment is parallel to them. Thus, MAGI-1 cleavage appears to be an important step in the disassembly of cell-cell contacts during apoptosis.

3T3 Cells↗
PubMed2003

Interleukin-6 changes tight junction permeability and intracellular phospholipid content in a human enterocyte cell culture model.

Proinflammatory cytokines and secretory phospholipase A(2) (sPLA(2)) are elevated in patients with inflammatory bowel disease (IBD). We previously reported that the proinflammatory cytokine IL-6 increased the expression of sPLA(2) (a hydrolyzer of phosphatidylcholine) and decreased membrane integrity in an intestinal epithelial cell culture model. To determine the physiological effects of the IL-6 mediated increase in sPLA(2) on decreased epithelial layer integrity, we investigated alterations of intracellular/secretory phospholipid (PL) composition in a cell culture model. In addition, since other PLs may also mediate epithelial membrane activity, we investigated the effect of IL-6 on PL activity in a Caco-2 enterocyte culture model. Caco-2 cells were incubated for 72 h with IL-6 or media alone (control). Both media and cell lysate were analyzed for PL composition using thin-layer chromatography. The PL composition in the media did not show any differences between the two groups ( p>0.1). Total intracellular PL contents were also unchanged; however, IL-6 led to significant changes in PL composition including an increase in phosphatidylethanolamine (PE) and sphingomyelin (SM) and a decrease in phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) ( p<0.05). Both PE and SM are known as inflammatory signaling factors involved in human IBD. Our study suggests that the decreased membrane integrity seen with IL-6 application may occur via intracellular PL alterations, rather than through the direct effects of sPLA(2).

Caco-2 Cells↗