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PubMed2005

Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins.

Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.

Animals↗
PubMed2001

Enteropathogenic Escherichia coli activates ezrin, which participates in disruption of tight junction barrier function.

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic E. coli and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.

Animals↗
PubMed1998

Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells.

Medium chain fatty acids (MCFAs) are used to enhance the permeability of mucosal tissues to hydrophilic drugs, but their mechanism of action is largely unknown. In this study, the absorption-enhancing effects of the sodium salts of two MCFAs, capric acid (C10) and lauric acid (C12), were studied in monolayers of human intestinal epithelial Caco-2 cells. Both MCFAs induced a rapid increase in epithelial permeability to the hydrophilic marker molecule sodium fluorescein. Inhibition of phospholipase C and inhibition or activation of various kinases and buffering of intracellular calcium indicated that the effects on epithelial permeability were mediated through phospholipase C-dependent inositol triphosphate/diacylglycerol pathways. Surprisingly, the inositol triphosphate and diacylglycerol pathways were found to have opposing effects on paracellular permeability. Exposure to the MCFAs also resulted in a concentration dependent reduction of cellular dehydrogenase activity and ATP levels. C10, but not C12, induced redistribution of the tight junction proteins ZO-1 and occludin. These results indicate that the two MCFAs have partially different and more complex mechanisms than previously recognized, which has important implications for their use in vivo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2004

Zebrafish mosaic eyes is a novel FERM protein required for retinal lamination and retinal pigmented epithelial tight junction formation.

Polarization is a common feature of many types of cells, and we are beginning to understand how cells become polarized. The role of cell polarity in development and tissue morphogenesis, however, is much less well understood. Our previous analysis of the mosaic eyes (moe) mutations revealed that moe is required for retinal lamination and also suggested that zebrafish moe function is required in the retinal pigmented epithelium (RPE) for the proper localization of adjacent retinal cell divisions at the apical neuroepithelial surface. To understand the function of moe in the RPE, we cloned the moe locus and show that it encodes a novel FERM (for 4.1 protein, ezrin, radixin, moesin) domain-containing protein. Expression of moe in the eye, kidney, and brain reflects phenotypes found in moe(-) mutants, including RPE and retinal lamination defects, edema, and small or absent brain ventricles. We show that moe function is required for tight junction formation in the RPE. We suggest that moe may be a necessary component of the crumbs pathway that regulates apical cell polarity and also may play a role in photoreceptor morphogenesis.

Amino Acid Sequence↗
PubMed2005

Glucocorticoid regulation of endothelial cell tight junction gene expression: novel treatments for diabetic retinopathy.

Loss of blood-retinal barrier (BRB) integrity and vascular permeability characterizes diabetic retinopathy, and new therapies to reverse or prevent vascular permeability are needed to treat this debilitating disease. Glucocorticoids are currently under investigation for use as a local therapeutic treatment for diabetic retinopathy. This review examines the changes that occur to barrier properties in diabetic retinopathy and the potential to use glucocorticoids to restore vascular barrier properties in the retina. Glucocorticoids are useful in preserving the integrity of the blood-brain barrier in the treatment of brain tumors, and these steroids show similar effects on the retinal vasculature suggesting their potential usefulness in treating diabetic retinopathy. Recent progress has been made toward the goal of elucidating the precise mechanism underlying the protective effects of glucocorticoids on the retinal vasculature. Glucocorticoids may act by both suppressing inflammation and by directly affecting the endothelial cells by regulating phosphorylation, organization, and content of tight junction proteins. Further work will advance our understanding of glucocorticoid regulation of barrier properties allowing the ultimate goal of developing a specific and safe therapy to treat or prevent loss of the blood-neural barrier in a number of diseases, including brain tumors and diabetic retinopathy.

Animals↗
PubMed2005

Transforming growth factor-beta regulation of epithelial tight junction proteins enhances barrier function and blocks enterohemorrhagic Escherichia coli O157:H7-induced increased permeability.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an enteric pathogen that causes potentially fatal symptoms after intimate adhesion, modulation of intestinal epithelial signal transduction, and alteration of epithelial function (eg, barrier disruption). Although the epithelial barrier is critical to gut homeostasis, only a few agents, such as transforming growth factor (TGF)-beta, can enhance or protect epithelial barrier function. Our aims were to delineate the mechanism(s) behind TGF-beta-induced barrier enhancement and to determine whether TGF-beta could prevent EHEC-induced barrier disruption. Using monolayers of the human T84 colonic epithelial cell line, we found that TGF-beta induced a significant increase in transepithelial electrical resistance (a measure of paracellular permeability) through activation of ERK MAPK and SMAD signaling pathways and up-regulation of the tight junction protein claudin-1. Additionally, TGF-beta pretreatment of epithelia blocked the decrease in transepithelial electrical resistance and the increase in transepithelial passage of [(3)H]-mannitol caused by EHEC infection. EHEC infection was associated with reduced expression of zonula occludens-1, occludin, and claudin-2 (but not claudin-1 or claudin-4); TGF-beta pretreatment prevented these changes. These studies provide insight into EHEC pathogenesis by illustrating the mechanisms underlying TGF-beta-induced epithelial barrier enhancement and identifying TGF-beta as an agent capable of blocking EHEC-induced increases in epithelial permeability via maintenance of claudin-2, occludin, and zonula occludens-1 levels.

Cell Line, Tumor↗
PubMed2006

Tyrosine phosphorylated Par3 regulates epithelial tight junction assembly promoted by EGFR signaling.

The conserved polarity complex, comprising the partitioning-defective (Par) proteins Par3 and Par6, and the atypical protein kinase C, functions in various cell-polarization events and asymmetric cell divisions. However, little is known about whether and how external stimuli-induced signals may regulate Par3 function in epithelial cell polarity. Here, we found that Par3 was tyrosine phosphorylated through phosphoproteomic profiling of pervanadate-induced phosphotyrosine proteins. We also demonstrated that the tyrosine phosphorylation event induced by multiple growth factors including epidermal growth factor (EGF) was dependent on activation of Src family kinase (SFK) members c-Src and c-Yes. The tyrosine residue 1127 (Y1127) of Par3 was identified as the major EGF-induced phosphorylation site. Moreover, we found that Y1127 phosphorylation reduced the association of Par3 with LIM kinase 2 (LIMK2), thus enabling LIMK2 to regulate cofilin phosphorylation dynamics. Substitution of Y1127 for phenylalanine impaired the EGF-induced Par3 and LIMK2 dissociation and delayed epithelial tight junction (TJ) assembly considerably. Collectively, these data suggest a novel, phosphotyrosine-dependent fine-tuning mechanism of Par3 in epithelial TJ assembly controlled by the EGF receptor-SFK signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2001

EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption.

Enteropathogenic Escherichia coli (EPEC) alters many functions of the host intestinal epithelia. Inflammation is initiated by activation of nuclear factor (NF)-kappaB, and paracellular permeability is enhanced via a Ca2+- and myosin light-chain kinase (MLCK)-dependent pathway. The aims of this study were to identify signaling pathways by which EPEC triggers inflammation and to determine whether these pathways parallel or diverge from those that alter permeability. EPEC-induced phosphorylation and degradation of the primary inhibitor of NF-kappaB (IkappaBalpha) were tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta independent. In contrast to Salmonella typhimurium, EPEC-stimulated IkappaBalpha degradation and IL-8 expression did not require Ca2+. Instead, extracellular signal-regulated kinase (ERK)-1/2 was significantly and rapidly activated. ERK1/2 inhibitors attenuated IkappaBalpha degradation and IL-8 expression. Although ERK1/2 can activate MLCK, its inhibition had no impact on EPEC disruption of the tight junction barrier. In conclusion, EPEC-induced inflammation 1) is TNF-alpha and IL-1beta receptor independent, 2) utilizes pathways differently from S. typhimurium, 3) requires ERK1/2, and 4) employs signals that are distinct from those that alter permeability. This is the first time that EPEC-activated signaling cascades have been linked to independent functional consequences.

Calcium↗
PubMed2005

Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions.

Surface-modified DL-lactide/glycolide copolymer (PLGA) nanospheres with chitosan (CS) were prepared by the emulsion solvent diffusion method for pulmonary delivery of peptide, i.e., elcatonin. The nanosphere suspension was successfully aerosolized with a nebulizer similar to the drug solution, whereas the microsphere suspensions could not be aerosolized. After pulmonary administration, CS-modified PLGA nanospheres were more slowly eliminated from the lungs than unmodified PLGA nanospheres. CS-modified PLGA nanospheres loaded with elcatonin reduced blood calcium levels to 80% of the initial calcium concentration and prolonged the pharmacological action to 24 h, which was a significantly longer duration of action than that by CS-unmodified nanospheres. These results were attributed to the retention of nanospheres adhered to the bronchial mucus and lung tissue and sustained drug release at the adherence site. In addition, CS and CS on the surface of the nanospheres enhanced the absorption of drug. The rank order of the absorption of the model drugs with CS solution was carboxyfluorescein>FITC-dextran-4 (FD-4; Mw. 4000)>FD-21 (Mw. 21,000)>FD70 (Mw. 70,000), which corresponded to the molecular weights ([Mw.] given in parentheses). The absorption-enhancing effect may have been caused by opening the intercellular tight junctions.

Absorption↗
PubMed2002

Melibiose, difructose anhydride III and difructose anhydride IV enhance net calcium absorption in rat small and large intestinal epithelium by increasing the passage of tight junctions in vitro.

An Ussing chamber technique was used to determine the effects of three indigestible disaccharides on net Ca transport from the luminal side to the basolateral side of isolated preparations of jejunal, ileal, cecal and colonic epithelium in rats. Permeability of Lucifer Yellow (LY) and transepithelial electrical resistance (TEER), which are indicators of intercellular passage of the intestinal mucosa, were also determined. The concentrations of Ca in the serosal and mucosal media were 1.25 mmol/L and 10 mmol/L, respectively. After a 30-min incubation, the net Ca transport, LY passage and TEER were determined. In the control experiment, LY permeability was lowest, and TEER value was highest in the colon. The addition of 1-100 mmol/L melibiose, difructose anhydride (DFA)III, or DFAIV to the mucosal medium increased the net Ca absorption and LY permeability dose-dependently in the jejunum, ileum, cecum and colon preparations. Melibiose decreased TEER dose-dependently in the jejunum and cecum, but not in the ileum and colon. DFAIII decreased TEER dose-dependently in the jejunum, cecum and colon, but not in the ileum. DFAIV decreased TEER dose-dependently in all four intestinal portions. Positive linear relationships were found between net Ca transport and LY passage in all portions of the intestine, whereas negative linear relationships were found between net Ca absorption and TEER. We concluded that the three indigestible saccharides directly affect the epithelial tissue and activate the passage of tight junctions, thereby promoting Ca absorption in the small and large intestine in vitro.

Animals↗
PubMed1995

Effect of cholephilic dyes on hepatic tight junctional permeability in the rat.

Changes in biliary permeability during cholephilic dye-induced choleresis, as assessed by measuring the movement into bile of two permeability probes, [14C]sucrose and horseradish peroxidase, were analyzed following an i.v. infusion (60 nmol/min per 100 g body wt) of the model cholephilic organic anion sulfobromophthalein in rats. Dye infusion led to a progressive increase of the [14C]sucrose bile-to-plasma ratio, which reached a maximum value after 100 min of dye infusion (+97%). Paracellular entry of horseradish peroxidase, as evaluated by the early peak of its biliary appearance curve, was also selectively increased (+69%), without changes in the later (transcytotic) access of the protein. Additional dose-response studies of biliary permeability to [14C]sucrose, using sulfobromophthalein and rose bengal, showed that this effect was dose-dependent and rapidly reversed by interruption of dye administration. The influence of hydrophobic/hydrophilic balance on this effect was also studied by infusing four dyes covering a broad range of hydrophobicity (phenol red, bromocresol green, sulfobromophthalein, and rose bengal), so as to attain a similar value of dye hepatic content at the end of the experiment (approximately 150 nmol/g liver wt). Under these conditions, a strong positive correlation was found between the increase in biliary permeability to [14C]sucrose and dye hydrophobicity. These results suggest that cholephilic dyes increase tight junctional permeability in a reversible and dose-dependent manner, and that this effect depends on the hydrophobic/hydrophilic balance of the dye.

Animals↗
PubMed1988

Rapid formation of tight junctions in HT 29 human adenocarcinoma cells by hypertonic salt solutions.

The human colon adenocarcinoma cell line HT 29 grows virtually without tight junctions (TJ) under standard culture conditions. Earlier studies have shown that focal TJ (fasciae occludentes) can be rapidly assembled in this cell line under the influence of various proteases. Here we show that focal TJ can be induced in this cell line by a brief treatment with appropriate salt solutions. Induction by ammonium sulfate in Hanks' buffer reached a maximum value after 15 to 30 min. The amount of TJ increased with the salt concentration and reached a plateau value at a concentration of 160 mM ammonium sulfate. The amount and complexity of TJ induced by ammonium sulfate were similar to those in experiments using trypsin as inducing agent as shown by morphometric analysis. At 0 degrees C, no TJ were formed under the influence of the salt. A comparative study of TJ induction using a variety of inorganic and organic salts gave the following results. All alkali sulfates induced TJ, although with different yield. Both calcium and magnesium chloride were potent inducers. Ammonium and sodium salts encompassing a variety of anions covered a wide range from maximum induction (sulfate, citrate) to almost complete absence of induction (nitrate). Sodium chloride did not induce any TJ. It follows that the induction of TJ is a specific effect of individual ionic components of the solution as opposed to a general effect of osmolarity and ionic strength. The data suggest tentatively that antichaotropic but not chaotropic ions have the potential to trigger the formation of TJ in this experimental system.

Adenocarcinoma↗
PubMed1989

ZO-1 and cingulin: tight junction proteins with distinct identities and localizations.

The relative localization of ZO-1 and cingulin, the only two known components of the tight junction, was compared in Madin-Darby canine kidney (MDCK) cells, chicken small intestine, rat kidney distal convoluted tubule, and a hepatoma cell line. Immunoblot analysis demonstrated that cingulin and ZO-1 are immunologically unrelated and that, in the colon, cingulin is a single polypeptide with a molecular mass of 140 kDa. Immunofluorescent localization of cingulin and ZO-1 in confluent monolayers of MDCK cells showed identical staining patterns. However, subconfluent MDCK cells showed distinct localizations of the two proteins. Both cingulin and ZO-1 were found at the plasma membrane only at areas of cell-cell contact, but cingulin was diffusely distributed within the cytoplasm, whereas ZO-1 showed a more clustered internal arrangement. Cingulin and ZO-1 were identically localized at the plasma membrane of hepatoma tissue culture (HTC) cells at sites of cell-cell contact. In chicken intestine examined at the ultrastructural level, immunogold particles associated with cingulin were found approximately three times farther from the junctional membrane than those affiliated with ZO-1.

Animals↗
PubMed2001

The effect of phospholipids and fatty acids on tight-junction permeability and bacterial translocation.

The activity of phospholipase A2 (PLA2) is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). We recently reported that PLA2 mediates hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine (L-PC) when both are applied to the apical surface of cultured EC monolayers, resulting in increased bacterial translocation (BT) and decreased transepithelial electrical resistance (TEER). Free fatty acids (FFA) are the other products of this reaction, however, their effect on Caco-2 cell permeability has not been reported. In addition to PC, other luminal phospholipids are present at the surface of the enterocyte. PLA2 may also mediate the hydrolysis of luminal phospholipids other than PC. The aim of this study was to examine the effects of phospholipids other than PC and common FFA on intestinal epithelial permeability and BT. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured as TEER. First, common FFA released by PC hydrolysis were determined using thin-layer chromatography (TLC). In separate experiments, monolayers were treated with phosphatidylethanolamine (PE), lysophosphatidylethanolamine (L-PE), or palmitoleic acid, oleic acids, linoleic acids, and arachidonic acid solubilized in solution with PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli C25 to the apical chamber followed by quantitatively culturing basal-chamber samples. Statistical analysis was by the Kurosaki-Wallis test. TLC of PC samples incubated with PLA2 on the apical surface of Caco-2 monolayers demonstrated the production of palmitoleic acid, oleic acids, linoleic acids, and arachidonic acid. L-PE significantly decreased TEER compared to controls, but to a lesser degree than L-PC alone. L-PE had no effects on BT. Palmitoleic acid and oleic acid likewise significantly decreased TEER compared to controls, however, less than L-PC. All FFA tested had no effect on BT. Phospholipids applied to the apical surface of enterocytes, such as those found in vivo in mucus, can be hydrolyzed by the enzyme PLA2 resulting in lysophospholipid and FFA species that can alter enterocyte monolayer permeability. However, FFA and L-PL, other than L-PC, appear to have no effect to stimulate BT. This observation may have clinical implications in the pathogenesis and treatment strategies for IBD patients in whom enterocyte PLA2 activity has been shown to be elevated.

Bacterial Translocation↗
PubMed2006

A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.

Dendritic cells (DC) mediate airway Ag presentation and play key roles in asthma and infections. Although DC subsets are known to perform different functions, their occurrence in mouse lungs has not been clearly defined. In this study, three major lung DC populations have been found. Two of them are the myeloid and plasmacytoid DC (PDC) well-characterized in other lymphoid organs. The third and largest DC population is the integrin alpha(E) (CD103) beta(7)-positive and I-A(high)CD11c(high)-DC population. This population was found to reside in the lung mucosa and the vascular wall, express a wide variety of adhesion and costimulation molecules, endocytose avidly, present Ag efficiently, and produce IL-12. Integrin alpha(E)beta(7)(+) DC (alphaE-DC) were distinct from intraepithelial lymphocytes and distinguishable from CD11b(high) myeloid and mPDCA-1(+)B220(+)Gr-1(+) PDC populations in surface marker phenotype, cellular functions, and tissue localization. Importantly, this epithelial DC population expressed high levels of the Langerhans cell marker Langerin and the tight junction proteins Claudin-1, Claudin-7, and ZO-2. In mice with induced airway hyperresponsiveness and eosinophilia, alphaE-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that alphaE-DC is a major and distinct lung DC population and a prime candidate APC with the requisite surface proteins for migrating across the airway epithelia for Ag and pathogen capture, transport, and presentation. They exhibit an activated phenotype in allergen-induced lung inflammation and may play significant roles in asthma pathogenesis.

Animals↗
PubMed1999

Amitriptyline-induced loss of tight junction integrity in a human endothelial--smooth muscle cell bi-layer model.

Tricyclic antidepressants can, when taken in overdose, cause serious pulmonary failure such as the adult respiratory distress syndrome (ARDS). In this study we have examined the effects of some tricyclic antidepressants (amitriptyline, imipramine, nortriptyline and desipramine) on the viability and morphology of human endothelial and smooth muscle cells derived from umbilical cord. Effects of amitriptyline on endothelial cell fluidity, as well as permeability changes to an endothelial-smooth muscle cell bi-layer, were also studied. The tricyclic antidepressants induced acute, sub-lethal toxicity in both cell types above 100 microM as assessed by the MTT reduction assay. Morphological changes were also observed at these concentrations. Such changes were, however, absent at 33 microM and below. Amitriptyline did, however, cause a concentration-dependent fall in the electrical resistance of an endothelial-smooth muscle cell bi-layer, with significant effects already evident at 33 microM. All of these observed effects were fairly rapid and appeared within 5-15 min of exposure. The rapidity of these permeabilisation effects suggests potential membrane perturbations, since tricyclic antidepressants are lipophilic molecules with affinity for cell membranes. However, fluorescence anisotropy measurements showed no significant difference in membrane fluidity between amitriptyline-treated and control endothelial cells. Collectively, these data point to specific mechanisms of action of amitriptyline, and probably also the other tricyclic antidepressants studied, on endothelial permeability, which is a hallmark of ARDS. The data suggest that increased endothelial permeability could be due to impaired tight junction function.

Amitriptyline↗
PubMed1983

Lack of orthogonal particle assemblies and presence of tight junctions in astrocytes of the goldfish (Carassius auratus). A freeze-fracture study.

The cytoplasmic membranes of astrocytes in the optic nerve of the goldfish were investigated by means of freeze-fracture techniques. Astrocytes of normal and regenerating optic nerves did not differ in the fine structure of plasma membranes. Emphasis is placed on the following results: Astrocytic membranes of fish do not reveal the orthogonal particle assemblies that are believed to be generally characteristic for astrocytes in the white matter. Astrocytes reveal extensive membrane areas occupied by tight junctions, which to date have not been described as characteristic astrocytic structures. These junctions are astro-astrocytic and are frequently intercalated by gap junctions. Desmosomes are another characteristic type of astro-astrocytic junction. By means of freeze-fracture replicas it can be demonstrated that they occur in more or less close association with tight and gap junctions. Caveolae are also seen in the astrocytic membranes of fish: their density and distribution show distinct variations. Caveolae occur at the interface between astrocytes and the interstitial space, between astrocytes and myelin sheaths, and in astrocytic processes. It is suggested that the differences between the astrocytic membranes of fish and mammals reflect different physiological functions. They are discussed in relation to the problem of neuronal-glial interrelationships and the behavior of astrocytes during fiber regeneration in the CNS.

Animals↗
PubMed1998

Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line.

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-alpha (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40-50% reduction in TER and a 30% decrease in 1SC (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [14C]-D-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial 22Na+ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na+ and Cl-. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [3H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mM apical amiloride reduced the basolateral to apical flux of 22Na+, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na+ and Cl-. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity.

Caco-2 Cells↗