Integrating Continuous Transepithelial Flux Measurements into an Ussing Chamber Set-Up.

Fluorescently labelled compounds are often employed to study the paracellular properties of epithelia. For flux measurements, these compounds are added to the donor compartment and samples collected from the acceptor compartment at regular intervals. However, this method fails to detect rapid changes in permeability. For continuous transepithelial flux measurements in an Ussing chamber setting, a device was developed, consisting of a flow-through chamber with an attached LED, optical filter, and photodiode, all encased in a light-impermeable container. The photodiode output was amplified and recorded. Calibration with defined fluorescein concentration (range of 1 nM to 150 nM) resulted in a linear output. As proof of principle, flux measurements were performed on various cell lines. The results confirmed a linear dependence of the flux on the fluorescein concentration in the donor compartment. Flux depended on paracellular barrier function (expression of specific tight junction proteins, and EGTA application to induce barrier loss), whereas activation of transcellular chloride secretion had no effect on fluorescein flux. Manipulation of the lateral space by osmotic changes in the perfusion solution also affected transepithelial fluorescein flux. In summary, this device allows a continuous recording of transepithelial flux of fluorescent compounds in parallel with the electrical parameters recorded by the Ussing chamber.

Myrrh protects against IL-13-induced epithelial barrier breakdown in HT-29/B6 cells.

The oleoresin myrrh has been used for centuries as an anti-inflammatory remedy for a variety of diseases and is said to have a protective effect on the intestinal epithelium. An intact epithelial barrier function is the prerequisite for a healthy gut. Inflammatory and infectious diseases of the intestine, in particular, lead to barrier impairment resulting in leak-flux diarrhea and mucosal immune responses. Therefore, the aim of the present study was to investigate the protective effect of myrrh in an experimental inflammatory situation, namely, under the influence of IL-13, one of the key cytokines in ulcerative colitis. We used human intestinal epithelial HT-29/B6 cell monolayers for functional and molecular assessment of the epithelial barrier under IL-13 and myrrh treatment. IL-13 induced a loss in barrier function that was fully restored with myrrh treatment, as shown by transepithelial electrical resistance measurements. The molecular correlate of the IL-13-mediated barrier dysfunction could be assigned to an upregulation of the channel-forming tight junction (TJ) protein claudin-2 and to a subcellular redistribution of the TJ protein tricellulin, loosening the sealing of tricellular TJs. Moreover, IL-13 exposure leads to an increase in the number of apoptotic cells, contributing to the leak pathway of barrier dysfunction. Myrrh protected against changes in TJ deregulation and decreased the elevated apoptotic ratio under IL-13. The protective effects are mediated through the inhibition of the STAT3 and STAT6 pathway. In conclusion, our results demonstrate that myrrh exhibits antagonizing effects against IL-13-induced barrier impairment in a human intestinal cell model. These data suggest the use of myrrh as a promising option in the treatment of inflammatory bowel disease.

Clinical Significance of Claudin Expression in Oral Squamous Cell Carcinoma.

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC.

Tight junction channels claudin-10b and claudin-15: Functional mapping of pore-lining residues.

Although functional and structural models for paracellular channels formed by claudins have been reported, mechanisms regulating charge and size selectivity of these channels are unknown in detail. Here, claudin-15 and claudin-10b cation channels showing high-sequence similarity but differing channel properties were analyzed. Mutants of pore-lining residues were expressed in MDCK-C7 cells. In claudin-15, proposed ion interaction sites (D55 and E64) conserved between both claudins were neutralized. D55N and E64Q substitutions decreased ion permeabilities, and D55N/E64Q had partly additive effects. D55N increased cation dehydration capability and decreased pore diameter. Additionally, residues differing between claudin-15 and -10b close to pore center were analyzed. Claudin-10b-mimicking W63K affected neither assembly nor function of claudin-15 channels. In contrast, in claudin-10b, corresponding (claudin-15b-mimicking) K64W and K64M substitutions disturbed integration into tight junction and slightly altered relative permeabilities for differently sized monovalent cations. Removal of claudin-10b-specific negative charge (D36A substitution) was without effect. The data suggest that a common tetra-aspartate ring (D55/D56) in pore center of claudin-15/-10b channels directly attracts cations, while E64/D65 may be at least partly shielded by W63/K64. Charge at position W63/K64 affects assembly and properties for claudin-10b but not for claudin-15 channels. Our findings add to the mechanistic understanding of the determinants of paracellular cation permeability.

Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells.

Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (alias lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines.

The Punicalagin Metabolites Ellagic Acid and Urolithin A Exert Different Strengthening and Anti-Inflammatory Effects on Tight Junction-Mediated Intestinal Barrier Function In Vitro.

Scope: Ellagitannins are polyphenols found in numerous fruits, nuts and seeds. The elagitannin punicalagin and its bioactive metabolites ellagic acid and urolithins are discussed to comprise a high potential for therapeutically or preventive medical application such as in intestinal diseases. The present study characterizes effects of punicalagin, ellagic acid and urolithin A on intestinal barrier function in the absence or presence of the proinflammatory cytokine tumor necrosis factor-α (TNFα). Methods and Results: Transepithelial resistance (TER), fluorescein and ion permeability, tight junction protein expression and signalling pathways were examined in Caco-2 and HT-29/B6 intestinal epithelial cell models. Punicalagin had less or no effects on barrier function in both cell models. Ellagic acid was most effective in ileum-like Caco-2 cells, where it increased TER and reduced fluorescein and sodium permeabilities. This was paralleled by myosin light chain kinase two mediated expression down-regulation of claudin-4, -7 and -15. Urolithin A impeded the TNFα-induced barrier loss by inhibition of claudin-1 and -2 protein expression upregulation and claudin-1 delocalization in HT-29/B6. Conclusion: Ellagic acid and urolithin A affect intestinal barrier function in distinct ways. Ellagic acid acts preventive by strengthening the barrier per se, while urolithin A protects against inflammation-induced barrier dysfunction.

Differential day-night expression of tight junction components in murine retinal pigment epithelium.

Strong communication and interaction between the retinal pigment epithelium (RPE) and the photoreceptor (PR) cells is essential for vision. RPE cells are essential for supporting and maintaining PR cells by transporting nutrients, waste products and ions, and phagocytosing photoreceptor outer segments (POS). POS phagocytosis follows a circadian pattern, taking place in the morning in human, mice and other organisms. However, it remains unknown whether other RPE processes follow a daily rhythm. To study the daily rhythm of RPE cells, we isolated murine RPE cells at six different time points during a 24 h period, after which RNA was isolated and sequenced. Murine RPE flatmounts were isolated at four different time points to study daily rhythm in protein abundance and localisation. EnrichR pathway analysis resulted in 13 significantly-enriched KEGG pathways (p < 0.01) of which seven showed a large number of overlapping genes. Several genes were involved in intracellular trafficking, possibly playing a role in nutrient transport, POS phagocytosis or membrane protein trafficking, with different expression patterns during the day-night cycle. Other genes were involved in actin cytoskeleton building, remodelling and crosslinking and showed a high expression in the morning, suggesting actin cytoskeleton remodelling at this time point. Finally, tight junction proteins Cldn2 and Cldn4 showed a difference in RNA and protein expression and tight junction localisation over time. Our study suggests that several important processes in the RPE follow a day-night rhythm, including intracellular trafficking, and processes involving the actin cytoskeleton and tight junctions. The differential protein localisation of Cldn2 in the RPE during the day-night cycle suggest that Cldn2 may facilitate paracellular water and sodium transport during the day.

Claudin-15 forms a water channel through the tight junction with distinct function compared to claudin-2.

AIM: Claudin-15 is mainly expressed in the small intestine and indirectly involved in glucose absorption. Similar to claudin-2 and -10b, claudin-15 is known to form a paracellular channel for small cations. Claudin-2, but not claudin-10b, also forms water channels. Here we experimentally tested whether claudin-15 also mediates water transport and if yes, whether water transport is Na+ -coupled, as seen for claudin-2. METHODS: MDCK C7 cells were stably transfected with claudin-15. Ion and water permeability were investigated in confluent monolayers of control and claudin-15-expressing cells. Water flux was induced by an osmotic or ionic gradient. RESULTS: Expression of claudin-15 in MDCK cells strongly increased cation permeability. The permeability ratios for monovalent cations indicated a passage of partially hydrated ions through the claudin-15 pore. Accordingly, its pore diameter was determined to be larger than that of claudin-2 and claudin-10b. Mannitol-induced water flux was elevated in claudin-15-expressing cells compared to control cells. In contrast to the Na+ -coupled water flux of claudin-2 channels, claudin-15-mediated water flux was inhibited by Na+ flux. Consequently, water flux was increased in Na+ -free solution. Likewise, Na+ flux was decreased after induction of water flux through claudin-15. CONCLUSION: Claudin-15, similar to claudin-2, forms a paracellular cation and water channel. In functional contrast to claudin-2, water and Na+ fluxes through claudin-15 inhibit each other. Claudin-15 allows Na+ to retain part of its hydration shell within the pore. This then reduces the simultaneous passage of additional water through the pore.

Tricellulin Effect on Paracellular Water Transport.

In epithelia, large amounts of water pass by transcellular and paracellular pathways, driven by the osmotic gradient built up by the movement of solutes. The transcellular pathway has been molecularly characterized by the discovery of aquaporin membrane channels. Unlike this, the existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until two molecular components of paracellular water transport, claudin-2 and claudin-15, were identified. A main protein of the tricellular TJ (tTJ), tricellulin, was shown to be downregulated in ulcerative colitis leading to increased permeability to macromolecules. Whether or not tricellulin also regulates water transport is unknown yet. To this end, an epithelial cell line featuring properties of a tight epithelium, Madin-Darby canine kidney cells clone 7 (MDCK C7), was stably transfected with small hairpin RNA (shRNA) targeting tricellulin, a protein of the tTJ essential for the barrier against passage of solutes up to 10 kDa. Water flux was induced by osmotic gradients using mannitol or 4 and 40 kDa-dextran. Water flux in tricellulin knockdown (KD) cells was higher compared to that of vector controls, indicating a direct role of tricellulin in regulating water permeability in a tight epithelial cell line. We conclude that tricellulin increases water permeability at reduced expression.

HDAC inhibitors promote intestinal epithelial regeneration via autocrine TGFß1 signalling in inflammation.

Intact epithelial barrier function is pivotal for maintaining intestinal homeostasis. Current therapeutic developments aim at restoring the epithelial barrier in inflammatory bowel disease. Histone deacetylase (HDAC) inhibitors are known to modulate immune responses and to ameliorate experimental colitis. However, their direct impact on epithelial barrier function and intestinal wound healing is unknown. In human and murine colonic epithelial cell lines, the presence of the HDAC inhibitors Givinostat and Vorinostat not only improved transepithelial electrical resistance under inflammatory conditions but also attenuated the passage of macromolecules across the epithelial monolayer. Givinostat treatment mediated an accelerated wound closure in scratch assays. In vivo, Givinostat treatment resulted in improved barrier recovery and epithelial wound healing in dextran sodium sulphate-stressed mice. Mechanistically, these regenerative effects could be linked to an increased secretion of transforming growth factor beta1 and interleukin 8, paralleled by differential expression of the tight junction proteins claudin-1, claudin-2 and occludin. Our data reveal a novel tissue regenerative property of the pan-HDAC inhibitors Givinostat and Vorinostat in intestinal inflammation, which may have beneficial implications by repurposing HDAC inhibitors for therapeutic strategies for inflammatory bowel disease.

Lactoferrin protects against intestinal inflammation and bacteria-induced barrier dysfunction in vitro.

The iron-binding glycoprotein lactoferrin (LF) is naturally present in human breast milk. Several studies suggest that LF contributes to infant health and development owing to a variety of protective effects, including antimicrobial and anti-inflammatory features. Therefore, we aimed to elucidate its protective properties on intestinal epithelial barrier dysfunction induced by infection or inflammation using the human epithelial cell culture models HT-29/B6 and T84. During barrier perturbation induced by the proinflammatory cytokine tumor necrosis factor α (TNF-α), bovine LF restored tight junction (TJ) morphometry and inhibited TNF-α-induced epithelial apoptosis. This resulted in an attenuation of the TNF-α-induced decrease in transepithelial resistance (TER) and increases in permeability of fluorescein and FITC-dextran (4 kDa) and was as effective as the apoptosis inhibitor Q-VD-Oph. The enteropathogenic bacterium Yersinia enterocolitica is a frequent cause of diarrhea in early childhood. This involves focal changes in TJ protein expression and localization. LF diminished the Y. enterocolitica-induced drop in TER in the present in vitro model, which was paralleled by an inhibition of the Yersinia-induced reduction of claudin-8 expression via c-Jun kinase signaling. In conclusion, LF exerts protective effects against inflammation- or infection-induced barrier dysfunction in human intestinal cell lines, supporting its relevance for healthy infant development.

Water channels and barriers formed by claudins.

Physiological studies in leaky epithelia, like kidney proximal tubules and the small intestine, have documented water transport via both transcellular and paracellular pathways. The discovery of aquaporin water channels provided a molecular basis for transcellular water movement. In contrast, the contribution, or even existence, of a specific paracellular water pathway has been disputed for a long time, until the cation channel-forming tight junction protein claudin-2 was shown to also permit the paracellular passage of water through its pore. In proximal kidney tubules, claudin-2-based water transport contributes 23-30% of the total water transport. Other paracellular ion channels (claudin-10a, -10b, and -17) proved to be impermeable to water, although their pore size would be sufficient for water molecules to pass. Studies of barrier-forming claudins, like claudin-1 and claudin-3, which tighten the paracellular pathway against ions and larger solutes, indicate that changes in the expression of these sealing claudins do not influence transepithelial water permeability. The present genetic, molecular, computational, and physiological studies are just now beginning to probe the mechanisms and regulation of paracellular permeation.

Tight junctions of the proximal tubule and their channel proteins.

The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na+ and K+. Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na+ and water share the same pore. Already solved claudin crystal structures reveal a characteristic ß-sheet, comprising ß-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.

Zinc treatment is efficient against Escherichia coli α-haemolysin-induced intestinal leakage in mice.

Zinc homoeostasis exerts protective effects in inflammatory intestinal diseases and zinc supplementation has been successfully used for treating infectious diarrhoea. This study aimed at a characterisation of zinc effects on focal leak induction by α-haemolysin (HlyA)-producing Escherichia coli (E. coli) as protective mechanism for colitis. We conducted in vivo experiments by oral challenge of gnotobiotic mice colonised with HlyA-expressing E. coli-536. Mice were either fed a defined normal or high zinc diet to analyse effects of zinc as a therapeutic regimen. HlyA-deficient E. coli-536 mutants were used as controls. Mice infected with HlyA-producing E. coli showed impaired barrier integrity when receiving normal zinc. High zinc supplementation in HlyA-producing E. coli-infected mice reduced epithelial dysfunction as indicated by ameliorated macromolecule permeability. Reduced size of focal leaks with diminished bacterial translocation was observed as inherent mechanisms of this zinc action. In human colon cell monolayers application of zinc rescued the HlyA-dependent decline in transepithelial electrical resistance via reduction of the calcium entry into HlyA-exposed cells. Calcium-dependent cell exfoliation was identified as mechanism for focal leak induction. In conclusion, zinc supplementation protects from HlyA-induced barrier dysfunction in vivo and in vitro, providing an explanation for the protective efficacy of zinc in intestinal disorders.

Myrrh exerts barrier-stabilising and -protective effects in HT-29/B6 and Caco-2 intestinal epithelial cells.

PURPOSE: Myrrh, the oleo-gum resin of Commiphora molmol, is well known for its anti-inflammatory properties. In different animal models, it protected against DSS-, TNBS- and oxazolone-induced colitis. To date, no information concerning the effect of myrrh on barrier properties are available. Thus, this study investigates the effect of myrrh on paracellular barrier function in the absence or presence of the pro-inflammatory cytokine TNFα. METHODS: Monolayers of human colon cell lines HT-29/B6 and Caco-2 were incubated with myrrh under control conditions or after challenge with the pro-inflammatory cytokine TNFα. Barrier function was analysed by electrophysiological and permeability measurements, Western blotting, immunostaining in combination with confocal microscopy, and freeze-fracture electron microscopy. RESULTS: In Caco-2 cells, myrrh induced an increase in transepithelial resistance (TER) which was associated with downregulation of the channel-forming tight junction (TJ) protein claudin-2 via inhibition of the PI3 kinase signalling pathway. In HT-29/B6 cells, myrrh had no effect on barrier properties under basic conditions, but protected against barrier damage induced by TNFα, as indicated by a decrease in TER and an increase in fluorescein permeability. The TNFα effect was associated with a redistribution of the sealing TJ protein claudin-1, an increase in the expression of claudin-2 and a change in TJ ultrastructure. Most importantly, all TNFα effects were inhibited by myrrh. The effect of myrrh on claudin-2 expression in this cell line was mediated via inhibition of the STAT6 pathway. CONCLUSIONS: This study shows for the first time that myrrh exerts barrier-stabilising and TNFα-antagonising effects in human intestinal epithelial cell models via inhibition of PI3K and STAT6 signalling. This suggests therapeutic application of myrrh in intestinal diseases associated with barrier defects and inflammation.

The ginger component 6-shogaol prevents TNF-α-induced barrier loss via inhibition of PI3K/Akt and NF-κB signaling.

SCOPE: Anti-inflammatory properties of the ginger-derived pungent component 6-shogaol (6-SG) have been studied intensively in recent years. Purpose of this study was to characterize the influence of 6-SG on inflammation-related intestinal barrier dysfunction, especially its paracellular component. METHODS AND RESULTS: The effect of 6-SG was studied in the human intestinal cell models HT-29/B6 and Caco-2 either under control conditions or challenged by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Electrophysiological measurements, freeze-fracture electron microscopy, and protein analyses were performed. 6-SG partially prevented both, the TNF-α-induced decrease in transepithelial resistance and the rise in fluorescein permeability. By inhibiting phosphatidylinositol-3-kinase/Akt signaling 6-SG prevented the TNF-α-induced increase in protein expression of claudin-2, a channel-forming tight junction protein. In addition, the TNF-α-induced disassembly of the sealing tight junction protein claudin-1 was attenuated, the latter of which was due to TNF-α-triggered phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB). CONCLUSION: 6-SG has barrier-protective effects by affecting TNF-α-induced claudin-2 upregulation and claudin-1 disassembly via inhibition of phoshatidylinositol-3-kinase/Akt and nuclear factor kappa light chain enhancer of activated B-cell signaling. Therefore, 6-SG-containing food might be beneficial for barrier preservation during intestinal inflammation.

Occludin is involved in adhesion, apoptosis, differentiation and Ca2+-homeostasis of human keratinocytes: implications for tumorigenesis.

Tight junction (TJ) proteins are involved in a number of cellular functions, including paracellular barrier formation, cell polarization, differentiation, and proliferation. Altered expression of TJ proteins was reported in various epithelial tumors. Here, we used tissue samples of human cutaneous squamous cell carcinoma (SCC), its precursor tumors, as well as sun-exposed and non-sun-exposed skin as a model system to investigate TJ protein alteration at various stages of tumorigenesis. We identified that a broader localization of zonula occludens protein (ZO)-1 and claudin-4 (Cldn-4) as well as downregulation of Cldn-1 in deeper epidermal layers is a frequent event in all the tumor entities as well as in sun-exposed skin, suggesting that these changes result from chronic UV irradiation. In contrast, SCC could be distinguished from the precursor tumors and sun-exposed skin by a frequent complete loss of occludin (Ocln). To elucidate the impact of down-regulation of Ocln, we performed Ocln siRNA experiments in human keratinocytes and uncovered that Ocln downregulation results in decreased epithelial cell-cell adhesion and reduced susceptibility to apoptosis induction by UVB or TNF-related apoptosis-inducing ligand (TRAIL), cellular characteristics for tumorigenesis. Furthermore, an influence on epidermal differentiation was observed, while there was no change of E-cadherin and vimentin, markers for epithelial-mesenchymal transition. Ocln knock-down altered Ca(2+)-homeostasis which may contribute to alterations of cell-cell adhesion and differentiation. As downregulation of Ocln is also seen in SCC derived from other tissues, as well as in other carcinomas, we suggest this as a common principle in tumor pathogenesis, which may be used as a target for therapeutic intervention.

Contribution of tight junction proteins to ion, macromolecule, and water barrier in keratinocytes.

Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na(+), Cl(-), and Ca(2+), and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.

Analysis of absorption enhancers in epithelial cell models.

A variety of chemical compounds are currently being discussed as novel drug delivery strategies. One promising strategy is to selectively open the paracellular pathway of epithelia for the passage of macromolecules. A prerequisite for this effect is a rapid and reversible action of these compounds, to allow a marked translocation of a drug, but also to avoid unwanted adverse effects, such as the translocation of noxious agents. Bioactive molecules that elevate paracellular permeability include Ca(2+) chelators, bacterial toxins, and other compounds, some of which perturb the structural basis of epithelial barrier function--the tight junction. Within the tight junction, organ- and tissue-specific barrier properties are determined mainly by claudins. The majority of members of the claudin protein family seal the paracellular pathway. This paper focuses on recent approaches concerning absorption-enhancing effects, with regard to selectivity and mechanism.