Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.
Aim Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle‐associated epithelium covering Peyer's patches (lymphoid follicles). Methods Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one‐path impedance spectroscopy were performed. Morphometry of HE‐stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. Results Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC–dextran were detected in follicle‐associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle‐associated epithelium, including claudin‐1, claudin‐4, claudin‐5 and claudin‐8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle‐associated epithelium vs. neighbouring villous epithelium. Conclusion We demonstrate that follicle‐associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions.
Milk production is modulated by the paracellular barrier function of tight junction (TJ) proteins located in the mammary epithelium. The aim of our study was the molecular analysis of TJs in native lactating murine mammary gland epithelium as this process may strongly challenge epithelial barrier properties and regulation. Mammary gland tissue specimens from lactating control mice and animals after a 20-h interruption of suckling were prepared; histological analyses were performed by light and electron microscopy; and expression of TJ proteins was detected by PCR, Western blotting, immunofluorescent staining, and confocal laser scanning microscopy. Discontinuation of suckling resulted in a substantial accumulation of milk in mammary glands, an increase of alveolar size, and a flattening of epithelial cells without effects on inflammatory indicators. In control tissues, PCR and Western blots showed signals for occludin, and claudin-1, -2, -3, -4, -5, -7, -8, -15, and -16. After a 20-h accumulation of milk, expression of two sealing TJ proteins, claudin-1 and -3, was markedly increased, whereas two TJ proteins involved in cation transport, claudin-2 and -16, were reduced. Real-time PCR validated increased transcripts of claudin-1 and claudin-3. During extension of mammary glands in the process of lactation, claudin-1 and -3 are markedly induced and claudin-2 and -16 are decreased. Volume and composition of milk might be strongly dependent on this counter-regulation of sealing claudins with permeability-mediating claudins, indicating a physiological process of a tightening of TJs against a back-leak of solutes and ions from the alveolar lumen.
Objective:The morphology and functions of the proximal and distal large intestine are not the same. The incidence of colorectal cancer in these regions is also different, as tumors more often appear in the descending colon than in the ascending colon. Inflammatory bowel disease and colorectal cancer can increase transepithelial permeability, which is a sign of reduced intestinal barrier function. However, there is not enough evidence to establish a connection between the difference in colorectal cancer incidence in the proximal and distal colon and intestinal permeability or the effects of carcinogenesis on the barrier properties in various areas of the colon. The aim of the study was to assess the permeability of different segments of the large intestine according to a developed mapping methodology in healthy rats and rats with 1,2-dimethylhydrazine (DMH)-induced colon adenocarcinoma.Methods:The short circuit current, the transepithelial electrical resistance and the paracellular permeability to fluorescein of large intestine wall of male Wistar rats were examined in the Ussing chambers. The optical density of the solution from the serosa side to assess the concentration of the diffused fluorescein from mucosa to serosa was analyzed by spectrophotometry. The morphometric and histological studies were performed by optical microscopy.Results:Rats with DMH-induced colon adenocarcinomas showed elevated transepithelial electrical resistance in the areas of neoplasm development. In contrast, there was no change in the electrophysiological properties of tumor adjacent areas, however, the paracellular permeability of these areas to fluorescein was increased compared to the control rats and was characterized by sharply reduced barrier function.Conclusions:The barrier properties of the colon vary depending on tumor location. The tumors were less permeable than the intact intestinal wall and probably have a negative influence on tumor-adjacent tissues by disrupting their barrier function.
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