The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 μg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [³H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.

The endogenous methylated derivative of ʟ-arginine, N^ω,N^ω′-dimethyl-ʟ-arginine (asymmetric dimethylarginine, ADMA), an independent risk factor in many diseases, inhibits the activity of nitric oxide synthases and, consequently, modulates the availability of nitric oxide. While most studies on the biological role of ADMA have focused on endothelial and inducible nitric oxide synthases modulation and its contribution to cardiovascular, metabolic, and renal diseases, a role in regulating neuronal nitric oxide synthases and pathologies of the central nervous system is less understood. The two isoforms of dimethylarginine dimethylaminohydrolase (DDAH), DDAH1 and DDAH2, are thought to be the main enzymes responsible for ADMA catabolism. A current impediment is limited knowledge on specific tissue and cellular distribution of DDAH enzymes within the brain. In this study, we provide a detailed characterization of the regional and cellular distribution of DDAH1 and DDAH2 proteins in the adult murine and human brain. Immunohistochemical analysis showed a wide distribution of DDAH1, mapping to multiple cell types, while DDAH2 was detected in a limited number of brain regions and exclusively in neurons. Our results provide key information for the investigation of the pathophysiological roles of the ADMA/DDAH system in neuropsychiatric diseases and pave the way for the development of novel selective therapeutic approaches.

The damaging effect of ionizing radiation (IR) exposure results in the disturbance of the gut natural barrier, followed by the development of severe gastrointestinal injury. However, the dose and application segment are known to determine the effects of IR. In this study, we demonstrated the dose- and segment-specificity of tight junction (TJ) alteration in IR-induced gastrointestinal injury in rats. Male Wistar rats were subjected to a total-body X-ray irradiation at doses of 2 or 10 Gy. Isolated jejunum and colon segments were tested in an Ussing chamber 72 h after exposure. In the jejunum, 10-Gy IR dramatically altered transepithelial resistance, short-circuit current and permeability for sodium fluorescein. These changes were accompanied by severe disturbance of histological structure and total rearrangement of TJ content (increased content of claudin-1, -2, -3 and -4; multidirectional changes in tricellulin and occludin). In the colon of 10-Gy irradiated rats, lesions of barrier and transport functions were less pronounced, with only claudin-2 and -4 altered among TJ proteins. The 2-Gy IR did not change electrophysiological characteristics or permeability in the colon or jejunum, although slight alterations in jejunum histology were noted, emphasized with claudin-3 increase. Considering that TJ proteins are critical for maintaining epithelial barrier integrity, these findings may have implications for countermeasures in gastrointestinal acute radiation injury.

Background: Many food components influence intestinal epithelial barrier properties and might therefore also affect susceptibility to the development of food allergies. Such allergies are triggered by increased antibody production initiated in Peyer’s patches (PP). Usually, the presentation of antigens in the lumen of the gut to the immune cells of the PP is strongly regulated by the follicle-associated epithelium (FAE) that covers the PP. As the food component caprate has been shown to impede barrier properties in villous epithelium, we hypothesized that caprate also affects the barrier function of the PP FAE, thereby possibly contributing a risk factor for the development of food allergies. Methods: In this study, we have focused on the effects of caprate on the barrier function of PP, employing in vitro and ex vivo experimental setups to investigate functional and molecular barrier properties. Incubation with caprate induced an increase of transepithelial resistance, and a marked increase of permeability for the paracellular marker fluorescein in porcine PP to 180% of control values. These effects are in accordance with changes in the expression levels of the barrier-forming tight junction proteins tricellulin and claudin-5. Conclusions: This barrier-affecting mechanism could be involved in the initial steps of a food allergy, since it might trigger unregulated contact of the gut lumen with antigens.

Ionizing radiation has a severe impact on the barrier and transport functions of the intestine triggering acute gastrointestinal radiation syndrome and limiting the dose and effectiveness of radiatio...