Dose- and Segment-Dependent Disturbance of Rat Gut by Ionizing Radiation: Impact of Tight Junction Proteins.

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.

Chronic Ouabain Targets Pore-Forming Claudin-2 and Ameliorates Radiation-Induced Damage to the Rat Intestinal Tissue Barrier.

Ionizing radiation (IR) causes disturbances in the functions of the gastrointestinal tract. Given the therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against IR-induced disturbances in the barrier and transport properties of the jejunum and colon of rats. Male Wistar rats were subjected to 6-day intraperitoneal injections of vehicle or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to total-body X-ray irradiation (10 Gy) or a sham irradiation. Isolated tissues were examined 72 h post-irradiation. Electrophysiological characteristics and paracellular permeability for sodium fluorescein were measured in an Ussing chamber. Histological analysis and Western blotting were also performed. In the jejunum tissue, ouabain exposure did not prevent disturbances in transepithelial resistance, paracellular permeability, histological characteristics, as well as changes in the expression of claudin-1, -3, -4, tricellulin, and caspase-3 induced by IR. However, ouabain prevented overexpression of occludin and the pore-forming claudin-2. In the colon tissue, ouabain prevented electrophysiological disturbances and claudin-2 overexpression. These observations may reveal a mechanism by which circulating ouabain maintains tight junction integrity under IR-induced intestinal dysfunction.

Prednisolone Targets Claudins in Mouse Brain Blood Vessels.

Endothelial cells in brain capillaries are crucial for the function of the blood-brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB's permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood-brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid-base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure.

Divergent Dimethylarginine Dimethylaminohydrolase Isoenzyme Expression in the Central Nervous System.

The endogenous methylated derivative of ʟ-arginine, Nω,Nω'-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.

Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats.

Recently it has been reported that the tumor adjacent colon tissues of 1,2-dymethylhydrazine induced (DMH)-rats revealed a high paracellular permeability. We hypothesized that the changes might be induced by cytokines. Colorectal cancer is accompanied by an increase in tumor necrosis factor alpha (TNFα) and interleukin 10 (IL10) that exert opposite regulatory effects on barrier properties of the colon, which is characterized by morphological and functional segmental heterogeneity. The aim of this study was to analyze the level of TNFα and IL10 in the colon segments of DMH-rats and to investigate their effects on barrier properties of the proximal and distal parts of the colon in healthy rats. Enzyme immunoassay analysis showed decreased TNFα in tumors in the distal part of the colon and increased IL10 in proximal tumors and in non-tumor tissues. Four-hour intraluminal exposure of the colon of healthy rats with cytokines showed reduced colon barrier function dependent on the cytokine: TNFα decreased it mainly in the distal part of the colon, whereas IL10 decreased it only in the proximal part. Western blot analysis revealed a more pronounced influence of IL10 on tight junction (TJ) proteins expression by down-regulation of the TJ proteins claudin-1, -2 and -4, and up-regulation of occludin only in the proximal part of the colon. These data may indicate a selective role of the cytokines in regulation of the barrier properties of the colon and a prominent role of IL10 in carcinogenesis in its proximal part.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle.

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1.

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.

Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells.

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.

Inflammatory Cell Infiltration of Adrenals in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was originated in November-December 2019 in Wuhan, China, and has rapidly spread around the world causing severe health and socioeconomical damage to the entire civilization. The key feature of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is upper respiratory tract infection, which may be complicated by bilateral pneumonia. Angiotensin converting enzyme 2 (ACE2) has been identified as a key host factor, required for virus entry into cells. Interestingly, ACE2 is expressed not only in the respiratory system, but also in the other organs and systems including adrenal glands. Here we provide the first description of the pathomorphological changes in adrenal glands in patients with severe COVID-19 characterized by perivascular infiltration of CD3+ and CD8+ T-lymphocytes. Due to the central role of the adrenals in the stress response of the organism, this finding is of potential clinical relevance, because infection with the SARS-CoV-2 virus might critically impair adrenal function under pathophysiological conditions.

Is Homoarginine a Protective Cardiovascular Risk Factor?

A series of recent epidemiological studies have implicated the endogenous nonproteinogenic amino acid l-homoarginine as a novel candidate cardiovascular risk factor. The association between homoarginine levels and the risk of adverse cardiovascular outcomes is inverse (ie, high cardiovascular risk is predicted by low rather than high homoarginine levels), which makes it plausible to normalize systemic homoarginine levels via oral supplementation. The emergence of homoarginine as a potentially treatable protective cardiovascular risk factor has generated a wave of hope in the field of cardiovascular prevention. Herein, we review the biochemistry, physiology, and metabolism of homoarginine, summarize the strengths and weaknesses of the epidemiological evidence linking homoarginine to cardiovascular disease and its potential protective cardiovascular effects, and identify priorities for future research needed to define the clinical utility of homoarginine as a prognostic factor and therapeutic target in cardiovascular disease.

Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels.

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.

Cholera toxin perturbs the paracellular barrier in the small intestinal epithelium of rats by affecting claudin-2 and tricellulin.

Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 µg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 µg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.

Caprate Modulates Intestinal Barrier Function in Porcine Peyer's Patch Follicle-Associated Epithelium.

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.

Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro.

During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation.

The epithelial barrier and beyond: Claudins as amplifiers of physiological organ functions.

Epithelial cell layers are interconnected by a meshwork of tight junction (TJ) protein strands, which are localized within apicolateral membranes. The proteins that form TJs are regarded to provide a static barrier, determining epithelial properties. However, recent findings in the field of barriology suggest that TJs contribute to more physiological aspects than indicated by the sum of the qualities of the single TJ proteins. Generally, TJs exhibit four major functions: (i) a "gate function," defining transepithelial permeability (i.e., barrier) properties, (ii) a "fence function" determining epithelial cell polarity, (iii) a "signaling function," affecting regulatory pathways, and (iv) a "stabilizing function," maintaining the integrity of the epithelium. This review presents a critical view on how the efficacy of physiological processes in epithelia and thus organ function might be improved by changes in the expression of claudins, the latter representing the largest and most variable family of TJ proteins. Major focus is set on (i) the coordinated regulation of transport and barrier in the intestine, (ii) the role of TJs in defining the route for antigen uptake and presentation in intestinal Peyer's patches, and (iii) the TJ function in mammary glands in response to milk accumulation, which represent impressive examples to highlight the amplification of epithelial functions by TJ proteins. © 2017 IUBMB Life, 69(5):290-296, 2017.

Claudin clusters as determinants of epithelial barrier function.

Claudins are tetraspan tight junction proteins which have been attributed to primarily determine epithelial barrier function in a wide variety of different organs and tissues. Among this protein family with currently 27 members, single claudins contribute in an organ- and tissue-specific manner to defined properties such as cation-, anion- or water-selective pore functions, sealing functions or ambiguous functions. As the size of tight junction strand particles visualized by freeze-fracture electron microscopy have a diameter of approximately 10 nm, multimeric assembly of tight junction proteins appears to be a basic principle for barrier formation. Moreover, expression patterns of different tissues showed that single claudins appear to specifically co-localize with other claudins, which indicates a cluster formation within tight junction strand particles with a fixed stoichiometry. This review provides a critical view on the current understanding of tight junction protein co-localization within strands. We analyze how tissue specific differences of claudin functions could be dependent on their specific partners for barrier formation. Furthermore, a model of claudin clusters as structural and functional units within tight junction strands is provided.

Inhibitory effects of Lactobacillus fermentum on microbial growth and biofilm formation.

Beneficial effects of Lactobacilli have been reported, and lactic bacteria are employed for conservation of foods. Therefore, the effects of a Lactobacillus fermentum strain were analyzed regarding inhibitory effects on staphylococci, Candida albicans and enterotoxigenic enterobacteria by transmission electron microscopy (TEM). TEM of bacterial biofilms was performed using cocultures of bacteriocin-producing L. fermentum 97 with different enterotoxigenic strains: Staphylococcus epidermidis expressing the ica gene responsible for biofilm formation, Staphylococcus aureus producing enterotoxin type A, Citrobacter freundii, Enterobacter cloaceae, Klebsiella oxytoca, Proteus mirabilis producing thermolabile and thermostable enterotoxins determined by elt or est genes, and Candida albicans. L. fermentum 97 changed morphological features and suppressed biofilm formation of staphylococci, enterotoxigenic enterobacteria and Candida albicans; a marked transition to resting states, a degradation of the cell walls and cytoplasm, and a disruption of mature bacterial biofilms were observed, the latter indicating efficiency even in the phase of higher cell density.

Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.

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.

Laurate permeabilizes the paracellular pathway for small molecules in the intestinal epithelial cell model HT-29/B6 via opening the tight junctions by reversible relocation of claudin-5. [Corrected].

PURPOSE: To mechanistically analyze effects of the medium-chain fatty acid laurate on transepithelial permeability in confluent monolayers of the intestinal epithelial cell line HT-29/B6, in context with an application as an absorption enhancer improving transepithelial drug permeation. METHODS: Transepithelial resistance and apparent permeability for paracellular flux markers was measured using Ussing-type chambers. Two-path impedance spectroscopy was employed to differentiate between transcellular and paracellular resistance, and confocal imaging and Western blotting was performed. RESULTS: Laurate resulted in a substantial and reversible decrease in transepithelial resistance by 50% which was attributed to a decrease in paracellular resistance. Simultaneously, an increase in permeability for fluorescein (330 Da) was detected, while permeabilities for 4 kDa FITC-dextran and sulpho-NHS-SS-biotin (607 Da) remained unaltered. Confocal laser-scanning microscopy revealed a marked reduction of claudin-5, while other tight junction proteins including tricellulin, a protein preventing the paracellular passage of macromolecules, were not affected. CONCLUSIONS: Laurate induces an increase in paracellular permeability for molecules up to a molecular mass of 330 Da by retrieval of claudin-5 from tight junctions without affecting tricellular contacts and the paracellular passage of macromolecules. We hereby provide, for the first time, a mechanistical explanation of laurate-induced permeability enhancement on molecular level.

Altered expression of tight junction proteins in mammary epithelium after discontinued suckling in mice.

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.