functional-assessment-intestinal-tight-junction-barrier-ion

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

University of Shizuoka

In neural regulation of the endocrine pancreas, there is much evidence to suggest that vagal efferents alter insulin and glucagon secretion, but less information on the effects of vagal afferents. In this study, we investigated the role and function of afferent fibers of the vagus nerve in normal and ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia. In normal rats, hepatic vagotomy was associated with intraperitoneal (ip) arginine-induced enhancement of insulin and glucagon secretion without an accompanying change in blood glucose levels, ip leucine induced enhancement of insulin secretion accompanied by a decrease in blood glucose levels, and ip alanine-induced enhancement of glucagon secretion accompanied by an increase in blood glucose levels. In VMH lesioned rats with marked hyperinsulinemia, none of these amino acids caused significant changes in insulin and glucagon secretion. We conclude that amino acid sensors in normal rats inhibit excess release of pancreatic hormones induced directly by intake of amino acids, such as that in excess protein ingestion, and maintain blood glucose levels within the normal range. In contrast, in VMH lesioned rats with marked hyperinsulinemia, the function of the amino acid sensors is masked due to the marked hyperinsulinemia in these rats.:

Masked function of amino acid sensors on pancreatic hormone secretion in ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia.

The role of mucosal layer thickness on prevention of acute gastric mucosal lesions (AGMLs) was examined in ventromedial hypothalamic (VMH)-lesioned rats.

Cell proliferation in ventromedial hypothalamic lesioned rats inhibits acute gastric mucosal lesions.

Ventromedial hypothalamic lesions enhance small intestinal cell proliferation in mice.

Dietary NaCl depletion increases Na absorption and K secretion in the colon, but the mechanisms are not fully understood. In mice fed with NaCl-depleted diets, the expression of claudin-2 and -7 increased compared to those in control mice. Aldosterone (ALD) concentration was also increased. We examined the regulatory mechanism of claudin expression by ALD using the murine colonic epithelial MCE301 cells. ALD dose-dependently increased claudin-2 expression without affecting the expression of claudin-4, -7, -8, and -15. ALD increased nuclear distribution of mineralocorticoid receptor (MR), which was inhibited by spironolactone, an MR antagonist. The ALD-induced elevation of claudin-2 mRNA and protein expression was inhibited by spironolactone, but not by RU-486, a glucocorticoid receptor antagonist. Luciferase reporter assay showed that ALD interacts with the promoter region between -2,021 and -2,008 of human claudin-2. The binding of MR on the promoter region of claudin-2 was increased by ALD, which was inhibited by spironolactone in chromatin immunoprecipitation assay. Our data suggest that ALD acts on MR and increases paracellular permeability to ions mediated by the elevation of claudin-2 expression in the colon. NaCl depletion may increase ALD secretion from adrenal cortex, resulting in the elevation of paracellular permeability to cations in the colon.

Up-regulation of claudin-2 expression by aldosterone in colonic epithelial cells of mice fed with NaCl-depleted diets.

Intestinal cell line studies indicated luminal Na homeostasis is essential for proton-coupled peptide absorption, because the driving force of PepT1 activity is supported by the apical Na/H exchanger NHE3. However, there is no direct evidence demonstrating the importance of in vivo luminal Na for peptide absorption in animal experiments. To investigate the relationship between luminal Na homeostasis and peptide absorption, we took advantage of claudin 15-deficient (cldn15) mice, whereby Na homeostasis is disrupted. We quantitatively assessed the intestinal segment responsible for peptide absorption using radiolabeled nonhydrolyzable dipeptide (glycylsarcosine, Gly-Sar) and nonabsorbable fluid phase marker polyethylene glycol (PEG) 4000 in vivo. In wild-type (WT) mice, the concentration ratio of Gly-Sar to PEG 4000 decreased in the upper jejunum, suggesting the upper jejunum is responsible for peptide absorption. Gly-Sar absorption was decreased in the jejunum of cldn15 mice. To elucidate the mechanism underlining these impairments, a Gly-Sar-induced short-circuit ( I) current was measured. In WT mice, increments of Gly-Sar-induced I were inhibited by the luminal application of a NHE3-specific inhibitor S3226 in a dose-dependent fashion. In contrast to in vivo experiments, robust Gly-Sar-induced I increments were observed in the jejunal mucosa of cldn15 mice. Gly-Sar-induced I was inhibited by S3226 or a reduction of luminal Na concentration, which mimics low luminal Na concentrations in vivo . Our study demonstrates that luminal Na homeostasis is important for peptide absorption in native epithelia and that there is a cooperative functional relationship between PepT1 and NHE3. NEW & NOTEWORTHY Our study is the first to demonstrate that luminal Na homeostasis is important for proton-coupled peptide absorption in in vivo animal experiments.

Luminal Na homeostasis has an important role in intestinal peptide absorption in vivo.

Many nutrients are absorbed via Na cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na. It is thought that Na diffuses back into the lumen via paracellular pathways to support Na cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient () mice, which have been shown to have decreased paracellular Na permeability. We measured glucose-induced currents (Δ) under open- and short-circuit conditions and simultaneously measured changes in unidirectional Na fluxes (Δ) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in Δ and unidirectional mucosal to serosal Na (∆) flux in both wild-type and  mice. However, under open-circuit conditions, Δ was observed but ∆ was strongly inhibited in wild-type but not in  mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na conductance was decreased and glucose-induced ∆ increment was observed under open-circuit conditions. We concluded that the Na which is absorbed by Na-dependent glucose cotransport is recycled back into the lumen via paracellular Na conductance through claudin-15, which is driven by Na cotransport induced luminal negativity.

Na-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na Recycling in Mouse Small Intestine.

Dietary NaCl depletion increases Na and Cl absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1 without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl mediated by the elevation of CLDN7 expression in the colon.

Upregulation of Claudin-7 Expression by Angiotensin II in Colonic Epithelial Cells of Mice Fed with NaCl-Depleted Diets.

Angulin-2/ILDR1 is a member of the angulin protein family, which is exclusively expressed at tricellular tight junctions in epithelia. Tricellular tight junctions are found where three cells meet and where three bicellular tight junction strands converge. Tricellular tight junctions are thought to be important for paracellular permeability of ions and water in epithelial tissues. It was recently reported that angulin-2/ILDR1 knockout mice have water transport abnormalities in the kidney. Since angulin-2/ILDR1 is the main tricellular tight junction protein in the large intestine, the goal of this research was to examine the effect of angulin-2/ILDR1 knockout on large intestinal paracellular water transport. We found that Ildr1 knockout mice showed no detectable phenotype other than deafness. In addition, paracellular transport as assessed by Ussing chamber was unchanged in Ildr1 knockout mice. However, we found that in the colon and the kidney of Ildr1 knockout mice, another tricellular tight junction protein, angulin-1/LSR, changes its expression pattern. We propose that with this replacement in tissue localization, angulin-1/LSR compensates for the loss of angulin-2/ILDR1 and maintains the barrier and function of the epithelia in the large intestine as well as the kidney.

Angulin-2/ILDR1, a tricellular tight junction protein, does not affect water transport in the mouse large intestine.

Colonic epithelial cells move up along the crypt villus axis and are differentiated into absorptive or secretory cells. Claudin-7 (CLDN7), a tight junctional protein, is mainly located at the surface of crypt, whereas CLDN2 is located at the bottom. However, the expression mechanism and function of these CLDNs are not fully understood. The expression levels of CLDN2 and CLDN7 were altered depending on the culture days in MCE301 cells derived from mouse colon. The nuclear levels of transcriptional factors p53 and hepatocyte nuclear factor 4α (HNF4α) at day 21 were higher than those at day 7. Tenovin-1 (TEN), a p53 activator, increased the nuclear levels of p53 and HNF4α. The mRNA level and promoter activity of CLDN7 were increased by TEN, whereas those of CLDN2 were decreased. The changes of CLDNs expression were inhibited by p53 and HNF4α siRNAs. The association between p53 and HNF4α was elevated by TEN. In addition, the binding of p53 and HNF4α to the promoter region of CLDN2 and CLDN7 was enhanced by TEN. Transepithelial electrical resistance was decreased by TEN, but paracellular fluxes of lucifer yellow and dextran were not. In the Ussing chamber assay, TEN increased dilution potential and the ratio of permeability of Cl to Na. Both p53 and HNF4α were highly expressed at the surface of mouse colon crypt. We suggest that p53 and HNF4α alter the paracellular permeability of Cl to Na mediated by the inverse regulation of CLDN2 and CLDN7 expression in the colon.

Article	Year
Masked function of amino acid sensors on pancreatic hormone secretion in ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia. Obesity research & clinical practice\| PubMed ID: 24331525	2012
Cell proliferation in ventromedial hypothalamic lesioned rats inhibits acute gastric mucosal lesions. Obesity research & clinical practice\| PubMed ID: 24331526	2012
Ventromedial hypothalamic lesions enhance small intestinal cell proliferation in mice. Obesity research & clinical practice\| PubMed ID: 24331527	2012
Up-regulation of claudin-2 expression by aldosterone in colonic epithelial cells of mice fed with NaCl-depleted diets. Scientific reports\| PubMed ID: 28939904	2017
Luminal Na homeostasis has an important role in intestinal peptide absorption in vivo. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 30138575	2018
Na-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na Recycling in Mouse Small Intestine. International journal of molecular sciences\| PubMed ID: 31936130	2020
Upregulation of Claudin-7 Expression by Angiotensin II in Colonic Epithelial Cells of Mice Fed with NaCl-Depleted Diets. International journal of molecular sciences\| PubMed ID: 32093310	2020
Angulin-2/ILDR1, a tricellular tight junction protein, does not affect water transport in the mouse large intestine. Scientific reports\| PubMed ID: 32587380	2020
Inverse regulation of claudin-2 and -7 expression by p53 and hepatocyte nuclear factor 4α in colonic MCE301 cells. Tissue barriers\| PubMed ID: 33356822	2021

Noriko Ishizuka

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