Inter-claudin antagonism of paracellular pore function: mechanism and beyond.

Claudin-2-dependent pore function mediates paracellular cation permeability and can result in pathogenesis of many diseases. Although existing various types of claudins, including barrier-forming and pore-forming claudins, their heterodimeric interaction affecting barrier and pore functions has never been fully elucidated yet. Recently, Shashikanth and colleagues demonstrated that expression of claudin-4 was able to antagonize paracellular pore activity of claudin-2. This commentary will emphasize the mechanism underlying claudin-4-mediated claudin-2-dependent pore inhibition and discuss its potential therapeutic and prognostic applications.

Matrix metalloproteinase-7 and claudin-7 as novel identified therapeutic targets for restoration of intestinal epithelial barrier in inflammatory bowel diseases.

Intestinal tight junction disruption and mucosal immune dysregulation contribute to pathogenesis and progression of inflammatory bowel diseases (IBD). A proteolytic enzyme matrix metalloproteinase 7 (MMP-7), which is highly expressed in intestinal tissue, is implicated to IBD and other immune overactivation-associated diseases. In the issue of the Frontiers in Immunology, Ying Xiao and colleagues demonstrate that MMP-7-mediated claudin-7 degradation promotes IBD pathogenesis and disease progression. Therefore, inhibition of MMP-7 enzymatic activity can be a therapeutic strategy for the treatment of IBD.

Discovery of Fungus-Derived Nornidulin as a Novel TMEM16A Inhibitor: A Potential Therapy to Inhibit Mucus Secretion in Asthma.

Introduction: Inhibition of Ca2+-activated transmembrane protein 16A (TMEM16A) Cl- channels has been proposed to alleviate mucus secretion in asthma. In this study, we identified a novel class of TMEM16A inhibitors from natural sources in airway epithelial Calu-3 cells and determine anti-asthmatic efficacy of the most potent candidate in a mouse model of asthma. Methods: For electrophysiological analyses, IL-4-primed Calu-3 cell monolayers were mounted in Ussing chamber and treated with various fungus-derived depsidones prior to the addition of UTP, ionomycin, thapsigargin, or Eact to stimulate TMEM16A Cl- current. Ca2+-induced mucus secretion in Calu-3 cell monolayers was assessed by determining MUC5AC protein remaining in the cells using immunofluorescence staining. OVA-induced female BALB/c mice was used as an animal model of asthma. After the course of induction, cellular and mucus components in bronchoalveolar lavage were analyzed. Lungs were fixed and undergone with H&E and PAS staining for the evaluation of airway inflammation and mucus production, respectively. Results: The screening of fungus-derived depsidones revealed that nornidulin completely abolished the UTP-activated TMEM16A current in Calu-3 cell monolayers with the IC50 and a maximal effect being at ~0.8 µM and 10 µM, respectively. Neither cell viability nor barrier function was affected by nornidulin. Mechanistically, nornidulin (10 µM) suppressed Cl- currents induced by ionomycin (a Ca2+-specific ionophore), thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ ATPase), and Eact (a putative TMEM16A activator) without interfering with intracellular Ca2+ ([Ca2+]i) levels. These results suggest that nornidulin exerts its effect without changing [Ca2+]i, possibly through direct effect on TMEM16A. Interestingly, nornidulin (at 10 µM) reduced Ca2+-dependent mucus release in the Calu-3 cell monolayers. In addition, nornidulin (20 mg/kg) inhibited bronchoalveolar mucus secretion without impeding airway inflammation in ovalbumin-induced asthmatic mice. Discussion and Conclusion: Our study revealed that nornidulin is a novel TMEM16A inhibitor that suppresses mucus secretion without compromising immunologic activity. Further development of nornidulin may provide a new remedy for asthma or other diseases associated with allergic mucus hypersecretion without causing opportunistic infections.

Butylene Glycol Used as a Sustainable Solvent for Extracting Bioactive Compounds from Camellia sinensis Flowers with Ultrasound-Assisted Extraction.

The research aims to assess the yield of bioactive compounds and their antioxidant activities obtained from tea flowers using an ultrasound-assisted extraction method with butylene glycol (BG-UAE) through Box-Behnken design. It investigates the bioactive compounds including the total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC) and analyzes their antioxidant activities, bioactive compound composition by liquid chromatography triple quadrupole tandem mass spectrometry, and their cellular activities via UAE and maceration using BG or ethanol as the solvent. Under optimal conditions, the values of the TPC, TFC, TTC, 1,1-diphenyl-2-picrylhydrazil radical scavenging assay, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical scavenging assay, and ferric reducing antioxidant power assay (FRAP) of the BG-UAE extract were 54.00 ± 1.19 mg GAE/g sample, 291.47 ± 3.34 mg QE/g sample, 65.37 ± 1.78 mg TAE/g sample, 106.45 ± 1.21 mg TEAC/g sample, 163.58 ± 2.76 mg TEAC/g sample, and 121.31 ± 4.75 mg FeSO4/g sample, respectively. Except for FRAP, BG-UAE exhibited the highest values in all parameters compared to the other extraction methods. Catechins and caffeine were predominantly detected in tea flower extracts through UAE with BG and ethanol (EtOH-UAE). BG-UAE exhibited greater cell viability and cellular antioxidant activity than EtOH-UAE. The researcher expects that this research will contribute to the emergence of a green extraction technique that will offer larger functional components with economic and environmental benefits and minimal chemicals and energy use.

Tight junctions: from molecules to gastrointestinal diseases.

Intestinal epithelium functions as a tissue barrier to prevent interaction between the internal compartment and the external milieu. Intestinal barrier function also determines epithelial polarity for the absorption of nutrients and the secretion of waste products. These vital functions require strong integrity of tight junction proteins. In fact, intestinal tight junctions that seal the paracellular space can restrict mucosal-to-serosal transport of hostile luminal contents. Tight junctions can form both an absolute barrier and a paracellular ion channel. Although defective tight junctions potentially lead to compromised intestinal barrier and the development and progression of gastrointestinal (GI) diseases, no FDA-approved therapies that recover the epithelial tight junction barrier are currently available in clinical practice. Here, we discuss the impacts and regulatory mechanisms of tight junction disruption in the gut and related diseases. We also provide an overview of potential therapeutic targets to restore the epithelial tight junction barrier in the GI tract.

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,ß-dehydrolovastatin (DHLV), α,ß-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 µM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 µM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 µM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

Relief Effects of Icariin on Inflammation-Induced Decrease of Tight Junctions in Intestinal Epithelial Cells.

Inflammatory cytokines including TNF-α and IL-1ß impair intestinal barrier function in aging by disrupting intestinal tight junction integrity. Icariin (ICA) has a variety of pharmacological effects. Indeed, ICA produces anti-inflammatory, anti-oxidative stress, and inhibitory effects on microRNA (miRNA) expression. This study was to explore whether ICA could alleviate inflammation-associated intestinal barrier function impairment in aging and its underlying mechanism. Of particular interest, network pharmacology prediction indicated the potential therapeutic impacts of ICA for the treatment of colitis. Then, rats were used to study whether ICA has a protective effect on the reduction of tight junctions caused by inflammatory cytokines. Next, Caco-2 cell monolayers were used to explore the mechanism by which ICA alleviates the down-regulation of tight junctions. Network pharmacology prediction revealed that ICA alleviated colitis via suppressing oxidative stress. After ICA intervention, expressions of inflammatory cytokines were reduced, but tight junctions, antioxidant enzymes in aging rats were up-regulated. ICA reversed the TNF-α-induced decrease in abundance of Occludin protein in Caco-2 cell monolayers. Meanwhile, ICA alleviated the increase in permeability and expression of miR-122a. However, the protective effect of ICA was markedly attenuated after transfection with miR-122a mimics. In conclusion, ICA reduced the expressions of Occludin, Claudin1, and Claudin5 in colon, which were related to the reduction of TNF-α and IL-1ß and alleviation of colonic in vivore. And ICA attenuated TNF-α-induced Occludin disruption and epithelial barrier impairment by decreasing miR-122a expression in Caco-2 cell monolayers.

ß-eudesmol but not atractylodin exerts an inhibitory effect on CFTR-mediated chloride transport in human intestinal epithelial cells.

Oriental herbal medicine with the two bioactive constituents, ß-eudesmol (BE) and atractylodin (AT), has been used as a remedy for gastrointestinal disorders. There was no scientific evidence reporting their antidiarrheal effect and underpinning mechanisms. Therefore, we aimed to investigate the anti-secretory activity of these two compounds in vitro. The inhibitory effect of BE and AT on cAMP-induced Cl- secretion was evaluated by Ussing chamber in human intestinal epithelial (T84) cells. Short-circuit current (ISC) and apical Cl- current (ICl-) were measured after adding indirect and direct cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activator. MTT assay was used to determine cellular cytotoxicity. Protein-ligand interaction was investigated by in silico molecular docking analysis. BE, but not AT concentration-dependently (IC50 of ~1.05 µM) reduced cAMP-mediated, CFTRinh-172 inhibitable Cl- secretion as determined by transepithelial ISC across a monolayer of T84 cells. Potency of CFTR-mediated ICl- inhibition by BE did not change with the use of different CFTR activators suggesting a direct blockage of the channel active site(s). Pretreatment with BE completely prevented cAMP-induced ICl-. Furthermore, BE at concentrations up to 200 µM (24 h) had no effect on T84 cell viability. In silico studies indicated that BE could best dock onto dephosphorylated structure of CFTR at ATP-binding pockets in nucleotide-binding domain (NBD) 2 region. These findings provide the first evidence for the anti-secretory effect of BE involving inhibition of CFTR function. BE represents a promising candidate for the therapeutic or prophylactic intervention of diarrhea resulted from intestinal hypersecretion of Cl.

Electrophysiologic Analysis of Tight Junction Size and Charge Selectivity.

Tight junctions form selectively permeable barriers that limit paracellular flux across epithelial-lined surfaces. Rather than being absolute barriers, tight junctions in many tissues allow ions, water, and other small molecules to cross on the basis of size and charge selectivity via the high-capacity pore pathway. Most probes currently used to assess tight junction permeability exceed the maximum size capacity of the pore pathway. As a result, available analytical tools have generally been limited to measurement of transepithelial electrical resistances. These provide no information regarding size selectivity and, therefore, cannot be used to distinguish between the pore pathway and the leak pathway, a low-capacity route that accommodates larger macromolecules. This article describes use of dilution potential and bi-ionic potential measurements for analysis of tight junction size and charge selectivity within monolayers of cultured epithelial cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Culture of MDCK monolayers on semipermeable supports and induction of claudin-2 expression Basic Protocol 2: Configuring voltage/current clamp and other equipment Basic Protocol 3: Measuring dilution and bi-ionic potentials Basic Protocol 4: Calculating ion permeabilities and pore diameter Support Protocol: Preparation of agar bridges and electrophysiology rig setup.

Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly.

Intestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca2+ concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca2+ into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this principle, the Ca2+-switch assay was established to investigate tight junction assembly in fully differentiated intestinal epithelial cells. This chapter provides a stepwise protocol for culture of intestinal epithelial cell monolayers using T84 cell line as an in vitro model and the Ca2+-switch assay for evaluating tight junction assembly.

Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity.

Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction-independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage. Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay.

Myosin light chain kinase is a potential target for hypopharyngeal cancer treatment.

Hypopharyngeal cancer is squamous cell carcinoma (SCC) with the worst prognosis among the head and neck cancers. Overall, the 5-year survival rate remains poor although diagnostic imaging, radiation, chemotherapy, and surgical techniques have been improved. The mortality of patients with hypopharyngeal cancer is partly due to an increased likelihood of developing a second primary malignancy and metastasis. In this study, we found that MLCK expression, compared to healthy tissue, was up-regulated in hypopharyngeal tumor tissue. Of particular interest, a low 5-year survival rate was positively correlated with MLCK expression. We hypothesized that MLCK might be a target for hypopharyngeal cancer prognosis and treatment. In order to explore the function of MLCK in the development of cancer, we knockdown MLCK in hypopharyngeal cancer FaDu cells. The results showed that MLCK knockdown reduced the migration and invasion of FaDu cells. 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) is the derivative of all-trans retinoic acid (ATRA), which was able to reduce both MLCK expression and activity in FaDu cells. ATPR induced FaDu cells apoptosis in a dose-dependent manner and also inhibited cell growth both in vivo and in vitro. Further experiments showed that overexpression of MLCK reduced ATPR induced-migration inhibition while increase of ATPR induced apoptosis, which suggested that MLCK was involved in ATPR's anti-cancer function. In conclusion, MLCK is a novel prognostic marker and therapeutic target for hypopharyngeal cancer. By targeting MLCK, ATPR exhibits its potential application in the treatment of this type of cancer.

A prebiotic fructo-oligosaccharide promotes tight junction assembly in intestinal epithelial cells via an AMPK-dependent pathway.

Tight junctions play an important role in maintaining barrier integrity of intestinal epithelia. Activation of AMP-activated protein kinase (AMPK) promotes tight junction assembly in intestinal epithelial cells (IEC). Fructo-oligosaccharides (FOS), well-known prebiotics, have previously been shown to alleviate inflammation-associated intestinal epithelial disruption although the mechanisms were unclear. This study aimed to investigate any effect of FOS on AMPK activity and tight junction assembly under non-inflammatory and inflammatory conditions using T84 cells as an IEC model. As analyzed by western blot, FOS induced AMPK activation through a calcium sensing receptor (CaSR)-phospholipase C (PLC)- Ca2+/calmodulin-dependent protein kinase kinase-ß (CaMKKß) pathway. Calcium switch assays and immunofluorescence staining of zonula occludens-1 (ZO-1) revealed that FOS induced tight junction assembly via an CaMKKß-AMPK-dependent mechanism in IEC. Interestingly, FOS reversed the suppressive effect of lipopolysaccharide (LPS) on AMPK activity and tight junction assembly via a CaMKKß pathway. Taken together, these findings uncover a prebiotic-independent effect of FOS in promoting intestinal epithelial tight junction assembly through AMPK activation, which may have implications for the treatment of diseases whose pathogenesis involves impaired intestinal barrier function.

Inactivation of paracellular cation-selective claudin-2 channels attenuates immune-mediated experimental colitis in mice.

The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.

A G-protein coupled receptor 39 agonist stimulates proliferation of keratinocytes via an ERK-dependent pathway.

Keratinocyte proliferation serves as a crucial process in skin wound healing. The zinc-sensing G-protein coupled receptor 39 (GPR39), which is highly expressed in keratinocytes, has been shown to promote skin wound healing. The aim of this study was to investigate the effect of GPR39 activation on proliferation of keratinocytes and its underlying mechanism using immortalized human keratinocytes (HaCaT) as an in vitro model. GPR39 was functionally expressed in HaCaT cells. BrdU proliferation assays showed that treatment with GPR39 agonist TC-G 1008 (100 nM and 1 µM) increased cell proliferation. TC-G 1008 also enhanced ERK phosphorylation in time- and concentration-dependent manners. This effect was suppressed by co-treatment with wortmannin (PI3K inhibitor) and U0126 (MKK inhibitor). Of note, neither inhibition of Gαq-phospholipase C (PLC)-[Ca2+]i nor Gαs-PKA pathway affected GPR39 stimulation-induced ERK phosphorylation. Similarly, barbadin, an inhibitor of G-protein-independent ß-arrestin pathway, did not suppress ERK phosphorylation induced by GPR39 activation. Of particular importance, wortmannin, U0126, and FR180204 (ERK inhibitor) abrogated the effect of TC-G 1008-induced cell proliferation. Taken together, this study reveals novel insights into the role of GPR39 in regulating keratinocyte proliferation via a PI3K-MKK-ERK-dependent mechanism. GPR39 agonists may be used in enhancing keratinocyte proliferation, which may be beneficial for the cutaneous wound treatment.

Fructo-oligosaccharides alleviate inflammation-associated apoptosis of GLP-1 secreting L cells via inhibition of iNOS and cleaved caspase-3 expression.

Glucagon-like peptide 1 (GLP-1) released from enteroendocrine (L) cells regulates insulin secretion. Intestinal inflammation and impaired GLP-1 release have been found in type 2 diabetes mellitus (T2DM) patients. Fructo-oligosaccharides (FOS), a known prebiotic, improve GLP-1 release and glucose homeostasis in T2DM models. This study aimed to investigate the effect of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine associated with intestinal inflammation in T2DM, on L cell apoptosis and the effect of FOS on inflammation-associated impairment of GLP-1 secretion. Herein, using cell death assays, immunofluorescence staining, real time PCR and Western blot analyses, we found that TNF-α induced L cell apoptosis via nuclear factor kappa B (NF-κB)- inducible nitric oxide synthase (iNOS)-cleaved caspase-3-dependent pathways. Interestingly, FOS did not suppress TNF-α-induced NF-κB nuclear translocation, but inhibited expression of iNOS and cleaved caspase-3. In addition, FOS alleviated apoptosis and rescued impaired GLP-1 release in TNF-α-treated L cells. Altogether, our data indicate that TNF-α induces L cell apoptosis via an NF-κB-iNOS-caspase-3-dependent pathway. FOS may be useful in suppressing inflammation-associated L cell apoptosis and maintaining GLP-1 level in T2DM patients.

Galactomannan Pentasaccharide Produced from Copra Meal Enhances Tight Junction Integration of Epithelial Tissue through Activation of AMPK.

Mannan oligosaccharide (MOS) is well-known as an effective fed supplement for livestock to increase their nutrients absorption and health status. Pentasaccharide of mannan (MOS5) was reported as a molecule that possesses the ability to increase tight junction of epithelial tissue, but the structure and mechanism of action remains undetermined. In this study, the mechanism of action and structure of MOS5 were investigated. T84 cells were cultured and treated with MOS5 compared with vehicle and compound C, a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. The results demonstrated that the ability of MOS5 to increase tight junction integration was inhibited in the presence of dorsomorphine (compound C). Phosphorylation level of AMPK was elevated in MOS5 treated group as determined by Western blot analysis. Determination of MOS5 structure was performed using enzymatic mapping together with 1H, 13C NMR, and 2D-NMR analysis. The results demonstrated that the structure of MOS5 is a ß-(1,4)-mannotetraose with α-(1,6)-galactose attached at the second mannose unit from non-reducing end.

Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

Constitutive androstane receptor (CAR) belonging to the nuclear receptor superfamily plays an important role in the xenobiotic metabolism and disposition. It has been reported that CAR regulates the expression of the ATP-binding cassette (ABC) transporters in the intestine, such as multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2/3 (MRP2 and MRP3). In this study, we investigated the role of CAR in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport in T84 human colonic epithelial cells and mouse intestinal tissues. Treatments of T84 cell monolayers with specific CAR agonists (CITCO and phenytoin at concentrations of 1 µM and 5 µM, respectively) for 24 h decreased transepithelial Cl- secretion in response to cAMP-dependent agonist. This inhibition was abolished by coincubation of CITCO with a CAR antagonist, CINPA1. We confirmed that an inhibitory effect of CAR agonists was not due to their cytotoxicity. Basolateral membrane permeabilization experiments also revealed that activation of CAR decreased apical Cl- current stimulated by both CPT-cAMP and genistein (a direct CFTR activator). Such activation also reduced both mRNA and protein expression of CFTR. Furthermore, CITCO decreased cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers. In ICR mice, administration of TCPOBOP (3 mg/kgBW), a murine-specific CAR agonist, for 7 days produced significant decreases in CFTR mRNA and protein expressions in intestinal tissues. Interestingly, TCPOBOP also inhibited CT-induced intestinal fluid accumulation in mice. This is the first evidence showing that CFTR was downregulated by CAR activation in the intestine. Our findings suggest that CAR has potential as a new drug target for treatment of condition with hyperactivity/ hyperfunction of CFTR especially secretory diarrheas.

An agonist of a zinc-sensing receptor GPR39 enhances tight junction assembly in intestinal epithelial cells via an AMPK-dependent mechanism.

Intestinal barrier function depends on integrity of tight junctions, which serve as barriers to transepithelial influx of noxious substances/microorganisms from gut lumen. The G-protein coupled receptor 39 (GPR39) is a zinc-sensing receptor, which is expressed in several cell types including intestinal epithelial cells (IECs). The main objective of this study was to investigate the effect of GPR39 activation on tight junction assembly in IECs. Treatment with TC-G 1008 (1 µM -10 µM), a GPR39 agonist, and zinc (10 µM -100 µM) increased tight junction assembly in T84 cells. This effect was suppressed by pretreatment with compound C, an inhibitor of AMP-activated protein kinase (AMPK). In addition, western blot analysis revealed that treatment with TC-G 1008 induced AMPK activation in time- and concentration-dependent manners. Interestingly, inhibitors of phospholipase C (PLC) and calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) abrogated the effect of TC-G 1008 on inducing AMPK activation, tight junction assembly and zonula occludens-1 re-organization. Collectively, this study reveals a novel role of GPR39 in enhancing tight junction assembly in IECs via PLC-CaMKKß-AMPK pathways. GPR39 agonists may be beneficial in the treatment of diseases associated impaired intestinal barrier function.