Transepithelial Flux Research Articles

880 Acid Suppression Restores Impaired Esophageal Mucosal Integrity in Patients With Esophageal Eosinophilia

Introduction: Gastroesophageal reflux and allergies are both thought to play a role in the pathophysiology of eosinophilic esophagitis (EoE). It has been suggested that acid-induced esophageal mucosal damage promotes transepithelial allergen flux. Anecdotal evidence suggests that patients with typical signs and symptoms of EoE benefit from acid-suppressive therapy. The aim of our study was to evaluate esophageal mucosal integrity in patients with significant esophageal eosinophilia (SEE) and to study the effects of acid-suppressive therapy with PPI on symptoms, endoscopic signs, mucosal integrity, dilation of epithelial intercellular spaces (DIS), and peak eosinophilia and mastocytosis. Methods: We included 11 untreated adults with SEE (.15 eosinophils/hpf) and predominant symptoms of dysphagia and/or food impaction and typical endoscopic signs of EoE, and 11 controls. All study subjects underwent endoscopy at baseline; in SEE patients endoscopy was repeated after 8 weeks of esomeprazole 40 mg BID. Esophageal mucosal integrity was measured in vivo during endoscopy with a through-the-scope electrical tissue impedance spectroscopy probe (ETIS) at 5 cm proximal of the LES. At the same location, we obtained 2 biopsies for electron microscopic analysis of DIS and 4 biopsies for mucosal integrity experiments in Ussing chambers. In the Ussing chambers, we measured transepithelial electrical resistance (TER) and transmucosal flux of fluorescently-labelled molecules sized 0.3 and 40 kDa (size of food allergens) during 1 hour. In SEE patients, we additionally scored symptoms and endoscopic signs of EoE. Furthermore, eosinophilia and mastocytosis were scored in biopsies taken at the proximal, midand distal esophagus of SEE patients at each endoscopy. Results: Both structural (DIS) and in vivo and in vitro functional measurements (ETIS, TER, and molecule flux) of mucosal integrity showed significant impairment in SEE patients compared to HC (table 1). After acid-suppressive therapy, mucosal integrity was significantly improved in patients with SEE, but still did not reach the levels seen in controls. Esophageal peak eosinophilia andmastocytosis as well as dysphagia and endoscopic signs of EoE also decreased after acid-suppressive therapy. Conclusion: Patients with SEE have an impaired esophageal mucosal integrity, which is partially restored after acid-suppressive therapy. The observed reduction of transmucosal flux of molecules with a similar size as food allergens after acidsuppressive therapy supports the hypothesis that acid-induced mucosal damage facilitates transepithelial food allergen flux, which results in esophageal eosinophilia. Study parameters in HC and SEE patients

Tu2114 Visceral Pain-Related Responses Are Attenuated in Mice With Antibiotic-Induced Intestinal Dysbiosis

BACKGROUND: Toll-like Receptors (TLRs) are effector components in host-bacterial interactions. Within the gut, TLRs participate microbial-related local neuroimmune control, likely including the modulation of epithelial barrier function (EBF). AIMS: To assess changes in colonic EBF associated to the local stimulation of TLR7, with the selective agonist imiquimod, in rats. METHODS: Adult male SD rats were treated intracolonically with the TLR7 agonist imiquimod (100-300 μg/rat, 0.2 ml) or vehicle (0.2 ml/rat). 5 h later colonic samples were obtained to assess EBF in vitro (Ussing chambers). Basal epithelial electrical parameters (short-circuit current; potential difference and conductance) and effects of neural blockade with tetrodotoxin (TTX basolateral, 1 μM) on epithelial secretion were assessed. Paracellular permeability was determined assessing the flux of 4kD fluorescein isothiocyanate-dextran (FD4). Expression of tight-junction-related proteins (occludin, zona occludens-1 [ZO-1], claudin-3, tricellulin and Jam-1), the barrier-related marker proglucagon (precursor of Glucagon-like Peptide-2 [GLP-2]); cytokines (IL-6 and IFN α) and TLR7 was assessed by RTqPCR. Gene expression (RT-qPCR) and distribution (immunohistochemistry) of the poreforming protein claudin-2 were also assessed. RESULTS: Imiquimod (300 μg/rat) showed a tendency to decrease conductance (Table). TTX reduced transepithelial ionic fluxes and conductance, in a similar proportion in either non-treated or imiquimod-treated animals. Imiquimod reduced paracellular flux of FD4 by 17% and 65% at 100 and 300 μg, respectively, vs. vehicle-treated controls (Table). Gene expression of tight-junction-related proteins was not affected by imiquimod. Similarly, expression of proglucagon, the precursor of the barrierenhancer GLP-2, was not modified by imiquimod. Neither gene expression levels nor distribution of the pore-forming protein claudin-2 along the colonic crypts were affected by imiquimod. No inflammatory-like changes associated to imiquimod administration were observed either at the macroscopical, microscopical (histopathology) or molecular (gene expression of cytokines) levels. CONCLUSIONS: In rats, activation of colonic TLR7 enhanced epithelial barrier function, as indicated by the reduction in conductance and permeability to macromolecules. These effects are mediated through yet to defined mechanisms, but were independent of changes in the expression of the main tight-junction-related proteins, the GLP-2 precursor, proglucagon, or the pore-forming protein, claudin-2. Since TLRs are activated by the gut microbiota, increased epithelial tightness associated to their activation might represent a local defensive mechanism generated by dysbiosis and directed towards the prevention of bacterial translocation and the penetration of luminal antigens. Table Effects of activation of TLR7 with imiquimod on colonic EBF

Tu2113 Activation of Colonic Toll-Like Receptor 7 (TLR7) Enhances Epithelial Barrier Function in Rats

BACKGROUND: Toll-like Receptors (TLRs) are effector components in host-bacterial interactions. Within the gut, TLRs participate microbial-related local neuroimmune control, likely including the modulation of epithelial barrier function (EBF). AIMS: To assess changes in colonic EBF associated to the local stimulation of TLR7, with the selective agonist imiquimod, in rats. METHODS: Adult male SD rats were treated intracolonically with the TLR7 agonist imiquimod (100-300 μg/rat, 0.2 ml) or vehicle (0.2 ml/rat). 5 h later colonic samples were obtained to assess EBF in vitro (Ussing chambers). Basal epithelial electrical parameters (short-circuit current; potential difference and conductance) and effects of neural blockade with tetrodotoxin (TTX basolateral, 1 μM) on epithelial secretion were assessed. Paracellular permeability was determined assessing the flux of 4kD fluorescein isothiocyanate-dextran (FD4). Expression of tight-junction-related proteins (occludin, zona occludens-1 [ZO-1], claudin-3, tricellulin and Jam-1), the barrier-related marker proglucagon (precursor of Glucagon-like Peptide-2 [GLP-2]); cytokines (IL-6 and IFN α) and TLR7 was assessed by RTqPCR. Gene expression (RT-qPCR) and distribution (immunohistochemistry) of the poreforming protein claudin-2 were also assessed. RESULTS: Imiquimod (300 μg/rat) showed a tendency to decrease conductance (Table). TTX reduced transepithelial ionic fluxes and conductance, in a similar proportion in either non-treated or imiquimod-treated animals. Imiquimod reduced paracellular flux of FD4 by 17% and 65% at 100 and 300 μg, respectively, vs. vehicle-treated controls (Table). Gene expression of tight-junction-related proteins was not affected by imiquimod. Similarly, expression of proglucagon, the precursor of the barrierenhancer GLP-2, was not modified by imiquimod. Neither gene expression levels nor distribution of the pore-forming protein claudin-2 along the colonic crypts were affected by imiquimod. No inflammatory-like changes associated to imiquimod administration were observed either at the macroscopical, microscopical (histopathology) or molecular (gene expression of cytokines) levels. CONCLUSIONS: In rats, activation of colonic TLR7 enhanced epithelial barrier function, as indicated by the reduction in conductance and permeability to macromolecules. These effects are mediated through yet to defined mechanisms, but were independent of changes in the expression of the main tight-junction-related proteins, the GLP-2 precursor, proglucagon, or the pore-forming protein, claudin-2. Since TLRs are activated by the gut microbiota, increased epithelial tightness associated to their activation might represent a local defensive mechanism generated by dysbiosis and directed towards the prevention of bacterial translocation and the penetration of luminal antigens. Table Effects of activation of TLR7 with imiquimod on colonic EBF

Tu1840 Gastro-Esophageal Reflux Disease, Esophageal Dysmotility and Gastric Emptying in Patients Before and After Laparoscopic Sleeve Gastrectomy

G A A b st ra ct s Barrett patients than in controls and was similar to GERD patients (table 1). In vitro transepithelial electrical resistance of biopsies was also lower in Barrett than in controls and similar to GERD patients, while in vitro transepithelial fluorescein flux was higher in GERD and Barrett patients compared to controls, both with a trend towards significance (table 1). Conclusions: The hypersensitivity to acid observed in GERD patients without Barrett is not present in patients with Barrett's esophagus. Mucosal integrity is, however, equally impaired in patients with Barrett and GERD, indicating that a preserved esophageal mucosal barrier to acid is not an explanation for the lack of acid hypersensitivity in patients with Barrett's esophagus. Table 1. Esophageal sensitivity and mucosal integrity parameters

Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct

The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 μM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.

Mechanisms of interferon gamma‐induced suppression of aquaporin 1 in murine intestinal epithelium

Chronic diseases like IBD are characterized by high levels of IFNγ and abnormal intestinal transepithelial fluid flux. The water channel protein Aquaporin 1 (AQP1) is widely expressed on intestinal epithelial cells with the primary purpose of regulating transmembrane water flux. To investigate the deficiencies in water transport associated with IBD, we sought to determine the cellular signaling mechanisms by which IFNγ modulates AQP1 expression in the mouse intestinal epithelium.CMT93 cells treated with IFNγ significantly decreased constitutive AQP1 levels detected by immunofluorescence. Pre‐treatment with either the JAK1 inhibitor or STAT3 siRNA, prior to IFNγ administration resulted in the amelioration of the IFNγ‐induced suppression of AQP1 protein expression. Conversely, IRF‐2 siRNA pre‐treatment further decreased AQP1 protein levels following IFNγ treatment.Inflammatory cytokines present during IBD are able to influence AQP1 levels and may contribute to the water transport deficiencies associated with these disorders. In this study, the results suggest that decreased epithelial AQP1 expression exerted by IFNγ in CMT93 cells is regulated by JAK1 and STAT3. Of note, IRF‐2 which is normally considered to be a negative regulator of IFNγ signaling, acted in a positive regulatory fashion by further decreasing AQP1 protein signal.Source of funding: Crohn's & Colitis Foundation of Canada

Sublethal hypoxic injury increases intestinal permeability via disruption of sealing tight junction proteins, but not pore forming tight junction proteins in human intestinal epithelium

Intestinal ischemia induces loss of barrier function which is followed by epithelial repair. The tight junction (TJ) has recently been characterized as having both leak and pore properties. We hypothesized that hypoxia would disrupt the TJ seal prior to the pore function followed by barrier recovery. Caco‐2 BBe1 confluent monolayers were subjected to hypoxia for 2 hours followed by recovery in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and flux of 4 kDa FITC‐dextran. TJ proteins were studied by immunoblotting and immunolocalization. Following hypoxic injury, there was an increase in the paracellular permeability, as determined by dextran fluxes, but no change in TER compared to control cells. Permeability recovered with significant reduction in fluxes at 2 and 4 h. The sealing TJ proteins occludin and claudin‐4 increased expression after hypoxic injury. However, the occludin and claudin‐4 fluorescence was diffuse on confocal microscopy, but re‐localized their normal distribution after 4 h recovery. The expression and distribution of the pore forming TJ protein claudin‐7 was not changed. This correlated with a lack of change in TER. These data suggest that hypoxic injury increases paracellular permeability by disruption of sealing TJ proteins, creating a leak but not altering pore selectivity.

Frog skin function revisited; physiology of cutaneous surface fluid of Rana esculenta

Amphibians are adapted to living on dry land where body fluid turnover is governed by evaporative water loss. We aimed at testing the hypothesis [EH Larsen (2011) Acta Physiologica 202: 435–464] that water is evaporating from the cutaneous surface fluid (CSF) secreted by subepidermal glands. Samples of CSF contained, [Na] = 64.5±5.1 and [K] = 14.9±1.6 mM (mean±s.e.m., n = 16) with osmotic pressures of CSF and hemolymph, 168±4 (n = 22) and 238±1.7 (n = 25) mOsm/Kg. The relatively high [K] of CSF and an inwardly directed driving force for transepithelial water flux confirm that gland secretions characterized by high [K] constitute the source of water evaporating from the body surface. Thus, on land CSF is maintained by a balance between fluid secretion by subepidermal glands, water evaporation into the atmosphere, and reabsorption of water and Na by principal cells and Cl by mitochondria‐rich cells of the epidermis. These mechanisms have evolved pari passu with life alternating between aquatic and terrestrial habitats associated with permeabilities of the skin controlled by external ion‐ and osmotic concentrations (loc. cit.). This allows for fast switching of the cutaneous uptake of chloride between active and passive transport associated with dynamic electrical coupling of active sodium uptake by principal cells and passive chloride uptake by mitochondria‐rich cells. Supported by the Carlsberg Foundation.

Involvement of polyamines in iron(III) transport in human intestinal Caco-2 cell lines

Natural polyamines such as putrescine (Put), spermidine (Spd), and spermine (Spm), which are present in the human diet in large amounts, associated with their active transporter, are assumed to play a role in non-heme iron uptake and iron bioavailability from nutrients. Enterocytes and hepatocytes play pivotal roles in the regulation of body iron homeostasis. In this study, we report the effects of natural polyamines on iron transport in the Caco-2 cell line. In enterocyte-like Caco-2 cells, polyamines did not significantly modulate the transepithelial iron flux across the cell monolayer cultured on permeable membranes. In contrast, Spd, Spm, and to a lesser extent, Put were shown to activate Caco-2 cell iron uptake and to induce an increase in the ferritin level. This iron co-transport in enterocytes, which involved an interaction between iron and polyamine then cell uptake of the polyamine-iron complexes by the polyamine transport system, was more pronounced in proliferating than in differentiated Caco-2 cells. Moreover, it was observed at physiological concentrations of both polyamines and iron. It could thus play a role in the rapid renewal of enterocytes. These data suggest the involvement of polyamines as components of the pool of transferrin-independent iron-chelating vectors. Further investigations are needed to demonstrate their biological relevance in physiological situations.

Activation of TRPV4 Strengthens the Tight-Junction Barrier in Human Epidermal Keratinocytes

The transient receptor potential cation channel, subfamily V (TRPV), is expressed in the epidermis and considered to be a sensor of extrinsic stimuli such as temperature and other physical or chemical factors. In this study, we examined whether or not the activation of TRPVs by their agonists alters the epidermal tight junction (TJ) function in cultured human epidermal keratinocytes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA for TRPV1, 3 and 4 were expressed in differentiated keratinocytes in which TJs had formed. Stimulation of the keratinocytes with a TRPV4 agonist (4α-phorbol 12, 13-didecanoate, 4α-PDD) strengthened the TJ-associated barrier, analyzed by means of transepithelial electric resistance measurements and flux measurements of the paracellular tracer. Stimulation with TRPV1 and TRPV3 agonists did not have the same result. Simultaneously, the 4α-PDD-stimulated keratinocytes showed an upregulation of TJ structural proteins, occludin and claudin-4, and TJ regulatory factors, phospho-atypical PKCζ/ι. It was also observed that the amounts of occludin and phospho-atypical PKCζ/ι complex were higher in 4α-PDD stimulated keratinocytes. In conclusion, we demonstrated that the activation of TRPV4 strengthened the TJ-associated barrier of epidermal cells. It was also suggested that the upregulation of TJ structural proteins and/or the posttranslational modification of TJ structural proteins by phospho-atypical PKCζ/ι are responsible for the enhancement of TJ function. Our study supports the hypothesis that TJs change their function in response to a change in the external environment sensed through TRPVs.

Deletion of Slc26a6 alters the stoichiometry of apical Cl−/HCO3− exchange in mouse pancreatic duct

To define the stoichiometry and molecular identity of the Cl(-)/HCO(3)(-) exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO(3)(-) and Cl(-) were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO(3)(-) with luminal Cl(-) in cAMP-stimulated ducts. The flux ratio of Cl(-) absorption/HCO(3)(-) secretion was ∼0.7 in wild-type ducts and ∼1.4 in Slc26a6(-/-) ducts where a different Cl(-)/HCO(3)(-) exchanger, most likely SLC26A3, was found to be active. Interactions between Cl(-)/HCO(3)(-) exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO(3)(-) efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO(3)(-) efflux in Slc26a6(-/-) ducts. In microperfused wild-type ducts, removal of luminal Cl(-), or luminal application of dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6(-/-) ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl(-)/HCO(3)(-) exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl(-)/HCO(3)(-) exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange.

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30-120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [(14)C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased V(max) of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor.

Determining rates of epithelial solute transport by optical measurement of fluorochrome concentration gradients in the unstirred layer

We describe a method for calculating rates of fluorochrome transport from unstirred layer (USL) concentration gradients measured using confocal microscopy. Isolated Malpighian tubules or guts of Drosophila melanogaster were secured to depression slides and bathed in saline containing a fluorescent compound. By measuring the concentration gradient of fluorescent organic anions (fluorescein, Texas Red) or the P-glycoprotein substrate daunorubicin in the USL adjacent to the epithelium we were able to calculate the transepithelial flux of the fluorochrome using Fick's equation. Dose-response curves for fluorescein and Texas Red based on USL concentration gradients near the surface of the Malpighian tubule were comparable to those based on collection and analysis of secreted fluid droplets. Rates of Texas Red and daunorubicin secretion were also calculated for the gut of second instar D. melanogaster larvae, a tissue that is too small for measurement of transport rates by other in vitro techniques such as cannulation and perfusion. Our results suggest that measurement of USL concentration gradients by confocal microscopy may be applicable to any fluorescent indicator of rapidly transported compounds.

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced byBlastocystiscysteine proteases

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors

BackgroundLinaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out.ResultsIn ischemia-damaged pig jejunum, using measurements of transepithelial resistance, 3H-mannitol fluxes, short-circuit current (Cl− secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 μM linaclotide and 1 μM lubiprostone both caused similar increases in short-circuit current (Cl− secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca2+]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear.ConclusionsConsidering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.

Mo1103 Blast-Type Traumatic Brain Injury Alters Intestinal Contractility and Barrier Integrity in Rats

Pathophysiologic and clinicalmanifestations of stress-induced intestinal disorders are initiated by disturbances in intestinal barrier function. Corticotropin releasing factor (CRF) receptor signaling pathways have been shown to be a central pathway by which stress mediates its deleterious effects on the intestinal barrier; however, the precise mechanisms remain poorly understood. In addition, few studies exist that utilize translational large animal models to study CRF pathways in intestinal dysfunction. Here we utilized In Vivo and ex vivo porcine models to investigate the mechanism of intestinal barrier dysfunction mediated by CRF. To determine the role of CRF signaling in stress-induced barrier dysfunction, 16-d old piglets were administered the dual CRF receptor 1 (CRF1)/CRF receptor 2(CRF2) antagonist Astressin B (30 μg/kg), 30 min prior to subjecting pigs to early weaning stress. At 24 h post-weaning stress, ileum was harvested and mounted on Ussing chambers for measurement of intestinal barrier function as determined by transepithelial electrical resistance (TER) and mucosalto-serosal flux of 3H-mannitol. Weaning stress caused significant reductions (p<0.05) in TER and elevations (p<0.01) in 3H-mannitol flux in control pigs; however, pre-treatment with Astressin B ameliorated deleterious changes in barrier function in weaned pigs. In an ex vivo Ussing chamber model, application of CRF to porcine ileum caused elevations in FITC dextran (4 kDa) flux that were blocked by pre-treating tissues with the CRF1 antagonist antalarmin and the CRF1/CRF2 antagonist Astressin B. Application of the selective CRF1 agonist stressin1 to porcine ileum increased FD4 flux and reduced TER compared with CRF and vehicle treatments (p<0.05). CRF and stressin1 were shown to induce mast cell activation and release of mast cell-derived TNF-α and tryptase. CRF and stressin1 mediated increases in FD4 flux were inhibited by pre-treating tissues with protease inhibitors and anti-TNF-α antibodies. These studies demonstrate that in the porcine ileum, stress induces increases in paracellular permeability that is mediated by CRF1. This pathway involves the release of mast cell-derived proteases and TNF-α that contribute to disturbances in intestinal barrier function.

Mo1102 Mechanisms of Stress-Induced Intestinal Barrier Dysfunction in the Porcine Intestine

Pathophysiologic and clinicalmanifestations of stress-induced intestinal disorders are initiated by disturbances in intestinal barrier function. Corticotropin releasing factor (CRF) receptor signaling pathways have been shown to be a central pathway by which stress mediates its deleterious effects on the intestinal barrier; however, the precise mechanisms remain poorly understood. In addition, few studies exist that utilize translational large animal models to study CRF pathways in intestinal dysfunction. Here we utilized In Vivo and ex vivo porcine models to investigate the mechanism of intestinal barrier dysfunction mediated by CRF. To determine the role of CRF signaling in stress-induced barrier dysfunction, 16-d old piglets were administered the dual CRF receptor 1 (CRF1)/CRF receptor 2(CRF2) antagonist Astressin B (30 μg/kg), 30 min prior to subjecting pigs to early weaning stress. At 24 h post-weaning stress, ileum was harvested and mounted on Ussing chambers for measurement of intestinal barrier function as determined by transepithelial electrical resistance (TER) and mucosalto-serosal flux of 3H-mannitol. Weaning stress caused significant reductions (p<0.05) in TER and elevations (p<0.01) in 3H-mannitol flux in control pigs; however, pre-treatment with Astressin B ameliorated deleterious changes in barrier function in weaned pigs. In an ex vivo Ussing chamber model, application of CRF to porcine ileum caused elevations in FITC dextran (4 kDa) flux that were blocked by pre-treating tissues with the CRF1 antagonist antalarmin and the CRF1/CRF2 antagonist Astressin B. Application of the selective CRF1 agonist stressin1 to porcine ileum increased FD4 flux and reduced TER compared with CRF and vehicle treatments (p<0.05). CRF and stressin1 were shown to induce mast cell activation and release of mast cell-derived TNF-α and tryptase. CRF and stressin1 mediated increases in FD4 flux were inhibited by pre-treating tissues with protease inhibitors and anti-TNF-α antibodies. These studies demonstrate that in the porcine ileum, stress induces increases in paracellular permeability that is mediated by CRF1. This pathway involves the release of mast cell-derived proteases and TNF-α that contribute to disturbances in intestinal barrier function.

Bin1 Attenuation Suppresses Experimental Colitis by Enforcing Intestinal Barrier Function

Inflammatory bowel disease (IBD) is associated with defects in intestinal barriers that rely upon cellular tight junctions. Thus, identifying genes that could be targeted to enforce tight junctions and improve barrier function may lead to new treatment strategies for IBD. This preclinical study aimed to evaluate an hypothesized role for the tumor suppressor gene Bin1 as a modifier of the severity of experimental colitis. We ablated the Bin1 gene in a mosaic mouse model to evaluate its effects on experimental colitis and intestinal barrier function. Gross pathology, histology and inflammatory cytokine expression patterns were characterized and ex vivo physiology determinations were conducted to evaluate barrier function in intact colon tissue. Bin1 attenuation limited experimental colitis in a sexually dimorphic manner with stronger protection in female subjects. Colitis suppression was associated with an increase in basal transepithelial electrical resistance (TER) and a decrease in paracellular transepithelial flux, compared to control wild-type animals. In contrast, Bin1 attenuation did not affect short circuit current, nor did it alter the epithelial barrier response to non-inflammatory permeability enhancers in the absence of inflammatory stimuli. Bin1 is a genetic modifier of experimental colitis that controls the paracellular pathway of transcellular ion transport regulated by cellular tight junctions. Our findings offer a preclinical validation of Bin1 as a novel therapeutic target for IBD treatment.

Methotrexate Modulates Tight Junctions Through NF‐κB, MEK, and JNK Pathways

Chemotherapy often induces intestinal mucositis, which is associated with an increase in intestinal permeability; however, underlying mechanisms remain incompletely understood. Thus, we aimed to study the regulation of 3 tight junction (TJ) proteins, claudin-1, occludin, and zonula occludens-1, after anticancer treatment. Methotrexate (MTX) was subcutaneously injected for 3 consecutive days in Sprague-Dawley rats to induce intestinal mucositis and was applied on Caco-2 cell monolayers. TJ protein expression and cellular distribution were studied by Western blot and microscopy, respectively. In Caco-2 cells, the paracellular permeability was evaluated by both transepithelial electrical resistance and flux of fluorescein isothiocyanate-dextran marker. Cytokine production and signaling pathways were also assessed. In MTX-treated rats, the cellular distribution of the 3 TJ proteins was altered and claudin-1 and occludin expression was reduced during the acute phase of mucositis compared with controls. During the recovery phase, these parameters were restored. In vitro, MTX treatment led to an increase in proinflammatory cytokine production at the apical side but did not affect Caco-2 cell apoptosis and necrosis. Increase in paracellular permeability was associated with altered occludin and zonula occludens-1 expression and cellular distribution. All of these alterations were prevented by MEK1 and 2, JNK, and NF-κB inhibitors. MTX treatment induced an increase in intestinal permeability partially related to alteration of TJs protein expression and cellular distribution that may be mediated by MAPK and NF-κB pathways. These are potential targets to limit the adverse effects of chemotherapy.

Physiologic Role of the Menkes Copper ATPase (Atp7a) in Intestinal Iron Transport

Atp7a is a copper transporting ATPase involved in intestinal copper export that is strongly induced in the intestine of iron deficient rats. We evaluated the possible role of Atp7a in iron homeostasis in Atp7a knock down (KD) rat intestinal epithelial (IEC‐6) cells and Brindle mice that express a mutant, inactive form of Atp7a. In KD cells with &gt;90% decrease in Atp7a protein levels, copper loading led to significantly increased intracellular Cu levels, demonstrating a reduction in the copper export function of Atp7a. Iron (59Fe) transport studies in IEC‐6 cells grown in bicameral cell culture inserts revealed significant transepithelial iron flux when cells were deprived of iron. Transport in Atp7a KD cells showed that 59Fe transported into cells and transferred to the basolateral chamber was reduced. Next, Brindled mice were rescued by perinatal copper injection and studies were done to consider the in vivo role of Atp7a in iron absorption. Brindled mice were anemic compared to wild type littermates, and Dmt1, TfR1 and Atp7a expression was induced by dietary iron deprivation. Furthermore, serum ferroxidase activity, reflecting ceruloplasmin, was decreased in Brindled mice as was activity of a novel cytosolic ferroxidase in enterocytes. These data demonstrate a physiologic role for Atp7a in intestinal iron transport possibly related to copper delivery to multi‐copper ferroxidases involved in iron homeostasis.Grant Funding Source : NIH