Ussing Chamber Method Research Articles

Modeling ocular surface ion and water transport by generation of lipid- and mucin-producing human Meibomian gland and conjunctival epithelial cells.

Despite the importance of ocular surface in human physiology and diseases, little is known about ion channel expression, properties and regulation in ocular epithelial cells. Furthermore, human primary epithelial cells have rarely been studied in favor of rat, mouse and especially rabbit animal models. Here, we developed primary human Meibomian gland (hMGEC) and conjunctival (hConEC) epithelial cells. We show that hConEC and hMGEC produce MUC5AC and lipids, respectively. With cell cultures maintained at the air-liquid interface, we recorded transepithelial short-circuit currents by the Ussing chamber method. We identified in the apical membrane Na+, Cl- and K+ ion channels; amiloride-sensitive ENaC, cAMP-dependent CFTR, UTP-dependent TMEM16a, and chromanol 293B-sensitive KCNQ1. At the basolateral membrane we identified bumetanide-sensitive NKCC and barium-sensitive K+ channels. We also found that VIP, concentration-dependent (EC50 of 1-8 nM), stimulates the CFTR-dependent Isc in both cells. Western blot analysis confirms the expression in both cell cultures of βENaC subunit, CFTR, TMEM16a and KCNQ1 proteins. We recorded water influx by quantitative phase microscopy and identified a cAMP-dependent and mercury sensitive water flux and identified by western blot AQP3 and AQP5 proteins in hConEC and hMGEC. Taken together, we propose a model of the ion transports of human conjunctival and Meibomian gland epithelial cells that will set the stage for a future molecular dissection of the regulation of these transport proteins in the context of tear secretion and related diseases.

Functional in vivo effects of an ENaC Na+ self inhibition-enhancing mutation

Background: Epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron is a critical regulator of extracellular fluid volume and K+ homeostasis. Physiologic concentrations of extracellular Na+ reduce channel activity (open probability; PO) via an allosteric mechanism termed “Na+ self-inhibition,” which involves Na+ ions binding to specific sites in the channel’s extracellular domains. Recent studies have identified human ENaC mutations that alter the Na+ self-inhibition response when assayed in vitro. Such mutations may confer sensitivity or resistance to salt-induced blood pressure changes. Using CRISPR-Cas9 technology, we generated mice carrying a mutation previously shown to enhance Na+ self-inhibition (αH282R) to assess the effects on ENaC-dependent Na+ transport in vivo. We predicted that ENaC activity would be reduced in these mice, leading to enhanced sensitivity to Na+ deprivation or K+ loading, along with accompanying compensatory changes to overcome the predicted loss of function. Methods: We measured urinary electrolyte handling in wild type (WT) and αH282R mice maintained on normal, low Na+, or high K+ diets using metabolic cages. We assessed ENaC activity ex vivo using patch clamp and Ussing chamber methods. We also measured blood chemistry and aldosterone levels in WT and αH282R mice under the dietary conditions described above. Results: When maintained on a normal chow diet (0.3% NaCl), αH2832R mice expressed ENaCs in the distal nephron with reduced PO compared to WT littermates (0.026 vs. 0.067; p<0.01), while ENaC subunit protein expression was unaltered. Amiloride-sensitive short-circuit currents were reduced in the distal colons of αH283R mice versus WT littermates (-12.6 vs. -3.9 μA/cm2; p<0.01). Plasma aldosterone levels were also higher in αH283R versus WT mice on a control diet (610 vs 288 pg/mL; p=0.02), while no other differences in baseline blood chemistry were observed. When challenged with either dietary Na+ restriction (<0.01% NaCl) or K+ loading (10% KCl), WT and αH283R mice responded similarly in terms of body weight maintenance, urinary Na+ and K+ excretion, and systemic electrolyte and metabolic parameters, with the exception of higher aldosterone levels in Na+-restricted αH282R mice compared to WT littermates (2,759 vs. 1271 pg/mL; p<0.01). Conclusions: The αH282R mutation, which enhances Na+ self-inhibition, confers reduced ENaC activity in mice. However, this loss of function may be compensated by ENaC-independent Na+/K+ handling mechanisms and/or increased aldosterone signaling during adaptation to dietary salt stressors. P30DK079307, T32DK061296. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Microplastics in aquaculture - Potential impacts on inflammatory processes in Nile tilapia

Aquaculture is essential for meeting the growing global demand for fish consumption. However, the widespread use of plastic and the presence of microplastics in aquaculture systems raise concerns about their impact on fish health and the safety of aquaculture products. This study focused on the Nile tilapia (Oreochromis niloticus), one of the most important aquaculture fish species globally. The aim of this study was to investigate the effects of dietary exposure to a mixture of four conventional fossil fuel-based polymers (microplastics) on the health of adult and juvenile Nile tilapia. Two experiments were conducted, with 36 juvenile tilapia (10–40 g weight) exposed for 30 days and 24 adult tilapia (600–1000 g) exposed for 7 days, the former including a natural particle (kaolin) treatment. In the adult tilapia experiment, no significant effects on intestinal health (Ussing chamber method), oxidative stress, or inflammatory pathways (enzymatic and genetic biomarkers) were observed after exposure to the microplastic mixture. However, in the juvenile tilapia experiment, significant alterations in inflammatory pathways were observed following 30 days of exposure to the microplastic mixture, indicating potential adverse effects on fish health. These results highlight the potential negative impacts of microplastics on fish health and the economics and safety of aquaculture.

Development of optimal in vitro release and permeation testing method for rectal suppositories

Currently there are no compendial assays for testing drug release from rectal suppositories. It is therefore essential to study different in vitro release testing (IVRT) and in vitro permeation testing (IVPT) methods for identifying a suitable technique to compare in vitro drug release and to predict in vivo performance of rectal suppositories. In the present study, three different rectal suppository formulations of mesalamine (CANASA, Generic, and In-house) were studied for in vitro bioequivalence. All the different suppository products were characterized by performing weight variation, content uniformity, hardness, melting time, and pH tests. Viscoelastic behavior of the suppositories was also tested both in presence and absence of mucin. Four different IVRT techniques such as Dialysis, Horizontal Ussing Chamber, Vertical Franz cell, and USP apparatus 4. IVPT studies were performed using Horizontal Ussing chamber and Vertical Franz cell methods. Q1/Q2 equivalent products (CANASA, Generic) and a half-strength product were studied to understand the reproducibility, bio relevance, and discriminatory ability of the IVRT and IVPT methods. This study is the first of its kind where molecular docking studies were performed to determine the potential interactions of drug (mesalamine) with mucin, IVRT studies were conducted with and without the presence of mucin, and porcine rectal mucosa was used to perform IVPT tests. The USP 4 method and Horizontal Ussing chamber methods were found to be suitable IVRT and IVPT techniques, respectfully, for rectal suppositories. RLD (Reference Listed Drug) and Generic rectal suppositories were found to exhibit similar release rate and permeation profiles obtained from USP 4, and the IVPT studies, respectfully. Wilcoxon Rank Sum/Mann-Whitney rank test, conducted for the IVRT profiles obtained using USP 4 method, proved the sameness of RLD and Generic suppository products.

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 skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Permeability of P and K-nutrient through polystyrene membrane from aqueous solutions of urea + KH2PO4

Abstract With the polymer-coated fertilizer as background, the permeability of P- and K-nutrient through a representative polymer membrane-polystyrene membrane were investigated by measuring their permeability in the solutions of KH2PO4-water and urea-KH2PO4-water at nominal temperature of 298 K using the Ussing chamber method. To analyze and interpret the variation of permeability with solute concentration, the solubility of permeate in polymer membrane were determined experimentally and the permeate diffusion coefficient were assessed by the measurements of density and apparent molar volume of the aqueous fertilizer solutions. An interesting “increase-decrease” trend for the permeability of both phosphorous (P)-nutrient, and potassium (K)-nutrient fertilizer with permeate concentration was observed, in which the increases in permeability at low concentrations of permeate could be attributed to the increase in solubility of KH2PO4 in polymer while the decreases in permeability at high concentrations was due to the decrease in diffusion coefficient of permeate in polymer membrane. Finally, the release kinetics of these nutrients from a PS-coated urea-KH2PO4 compound fertilizer granule was predicted using the Shaviv’s model along with the permeability data of P- and K-nutrient generated.

Cyclodextrin based nanosponge of norfloxacin: Intestinal permeation enhancement and improved antibacterial activity

Nanosponges are a novel class of hyperbranched cyclodextrin-based nanostructures that exhibits remarkable potential as a drug host system for the improvement in biopharmaceutical properties. This work aims the development of cyclodextrin-based nanosponge of norfloxacin to improve its physicochemical characteristics. β-cyclodextrin was used as base and diphenyl carbonate as crosslinker agent at different proportions to produce nanosponges that were evaluated by in vitro and in vivo techniques. The proportion cyclodextrin:crosslinker 1:2 M/M was chosen due to its higher encapsulation efficiency (80%), revealing an average diameter size of 40 nm with zeta potential of −19 mV. Norfloxacin-loaded nanosponges exhibited higher passage of norfloxacin in comparison to norfloxacin drug alone by Ussing chamber method. The nanosponge formulation also revealed a mucoadhesive property that could increase norfloxacin absorption thus improving its antibiotic activity in an in vivo sepsis model. Therefore, nanosponges may be suitable carrier of norfloxacin to maximize and facilitate oral absorption.

Transport of valine across the small intestinal epithelium in pigs fed different valine levels andBacillus subtilis

Mutants of Bacillus subtilis overproducing valine (B.subtilis VAL) could be an approach to supply pigs dietary valine (Val). In the study, 18 gilts were fed: (i) negative diet with a standardized ileal digestible (SID) Val:Lys of 0.63:1 (Neg); (ii) Neg added B.subtilis VAL (1.28×1011 cfu/kg as-fed) or; (iii) Neg added L-Val to a Val:Lys of 0.69:1. Using the Ussing chamber method, the study aimed to investigate whether (i) the diets affect intestinal transport of additions of 0, 5, 10 or 20mmol Val/L from the mucosal to the serosal side and (ii) the B.subtilis VAL contributes to a net transport of Val produced in situ. The results showed that the Isc (ΔIscVal ) and release of Val to the serosal side solution (Srel ; μmolcm-2 min-1 ) increased with Val addition (linear and quadratic, p<.0001) but was similar for 5, 10 or 20mmol Val/L and not affected by diet. No net transport of in situ produced Val by B.subtilis VAL was detected. In conclusion, feeding a Val-deficient diet with or without B.subtilis VAL or a Val sufficient diet did not affect the Val transport across intestinal epithelia. No in situ Val production by B.subtilis VAL was observed in the Ussing chambers.

Esophageal Epithelial Resistance and Lower Esophageal Sphincter Muscle Contraction Increase in a Chronic Diabetic Rabbit Model.

Esophageal motility disorders and possibly gastroesophageal reflux disease are common in patients with diabetes mellitus. We aimed to investigate both the electrophysiological characteristics of the esophageal epithelium and the contractility of the lower esophageal sphincter (LES) muscle in alloxane-induced diabetic rabbits. Electrophysiological properties were measured using an Ussing chamber method. An acid-pepsin model was employed with pH 1.7 or weakly acidic (pH 4) Ringer and/or pepsin. Smooth muscle strips of the LES were mounted in an isolated organ bath. Contractile responses to an electrical field stimulation and cumulative concentrations of acetylcholine were recorded. Contractility of the muscle strips were tested in the presence of Rho-kinase inhibitor (Y-27632) and nonspecific nitric oxide inhibitor (L-NAME). The resistance of diabetic tissue perfused in the pH 1.7 Ringer decreased 17%; pepsin addition decreased it by 49%. The same concentrations caused a more distinct loss of resistance in the control tissues (22 and 76%, p<0.05). The perfusion of tissues in increased concentrations of luminal and serosal glucose did not change the tissue resistance and voltage. Diabetes significantly increased both the electrical field stimulation and acetylcholine-induced contractions in the LES muscle strips (p<0.01). Incubation with Y-27632 significantly decreased the acetylcholine-induced contractions in a concentration-dependent manner (p<0.01). The acid-pepsin model in the diabetic rabbit esophageal tissue had less injury compared with the control. The diabetic rabbit LES muscle had higher contractility, possibly because of the activation of the Rho-Rhokinase pathway. Our results show that in a chronic diabetic rabbit model the esophagus resists reflux by activating mechanisms of mucosal defense and increasing the contractility of the LES.

Permeation of fertilizer nutrients through polymer membrane: part I. Effect of P, K, and micronutrient fertilizer on permeability of urea

AbstractThe study aimed at investigating the addition effects of P, K, and micronutrient fertilizers on the permeability of N‐nutrient fertilizer urea through a representative polymer membrane, which is of great importance in polymer‐coated compound fertilizer. Specifically, the permeability of urea through polystyrene membrane was determined experimentally at the nominal temperature of 298 K using the Ussing chamber method for fertilizer solutions of urea–water, urea–NaH2PO4–water, urea–Ca(H2PO4)2–water, urea–KCl–water, and urea–NaCl–water. The experimental data showed that the addition effects of NaH2PO4, KCl, and NaCl on urea permeability were pronounced and could be promotion or suppression depending on the amounts added. The variation of urea permeability with additive concentration could be explained by the ‘solution–diffusion’ model, suggesting that the specific intermolecular and interionic interactions in the permeation solution were an important factor determining the permeability. Using the saturated urea–water solution as basis, the addition of saturated KCl increased the urea permeability by 2.5 times, while the addition of saturated Ca(H2PO4)2 and NaH2PO4 decreased the permeability by 41% and 76%, respectively. © 2016 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Nutrient-intake-level-dependent regulation of intestinal development in newborn intrauterine growth-restricted piglets via glucagon-like peptide-2

The objective of the present study was to investigate the intestinal development of newborn intrauterine growth-restricted (IUGR) piglets subjected to normal nutrient intake (NNI) or restricted nutrient intake (RNI). Newborn normal birth weight (NBW) and IUGR piglets were allotted to NNI or RNI levels for 4 weeks from day 8 postnatal. IUGR piglets receiving NNI had similar growth performance compared with that of NBW piglets. Small intestine length and villous height were greater in IUGR piglets fed the NNI than that of piglets fed the RNI. Lactase activity was increased in piglets fed the NNI compared with piglets fed the RNI. Absorptive function, represented by active glucose transport by the Ussing chamber method and messenger RNA (mRNA) expressions of two main intestinal glucose transporters, Na+-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2), were greater in IUGR piglets fed the NNI compared with piglets fed the RNI regimen. The apoptotic process, characterized by caspase-3 activity (a sign of activated apoptotic cells) and mRNA expressions of p53 (pro-apoptotic), bcl-2-like protein 4 (Bax) (pro-apoptotic) and B-cell lymphoma-2 (Bcl-2) (anti-apoptotic), were improved in IUGR piglets fed the NNI regimen. To test the hypothesis that improvements in intestinal development of IUGR piglets fed NNI might be mediated through circulating glucagon-like peptide-2 (GLP-2), GLP-2 was injected subcutaneously to IUGR piglets fed the RNI from day 8 to day 15 postnatal. Although the intestinal development of IUGR piglets fed the RNI regimen was suppressed compared with those fed the NNI regimen, an exogenous injection of GLP-2 was able to bring intestinal development to similar levels as NNI-fed IUGR piglets. Collectively, our results demonstrate that IUGR neonates that have NNI levels could improve intestinal function via the regulation of GLP-2.

The study of barrier characteristics of Peyer’s patches epithelium in rats

The aim of the study was to investigate the barrier function of Peyer’s patches epithelium, which covers the clusters of organized lymphoid tissues in the small intestinal. We used the Ussing chamber method to determine electrophysiological characteristics of epithelium and Western blot method and immunohistochemistry to investigate expression of tight junction proteins. We revealed that Peyer’s patches epithelium, which is specialized in sampling and transporting of antigen structures, has lower conductance compare to adjacent intestinal villous epithelium, the main function of which is to uptake ions, water and nutrients. On molecular level Peyer’s patch epithelium has lower expression of claudin-2 and claudin-7, which increases the permeability of the intercellular space, and higher expression of claudin-5 and -8, which decreases the paracellular pathway. Immunohistochemistry confirmed localization of claudins in the tight junction complex. We suggest that the restriction of paracellular transport is a prerequisite for the antigen presentation through specialized M-cells in Peyer’s patches epithelium. Refs 29. Figs 4.

202 Omeprazole and Fluticasone Inhibit IL-13-Stimulated Eotaxin-3 Expression by Esophageal Epithelial Cells Through Different Mechanisms and With Additive Effects: Rationale for Combining PPIs With Topical Steroids for EoE Patients

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

201 Claudin-7 Dysregulation in Pediatric Eosinophilic Esophagitis: A Role for TGF-β in Esophageal Epithelial Barrier Dysfunction

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

203 A STAT6 Inhibitor (AS1517499) and a Tyrosine Kinase Inhibitor (Leflunomide) Block IL-13/STAT6 Signaling and Eotaxin-3 Expression in Esophageal Fibroblasts and Epithelial Cells: Promising Agents to Improve Inflammation and Prevent Fibrostenosis in EoE

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

200 Allergen Challenge Sensitizes TRPA1 in Vagal Sensory Neurons and Afferent C-Fibers in Guinea Pig Esophagus

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

Glucagon-like peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

Glucagon-like peptide-1 (GLP-1) acts at the G protein-coupled receptor, GLP-1R, to stimulate secretion of insulin and to inhibit secretion of glucagon and gastric acid. Involvement in mucosal secretory physiology has received negligible attention. We aimed to study involvement of GLP-1 in mucosal chloride secretion in the small intestine. Ussing chamber methods, in concert with transmural electrical field stimulation (EFS), were used to study actions on neurogenic chloride secretion. ELISA was used to study GLP-1R effects on neural release of acetylcholine (ACh). Intramural localization of GLP-1R was assessed with immunohistochemistry. Application of GLP-1 to serosal or mucosal sides of flat-sheet preparations in Ussing chambers did not change baseline short-circuit current (I(sc)), which served as a marker for chloride secretion. Transmural EFS evoked neurally mediated biphasic increases in I(sc) that had an initial spike-like rising phase followed by a sustained plateau-like phase. Blockade of the EFS-evoked responses by tetrodotoxin indicated that the responses were neurally mediated. Application of GLP-1 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-1 receptor antagonist exendin-(9-39) suppressed this action of GLP-1. The GLP-1 inhibitory action on EFS-evoked responses persisted in the presence of nicotinic or vasoactive intestinal peptide receptor antagonists but not in the presence of a muscarinic receptor antagonist. GLP-1 significantly reduced EFS-evoked ACh release. In the submucosal plexus, GLP-1R immunoreactivity (IR) was expressed by choline acetyltransferase-IR neurons, neuropeptide Y-IR neurons, somatostatin-IR neurons, and vasoactive intestinal peptide-IR neurons. Our results suggest that GLP-1R is expressed in guinea pig submucosal neurons and that its activation leads to a decrease in neurally evoked chloride secretion by suppressing release of ACh at neuroepithelial junctions in the enteric neural networks that control secretomotor functions.

Systemic influence of immunosuppressive drugs on small and large bowel transport and barrier function

Immunosuppressive drug (ISD)-associated gastrointestinal disorders are a relevant risk factor for graft loss or patient death. The pathomechanisms and the incidence of post-transplantation diarrhea remain to be fully understood. The aim of this study was to characterize the impact of cyclosporine A, tacrolimus (TAC), mycophenolate mofetil (MMF), enteric coated mycophenolic acid (EC-MPA), sirolimus, everolimus (EVE) and fingolimod (FTY 720) on small and large bowel transport and barrier function. Functions of the small bowel and distal colon of Wistar rats treated for 14 days with one of the drug were analyzed using Ussing chamber method. In detail, the glucose and sodium absorption, chloride secretion, and barrier function were compared. Bowel functions were investigated by inhibition or activation of the electrogenic epithelial transport, as well as by measuring transepithelial H(3) -lactulose flux. TAC altered glucose absorption; EVE glucose absorption, small bowel barrier function and chloride secretion; MMF small bowel barrier function; and EC-MPA glucose absorption and the small bowel barrier function. Drug effects were partially dose-dependent. In conclusion, different ISD, such as TAC, EVE, MMF, or EC-MPA lead to different and specific patterns of pathophysiologic changes of small and large bowel barrier and transport function.

Early recombinant human epidermal growth factor treatment recovers the irradiation-induced decrease of Na+ absorption prior to the definite histological mucositis

Recombinant human epidermal growth factor (rhEGF) has potential benefit for the mucositis induced by radiation therapy as a therapeutic setting. In this study, we aimed to investigate the effects of rhEGF treatment on the radiation-induced changes in epithelial transport function, before the occurrence of the definitive histological mucosal changes. C3H/He mice received 0, 4, or 8 Gy irradiation and/or EGF treatment (rhEGF 0, 1 and 5 mg/kg, i.p., 5 days). At day 7, we recorded short circuit current ( I sc) of the upper tracheal epithelium using the flow-type Ussing chamber method, with histological analysis. As a result, there was no evident pathological change in the epithelium from the irradiated and/or rhEGF treated mice at day 7. The initial level of I sc and amiloride-sensitive I sc (Δ I sc,Amil) were decreased after 8 Gy irradiation, reflecting suppression of basal Na + absorption. The decreased Δ I sc,Amil was recovered by rhEGF treatment. In conclusion, epithelial Na + channel-dependent basal Na + absorption was primarily affected by irradiation, before the pathological changes. The recovery of basal Na + absorption (Δ I sc,Amil) suggested a potentially beneficial effect of early rhEGF treatment for irradiation-induced suppression of the upper aerodigestive epithelial functions.

A guide to Ussing chamber studies of mouse intestine

The Ussing chamber provides a physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues. One of the most studied epithelia is the intestine, which has provided several landmark discoveries regarding the mechanisms of ion transport processes. Adaptation of this method to mouse intestine adds the dimension of investigating genetic loss or gain of function as a means to identify proteins or processes affecting transepithelial transport. In this review, the principles underlying the use of Ussing chambers are outlined including limitations and advantages of the technique. With an emphasis on mouse intestinal preparations, the review covers chamber design, commercial equipment sources, tissue preparation, step-by-step instruction for operation, troubleshooting, and examples of interpretation difficulties. Specialized uses of the Ussing chamber such as the pH stat technique to measure transepithelial bicarbonate secretion and isotopic flux methods to measure net secretion or absorption of substrates are discussed in detail, and examples are given for the adaptation of Ussing chamber principles to other measurement systems. The purpose of the review is to provide a practical guide for investigators who are new to the Ussing chamber method.