mini-Ussing Chamber Research Articles

Simultaneous Prediction Method for Intestinal Absorption and Metabolism Using the Mini-Ussing Chamber System.

Many evaluation tools for predicting human absorption are well-known for using cultured cell lines such as Caco-2, MDCK, and so on. Since the combinatorial chemistry and high throughput screening system, pharmacological assay, and pharmaceutical profiling assay are mainstays of drug development, PAMPA has been used to evaluate human drug absorption. In addition, cultured cell lines from iPS cells have been attracting attention because they morphologically resemble human intestinal tissues. In this review, we used human intestinal tissues to estimate human intestinal absorption and metabolism. The Ussing chamber uses human intestinal tissues to directly assay a drug candidate's permeability and determine the electrophysiological parameters such as potential differences (PD), short circuit current (Isc), and resistance (R). Thus, it is an attractive tool for elucidating human intestinal permeability and metabolism. We have presented a novel prediction method for intestinal absorption and metabolism by utilizing a mini-Ussing chamber using human intestinal tissues and animal intestinal tissues, based on the transport index (TI). The TI value was calculated by taking the change in drug concentrations on the apical side due to precipitation and the total amounts accumulated in the tissue (Tcorr) and transported to the basal side (Xcorr). The drug absorbability in rank order, as well as the fraction of dose absorbed (Fa) in humans, was predicted, and the intestinal metabolism of dogs and rats was also predicted, although it was not quantitative. However, the metabolites formation index (MFI) values, which are included in the TI values, can predict the evaluation of intestinal metabolism and absorption by using ketoconazole. Therefore, the mini-Ussing chamber, equipped with human and animal intestinal tissues, would be an ultimate method to predict intestinal absorption and metabolism simultaneously.

Decreased Expression of NRF2 Target Genes after Alcohol Exposure in the Background Esophageal Mucosa of Patients with Esophageal Squamous Cell Carcinoma.

Patients with esophageal squamous cell carcinoma (ESCC) might have a specific mechanism for the carcinogenesis by alcohol consumption in the background esophageal mucosa, and nuclear factor erythroid 2-related factor 2 (NRF2), which plays a protective role against esophageal carcinogenesis, and barrier dysfunction might be associated with this phenomenon. This study aimed to confirm this hypothesis. Twenty patients with superficial ESCCs (ESCC patients) and 20 age- and sex-matched patients without ESCC (non-ESCC patients) were enrolled. Biopsy samples were obtained from non-neoplastic esophageal mucosa: one for histological evaluation, one for quantitative real-time polymerase chain reaction (PCR), and two for the mini-Ussing chamber system to measure transepithelial electrical resistance (TEER) and, thereafter, for PCR. The TEER after acetaldehyde or both acetaldehyde and ethanol exposure did not differ significantly between ESCC and non-ESCC patients. Unlike non-ESCC patients, mRNA levels of NRF2 target genes and claudin4 in ESCC patients tended to decrease after the exposure, with a significant difference between no exposure and both acetaldehyde and ethanol exposure in NRF2 target genes (p < 0.05). Furthermore, in ESCC patients, the decreased tendency of mRNA levels of NRF2 target genes after the exposure was more pronounced in high-risk states, such as aldehyde dehydrogenase 2 (ALDH2) Lys alleles (Glu/Lys + Lys/Lys), Lugol-voiding lesion grade C, and drinking history. In conclusion, the protective role of NRF2 against carcinogenesis from alcohol exposure might be disrupted in the background esophageal mucosa of ESCC patients, which might lead to a high incidence of metachronous ESCC.

Correlation of Probe-Based Confocal Laser Endomicroscopy (pCLE) and Mucosal Integrity Testing (MIT) with Epithelial Barrier Function and Presence of Gastroesophageal Reflux Disease (GERD).

Epithelial barrier function (EBF) disruption is a key mechanism underlying gastroesophageal reflux disease (GERD). Our aim was to assess whether two novel technologies, probe-based confocal laser endomicroscopy (pCLE) and mucosal integrity testing (MIT), could assess EBF. We prospectively enrolled patients undergoing upper endoscopy for refractory GERD or non-GERD conditions. Patients underwent esophagogastroduodenoscopy, pCLE, MIT, esophageal biopsy at 2 cm and 6 cm above the esophagogastric junction, and wireless pH testing. To assess EBF in vitro, biopsies were mounted in a mini-Ussing chamber, 1 ml of fluorescein was instilled on the mucosal side, and concentration of fluorescein on the serosal side was measured at 3h. We enrolled 54 subjects (28 GERD, 26 non-GERD based on Lyon consensus criteria). In vivo permeability assessed by pCLE did not differ significantly between GERD vs. non-GERD patients and did not correlate with in vitro permeability. Mean MIT at 2cm was lower in GERD compared to non-GERD (1914 vs. 3727 ohms). MIT correlated inversely with in vitro permeability at 2cm and at 6cm. Using a predictive model that used slope and intercept of MIT at 2 cm and 6 cm, sensitivity and specificity of MIT at identifying GERD was 76% and 72%, respectively. pCLE did not differentiate GERD vs non-GERD and did not correlate with EBF measured in vitro. MIT, on the other hand, may be more promising as it differentiated GERD vs non-GERD and correlated with EBF measured in vitro.

Impaired Mucosal Integrity in Proximal Esophagus Is Involved in Development of Proton Pump Inhibitor-Refractory Nonerosive Reflux Disease

Background and Objective: Weakly acidic reflux reaching to the proximal esophagus is closely related to the perception of gastroesophageal reflux in patients with nonerosive reflux disease despite treatment with a proton pump inhibitor (PPI). However, little is known about the involvement of the patients’ mucosal integrity of the proximal esophagus. Methods: We recruited 15 symptomatic nonerosive gastroesophageal reflux disease (GERD) patients with a positive symptom index despite PPI treatment and 11 healthy asymptomatic volunteers as controls. The biopsy specimens obtained from the proximal and distal esophagus were applied to a mini-Ussing chamber system to measure transepithelial electrical resistance (TEER) against a pH 4 weak acid. The esophageal biopsy samples were subjected to quantitative real-time PCR and immunohistochemical analysis. Results: In the proximal esophagus, the weak acid exposure reduced the TEER in the PPI-refractory patients compared to that in the controls. The frequency of the reflux extending to the proximal esophagus had a significant correlation with the reduction in the proximal esophageal TEER in the patients. The reduced TEER in the proximal esophagus was accompanied by an increase in IL-8 and IL-1β mRNA and a decrease in occludin mRNA levels. The proximal esophageal mucosa in the patients presented infiltration of CD3-positive lymphocytes and an increased expression of solute carrier organic anion transporter family member 2A1 (SLCO2A1), a passage gate of reflux symptom-evoking molecules. Conclusions: The reflux perception is related to an impairment of the proximal esophageal mucosal integrity in patients with nonerosive reflux disease despite PPI.

Comparison of the Intestinal Drug Permeation and Accumulation Between Normal Human Intestinal Tissues and Human Intestinal Tissues With Ulcerative Colitis

The purpose of this study was to examine drug absorption profile utilizing human intestinal tissues from ulcerative colitis (UC) patients and to compare with normal tissues from intestinal cancer patients. Human intestinal tissues from UC and cancer patients mounted in a mini-Ussing chamber were used to evaluate the permeation of drugs, including FD-4, a very low permeable marker, rebamipide, a low permeable marker, and metoprolol, a high permeable marker. The transport index, an index of sum of permeated and tissue-accumulated molecules, of the model drugs was in accordance with their absorption rank order, and was almost kept constant irrespective of autopsy grade based on tissue fibrosis. On the other hand, UC tissues of grade 2 showed the decreased Xcorr, an index of permeated amount of molecules and increased Tcorr, an index of tissue-accumulated molecules for every tested compound.Our finding of the transport characteristics in intestinal tissues of severe UC patients in mini-Ussing chamber system demonstrated that autopsy grade of UC patients did not drastically change membrane permeability of the tested compounds. Furthermore, it was suggested that morphological changes of intestinal tissues caused by fibrosis led to limited permeation and subsequently increased accumulation with little change of total absorption.

Simultaneous Prediction of Intestinal Absorption and Metabolism Using the Mini-Ussing Chamber System

The purpose of this study was to investigate the possibility of simultaneous prediction of the intestinal absorption and metabolism in a mini-Ussing chamber equipped with rat intestinal tissues, based on the transport index (TI). TI value was defined as the sum of drug amounts, by mass balance method, transported to the basal-side component and drug amounts accumulated in the tissue, which are normalized by area under the curve of the drug in the apical compartment. Midazolam and nifedipine with high permeability were used as typical P450 substrates to examine the possibility of simultaneous prediction of intestinal absorption and metabolism. The metabolite formation of both compounds was observed and ketoconazole strongly inhibited the metabolite formation of both compounds in rat intestinal tissues, leading to the improvement of the TI value to a statistically significant extent for both compounds. TI ratio of nifedipine between in the presence and absence of ketoconazole was larger than that of midazolam, which was consistent with the reported lower value of fraction absorbed multiplied by intestinal availability of nifedipine. Therefore, the mini-Ussing chamber, equipped with animal intestinal tissues, showed potential to predict the intestinal absorption and metabolism simultaneously.

Acidic bile salts induces mucosal barrier dysfunction through let-7a reduction during gastric carcinogenesis after Helicobacter pylori eradication.

Gastric cancer (GC) after eradication for Helicobacter pylori (H.pylori) increases, but its carcinogenesis is not elucidated. It is mainly found in acid non-secretion areas (ANA), as mucosal regeneration in acid secretory areas (AA) after eradication changes the acidity and bile toxicity of gastric juice. We aimed to clarify the role of barrier dysfunction of ANA by the stimulation of pH3 bile acid cocktail (ABC) during carcinogenesis. We collected 18 patients after curative endoscopic resection for GC, identified later than 24 months after eradication, and took biopsies by Congo-red chromoendoscopy to distinguish AA and ANA (UMIN00018967). The mucosal barrier function was investigated using a mini-Ussing chamber system and molecular biological methods. The reduction in mucosal impedance in ANA after stimulation was significantly larger than that in AA, 79.6% vs. 87.9%, respectively. The decrease of zonula occludens-1 (ZO-1) and let-7a and the increase of snail in ANA were significant compared to those in AA. In an in vitro study, the restoration of ZO-1 and let-7a as well as the induction of snail were observed after stimulation. High mobility group A2 (HMGA2)-snail activation, MTT proliferation, and cellular infiltration capacity were significantly increased in AGS transfected with let-7a inhibitor, and vice versa. Accordingly, using a mini-Ussing chamber system for human biopsy specimens followed by an in vitro study, we demonstrated for the first time that the exposure of acidic bile salts to ANA might cause serious barrier dysfunction through the let-7a reduction, promoting epithelial-mesenchymal transition during inflammation-associated carcinogenesis even after eradication.

Evaluation of intestinal metabolism and absorption using the Ussing chamber system equipped with intestinal tissue from rats and dogs

The purpose of this study was to evaluate the intestinal metabolism and absorption in a mini-Ussing chamber equipped with animal intestinal tissues, based on the transport index (TI). TI value was defined as the sum of drug amounts transported to the basal-side component (Xcorr) and drug amounts accumulated in the tissue (Tcorr), which are normalized by AUC of a drug in the apical compartment, as an index for drug absorption. Midazolam was used as a test compound for the evaluation of intestinal metabolism and absorption. The metabolite formulation of midazolam was observed in both rats and dogs. Ketoconazole inhibited the intestinal metabolism of midazolam in rats and improved its intestinal absorption to a statistically significant extent. Therefore, the mini-Ussing chamber, equipped with animal intestinal tissues, showed potential to use the evaluation of the intestinal metabolism and absorption, including the assessment of species differences.

Prediction of drug intestinal absorption in human using the Ussing chamber system: A comparison of intestinal tissues from animals and humans

An adequate evaluation system for drug intestinal absorption is essential in the pharmaceutical industry. Previously, we established a novel prediction system of drug intestinal absorption in humans, using the mini-Ussing chamber equipped with human intestinal tissues. In this system, the TI value was defined as the sum of drug amounts transported to the basal-side component (Xcorr) and drug amounts accumulated in the tissue (Tcorr), which are normalized by AUC of a drug in the apical compartment, as an index for drug absorption. In order to apply this system to the screening assay, it is important to understand the differences between animal and human tissues in the intestinal absorption of drugs. In this study, the transport index (TI) values of three drugs, with different levels of membrane permeability, were determined to evaluate the rank order of drug absorbability in intestinal tissues from rats, dogs, and monkeys. The TI values in small intestinal tissues in rats and dogs showed a good correlation with those in humans. On the other hand, the correlation of TI values in monkeys was lower compared to rats and dogs. The rank order of the correlation coefficient between human and investigated animal tissues was as follows: dog (r2=0.978), rat (r2=0.955), and monkey (r2=0.620). TI values in large intestinal tissues from rats (r2=0.929) and dogs (r2=0.808) also showed a good correlation. The obtained TI values in small intestinal tissues in rats and dogs were well correlated with the fraction of drug absorbed (Fa) in humans. From these results, the mini-Ussing chamber, equipped with intestinal tissues in rats and dogs, would be useful as a screening tool in the drug discovery stage. In addition, the obtained TI values can be used for the prediction of the Fa in humans.

Tu1874 A Low Mucosal Integrity in a Subgroup of Healthy Subjects Increases With Proton Pump Inhibitor Treatment

Background: Esophageal epithelial integrity is considered an important factor in the prevention of tissue damage by gastric refluxate.We hypothesized that in patients with gastroesophageal reflux disease (GERD) the esophageal epithelial barrier function is impaired and less resistant in response to acid exposure. We therefore investigated esophageal epithelial integrity in GERD patients and in healthy controls both in the basal state and in response to acid exposure Methods: 14 patients with chronic GERD (8 with erosive esophagitis, 6 with non-erosive reflux disease) and 10 healthy controls (HC) were enrolled. Before endoscopy, GERD patients discontinued PPI therapy for 7 days. Six esophageal biopsies frommacroscopically normal mucosa were obtained approximately 5 cm above the gastroesophageal junction and directly transferred to a mini-Ussing chamber system. After an equilibration period, baseline TEER was assessed. Half of the biopsies were then exposed at their luminal side to an acidic solution (pH1) for 30 minutes. During exposure and after removal of the acidic solution, changes in TEER were analyzed relative to baseline TEER. Permeation to the paracellular permeation marker fluorescein (375 DA 1 mg/ml) was assessed in all biopsies (previous acid-exposed and non-exposed) for 120 minutes. Only subjects with at least two adequate biopsies (one for acid exposure, one as a control) were included. Results: Esophageal epithelium of GERD patients showed lower baseline TEER (127.7±13.3 Ω vs. 174.3±17.6 Ω, p=0.04) and a trend toward higher transmucosal permeation of fluorescein in the nonexposed biopsies when compared to healthy controls (serosal concentration (pmol/ml) after 120 min: 48.2 (7.6-66.7) vs. 6.8 (3.6-20.2), p=0.09 and AUC: 79.8 (12.9-135.6) vs. 9.0 (5.5-31.3), p=0.07). Acid exposure provoked a fall in TEER that was equal for the biopsies of GERD patients and healthy controls (-52.1±2.5% vs. -50.0±4.4% of baseline TEER). After removal of the acidic solution, TEER recovered also to a similar extent in GERD patients and healthy controls (89.6±3.8% vs. 93.8±3.0% of baseline TEER). However, maximum TEER was reached earlier in biopsies of GERD patients (54±9 min vs. 83±6 min (HC), p= 0.02) and at the end of the experiment, TEER relative to baseline was lower in biopsies of GERD patients (73.7±5.8% vs. 89.7±3.3% (HC) of baseline TEER, p<0.05). Conclusion: The esophageal epithelial barrier function of GERD patients is impaired, reflected by lower baseline transepithelial electrical resistance and a trend toward higher fluorescein permeation, and seems to be less resistant in response to acid exposure ex-vivo.

Tu1873 Esophageal Epithelial Barrier Function in GERD Patients and Healthy Controls: An Ex-Vivo Study in the Basal State and in Response to Acid Exposure

Background: Esophageal epithelial integrity is considered an important factor in the prevention of tissue damage by gastric refluxate.We hypothesized that in patients with gastroesophageal reflux disease (GERD) the esophageal epithelial barrier function is impaired and less resistant in response to acid exposure. We therefore investigated esophageal epithelial integrity in GERD patients and in healthy controls both in the basal state and in response to acid exposure Methods: 14 patients with chronic GERD (8 with erosive esophagitis, 6 with non-erosive reflux disease) and 10 healthy controls (HC) were enrolled. Before endoscopy, GERD patients discontinued PPI therapy for 7 days. Six esophageal biopsies frommacroscopically normal mucosa were obtained approximately 5 cm above the gastroesophageal junction and directly transferred to a mini-Ussing chamber system. After an equilibration period, baseline TEER was assessed. Half of the biopsies were then exposed at their luminal side to an acidic solution (pH1) for 30 minutes. During exposure and after removal of the acidic solution, changes in TEER were analyzed relative to baseline TEER. Permeation to the paracellular permeation marker fluorescein (375 DA 1 mg/ml) was assessed in all biopsies (previous acid-exposed and non-exposed) for 120 minutes. Only subjects with at least two adequate biopsies (one for acid exposure, one as a control) were included. Results: Esophageal epithelium of GERD patients showed lower baseline TEER (127.7±13.3 Ω vs. 174.3±17.6 Ω, p=0.04) and a trend toward higher transmucosal permeation of fluorescein in the nonexposed biopsies when compared to healthy controls (serosal concentration (pmol/ml) after 120 min: 48.2 (7.6-66.7) vs. 6.8 (3.6-20.2), p=0.09 and AUC: 79.8 (12.9-135.6) vs. 9.0 (5.5-31.3), p=0.07). Acid exposure provoked a fall in TEER that was equal for the biopsies of GERD patients and healthy controls (-52.1±2.5% vs. -50.0±4.4% of baseline TEER). After removal of the acidic solution, TEER recovered also to a similar extent in GERD patients and healthy controls (89.6±3.8% vs. 93.8±3.0% of baseline TEER). However, maximum TEER was reached earlier in biopsies of GERD patients (54±9 min vs. 83±6 min (HC), p= 0.02) and at the end of the experiment, TEER relative to baseline was lower in biopsies of GERD patients (73.7±5.8% vs. 89.7±3.3% (HC) of baseline TEER, p<0.05). Conclusion: The esophageal epithelial barrier function of GERD patients is impaired, reflected by lower baseline transepithelial electrical resistance and a trend toward higher fluorescein permeation, and seems to be less resistant in response to acid exposure ex-vivo.

Tu1876 Factors Associated With Non Response to Proton Pump Inhibitors in Patients Referred for Esophageal pH-Impedance Monitoring

Background: Esophageal epithelial integrity is considered an important factor in the prevention of tissue damage by gastric refluxate.We hypothesized that in patients with gastroesophageal reflux disease (GERD) the esophageal epithelial barrier function is impaired and less resistant in response to acid exposure. We therefore investigated esophageal epithelial integrity in GERD patients and in healthy controls both in the basal state and in response to acid exposure Methods: 14 patients with chronic GERD (8 with erosive esophagitis, 6 with non-erosive reflux disease) and 10 healthy controls (HC) were enrolled. Before endoscopy, GERD patients discontinued PPI therapy for 7 days. Six esophageal biopsies frommacroscopically normal mucosa were obtained approximately 5 cm above the gastroesophageal junction and directly transferred to a mini-Ussing chamber system. After an equilibration period, baseline TEER was assessed. Half of the biopsies were then exposed at their luminal side to an acidic solution (pH1) for 30 minutes. During exposure and after removal of the acidic solution, changes in TEER were analyzed relative to baseline TEER. Permeation to the paracellular permeation marker fluorescein (375 DA 1 mg/ml) was assessed in all biopsies (previous acid-exposed and non-exposed) for 120 minutes. Only subjects with at least two adequate biopsies (one for acid exposure, one as a control) were included. Results: Esophageal epithelium of GERD patients showed lower baseline TEER (127.7±13.3 Ω vs. 174.3±17.6 Ω, p=0.04) and a trend toward higher transmucosal permeation of fluorescein in the nonexposed biopsies when compared to healthy controls (serosal concentration (pmol/ml) after 120 min: 48.2 (7.6-66.7) vs. 6.8 (3.6-20.2), p=0.09 and AUC: 79.8 (12.9-135.6) vs. 9.0 (5.5-31.3), p=0.07). Acid exposure provoked a fall in TEER that was equal for the biopsies of GERD patients and healthy controls (-52.1±2.5% vs. -50.0±4.4% of baseline TEER). After removal of the acidic solution, TEER recovered also to a similar extent in GERD patients and healthy controls (89.6±3.8% vs. 93.8±3.0% of baseline TEER). However, maximum TEER was reached earlier in biopsies of GERD patients (54±9 min vs. 83±6 min (HC), p= 0.02) and at the end of the experiment, TEER relative to baseline was lower in biopsies of GERD patients (73.7±5.8% vs. 89.7±3.3% (HC) of baseline TEER, p<0.05). Conclusion: The esophageal epithelial barrier function of GERD patients is impaired, reflected by lower baseline transepithelial electrical resistance and a trend toward higher fluorescein permeation, and seems to be less resistant in response to acid exposure ex-vivo.

Native Small Airways Secrete Bicarbonate

Since the discovery of Cl(-) impermeability in cystic fibrosis (CF) and the cloning of the responsible channel, CF pathology has been widely attributed to a defect in epithelial Cl(-) transport. However, loss of bicarbonate (HCO3(-)) transport also plays a major, possibly more critical role in CF pathogenesis. Even though HCO3(-) transport is severely affected in the native pancreas, liver, and intestines in CF, we know very little about HCO3(-) secretion in small airways, the principle site of morbidity in CF. We used a novel, mini-Ussing chamber system to investigate the properties of HCO3(-) transport in native porcine small airways (∼ 1 mm φ). We assayed HCO3(-) transport across small airway epithelia as reflected by the transepithelial voltage, conductance, and equivalent short-circuit current with bilateral 25-mM HCO3(-) plus 125-mM NaGlu Ringer's solution in the presence of luminal amiloride (10 μM). Under these conditions, because no major transportable anions other than HCO3(-) were present, we took the equivalent short-circuit current to be a direct measure of active HCO3(-) secretion. Applying selective agonists and inhibitors, we show constitutive HCO3(-) secretion in small airways, which can be stimulated significantly by β-adrenergic- (cAMP) and purinergic (Ca(2+)) -mediated agonists, independently. These results indicate that two separate components for HCO3(-) secretion, likely via CFTR- and calcium-activated chloride channel-dependent processes, are physiologically regulated for likely roles in mucus clearance and antimicrobial innate defenses of small airways.

Establishment of Novel Prediction System of Intestinal Absorption in Humans Using Human Intestinal Tissues

The objective of this study was to establish a novel prediction system of drug absorption in humans by utilizing human intestinal tissues. Based on the transport index (TI), a newly defined parameter, calculated by taking account of the change in drug concentrations because of precipitation on the apical side and the amounts accumulated in the tissue and transported to the basal side, the absorbability of drugs in rank order as well as the fraction of dose absorbed (Fa) in humans were estimated. Human intestinal tissues taken from ulcerative colitis or Crohn's disease patients were mounted in a mini-Ussing chamber and transport studies were performed to evaluate the permeation of drugs, including FD-4, a very low permeable marker, atenolol, a low permeable marker, and metoprolol, a high permeable marker. Although apparent permeability coefficients calculated by the conventional equation did not reflect human Fa values for FD-4, atenolol, and metoprolol, TI values were well correlated with Fa values, which are described by 100 · [1 - e (- f · (TI - α)) ]. Based on this equation, Fa values in humans for other test drugs were predicted successfully, indicating that our new system utilizing human intestinal tissues would be valuable for predicting oral drug absorption in humans.

856 Treatment of Eosinophilic Esophagitis With the CRTH2-Antagonist Oc000459: A Novel Therapeutic Principle

bent position for 30 min with one of the following saline solutions at pH 7.2, 1.0 and 5.0. Thereafter, recording continued for 1h and baseline esophageal impedance was measured for the last 15 min. Next, an esophagoscopy was performed and biopsies were taken from the perfused area. Then, endoscopic biopsies were used to determine In Vitro the transepithelial resistance (TER) and passage of fluorescein (FITC) by using a mini-Ussing chamber system. Impedance baseline was evaluated at 5 cm above the LES in 30 GERD patients ‘off' PPI and in 10 healthy volunteers. Results: No macroscopic alterations were observed during endoscopy after perfusion with the different solutions. After esophageal perfusion with pH 7.2 solution, impedance baseline was 3012±619 Ω. Solutions at a pH 5.0 did not affect baseline values (3686±768 Ω), while acidic solution (pH 1.0) decreased values to 725±228 Ω (p<0.05). Similar results were found using the gold standard mini-Ussing chambers technique. TER and FITC passage after perfusion with pH 7.2 were 206.9±17.1 Ω/cm2 and 16.3±6.2 pmols. Solution at pH 5.0 did not affect mucosal integrity (TER: 213.4±26.4Ω/cm2 and FITC passage 17.24±4.3 pmols) while a pH 1.0 solution decreased TER to 124.2±23.3Ω/ cm2 (p<0.05) and increased passage of FITC to 53.6±10.7 pmols (p<0.05). A highly significant correlation was found between FITC passage and TER (r=-0.83, p=0.0002) and between impedance baseline and TER (r=0.84, p=0.0001). Impedance baseline was significantly reduced in the distal esophagus of GERD patients (2094± 724Ω vs. 2757± 848Ω, p=0.01). Conclusions: Impedance baseline is closely correlated to established In Vitro methodologies to measure mucosal integrity, thereby establishing impedance baseline measurement as a useful tool for the evaluation of mucosal integrity In Vivo. Mucosal integrity is impaired at the distal esophagus of GERD patients ‘off' PPI.

Utility of endoscopic biopsy samples to quantitate human duodenal ion transport

Duodenal mucosal bicarbonate secretion (DMBS) prevents acid-peptic damage and facilitates nutrient absorption. DMBS is diminished in patients with duodenal ulcers and is normalized after Helicobacter pylori eradication. The measurement of DMBS in human patients in vivo requires intubation with a multi-lumen balloon tube and permits limited testing with putative agonists and antagonists. Our purpose was to develop a means to investigate transport events in human duodenal biopsy samples in vitro. After validation studies in a modified mini-Ussing chamber were performed, duodenal transport events were examined in proximal endoscopic biopsy samples from normal volunteers (n = 17). Tissues were mounted in modified mini-Ussing chambers (volume 2.5 ml, surface area 3.8 mm 2). Short circuit current (I sc), potential difference (PD), and bicarbonate secretion were determined under basal conditions and after stimulation with graded doses of prostaglandin E 2 (PGE 2)(10 −8 to 10 −4 mol/L) and dibutyryl cAMP (db-cAMP)(10 −4 to 10 −2 mol/L). Duodenal tissues remained viable for at least 2 hours and exhibited stable basal HCO 3-secretion and electrical parameters. Stimulation with PGE 2 and db-cAMP resulted in dose-related increases in both I SC and HCO 3-secretion ( P < .05) that were abolished by ouabain and anoxia. It is concluded (1) that human duodenal bulb biopsy samples maintain their inherent transport function in mini-Ussing chambers and (2) that by using this novel method it will be possible to define the transport events that modulate human duodenal secretion, in particular bicarbonate secretion, in both health and disease.

Loperamide Inhibits the Enhanced Intestinal Glucose Absorption of Cystic Fibrosis In Vitro

Enhanced Na(+)-linked nutrient absorption has been demonstrated in the cystic fibrosis bowel and may contribute to the dehydration of the luminal contents. The ability of loperamide to inhibit glucose transport was therefore assessed in jejunal biopsy specimens from children with cystic fibrosis by measuring the increased short-circuit current associated with active glucose absorption using a mini-Ussing chamber technique. The presence of loperamide in the luminal solution reduced the rise in short-circuit current induced by glucose over the range of concentrations tested (2.5 to 40 mmol.L-1). This finding suggests that the enhanced Na+ absorption, which is a feature of cystic fibrosis, is amenable to treatment and can be restored to normal levels. This result may have therapeutic implications for gastrointestinal function and may be applicable to other tissues where similar therapeutic approaches to reduce Na+ and thus water absorption are being pursued.

In vitro bioelectric properties of bronchial epithelium from transplanted lungs in recipients with cystic fibrosis.

BACKGROUND--Bronchial epithelial function after heart-lung transplantation (HLT) for cystic fibrosis (CF) may be affected by the original disease as well as other factors such as prolonged organ ischaemic time, the interruption of bronchial arterial and lymphatic supply, infection, rejection, and cyclosporin. In vitro measurement of the bioelectric properties of the bronchial mucosal lining may be an effective means of characterising the mucosal function of the lung allografts in response to pharmacological agents. METHODS--Bronchial mucosal tissues from explanted native lungs of CF and non-CF patients at transplantation were used to assess the possible application of a mini-Ussing chamber. With this technique, the bioelectric responses of bronchial mucosal biopsies from six patients with CF, one patient with congenital heart disease, four with primary pulmonary hypertension, and one with emphysema, all after HLT, were studied. The bioelectric and pharmacological responses of biopsies of bronchial mucosa from patients after HLT were compared with biopsies from non-CF non-HLT subjects. RESULTS--The altered bioelectric properties of CF tissues could be detected by the mini-Ussing chamber technique. The basal bioelectric values and the responses to amiloride and isoprenaline in CF patients were not different from those in non-CF patients two years after HLT. No significant difference in the basal bioelectric properties and responses to amiloride and isoprenaline was found between HLT recipients and non-CF non-HLT subjects. CONCLUSIONS--The mini-Ussing chamber is an effective means of characterising the typical CF bioelectric defect which was not found in the transplanted lungs of CF patients up to two years after HLT. Furthermore, values were unaltered in comparison with non-transplanted lungs, suggesting that bronchial epithelial function is maintained after HLT.

Chloride and potassium conductances of cultured human sweat ducts.

The purpose of this study was to characterize the ion conductances, in particular those for Cl- and K+, of human sweat duct cells grown in primary culture. Sweat duct cells from healthy individuals were grown to confluence on a dialysis membrane, which was then mounted in a mini-Ussing chamber and transepithelial and intracellular potentials were measured under open-circuit conditions. Under control conditions the epithelia developed mucosa-negative transepithelial potentials, Vte, of about -10 mV. The apical membrane potential, Va, was -25 mV to -30 mV (n = 97) in most cells, but several cells had a higher potential of about -55 mV (n = 29). Mucosal amiloride (10 mumol/l) hyperpolarized Va from -31 +/- 1 mV to a new sustained level of -46 +/- 2 mV (n = 36). These changes were accompanied by increase in the fractional resistance of the apical membrane, fRa, and decreases of Vte and the equivalent short-circuit current, Isc. In amiloride-treated tissues an increase in mucosal K+ concentration (5 mmol/l to 25 mmol/l) depolarized Va by 5 +/- 1 mV (n = 8), while the same step on the serosal side depolarized Va by 20 +/- 2 mV (n = 8). A Cl- channel blocker 3',5-dichlorodiphenylamine-2-carboxylate DCl-DPC; 10 mumol/l) depolarized Va by 5 +/- 1 mV (n = 6), an effect that was lost after amiloride application. The blocker had no effect from the serosal side. Reduction of mucosal Cl- (from 120 to 30 or 10 mmol/l) depolarized Va by 9-11 mV (n = 35), an effect that was often followed by a secondary hyperpolarization of 10-30 mV (n = 27).(ABSTRACT TRUNCATED AT 250 WORDS)