Ussing Chamber System Research Articles

Low edge damage container insert that adjusts intestinal forceps biopsies into Ussing chamber systems.

Ussing chamber experiments with human intestinal tissue are impeded by the small size of forceps biopsy specimens. Therefore, a miniaturized container insert featuring low edge damage was designed with an exposure area of only 0.05 cm2. It allows measurement of short-circuit current (ISC) and transmural resistance (Rt) on endoscopically obtained biopsy specimens, as well as alternating current impedance analysis and conductance scanning. Comparison with larger specimens mounted in a conventional Ussing chamber without the insert (exposure area 0.54 cm2) was made using rat jejunum and rectum. No differences in ISC, Rt, or secretory response were found, indicating proper sealing and prevention of edge damage, as well as tissue viability in the container system. If biopsy samples obtained from human rectum were mounted in the insert, the local resistance near the edge was almost the same as the overall resistance (52.3 Omega.cm2). Epithelial and subepithelial resistances of human rectum were 43+/-1 Omega.cm2 and 10+/-1 Omega.cm2, respectively. In conclusion, we present a tool that allows reliable Ussing-type, impedance, and conductance scanning measurements to be made from intestinal biopsy specimens.

Increase in endotoxin-induced mucosal permeability is related to increased nitric oxide synthase activity using the Ussing chamber.

To determine if nitric oxide production is associated with increased intestinal permeability after endotoxin challenge using the ex vivo Ussing chamber. Ileal mucosal membranes harvested from normal rats weighing 300 to 420 g. Endotoxin (lipopolysaccharide), 1, 10, 100 microg/ mL, or saline was placed on the serosal side of ileal mucosal membranes mounted in Ussing chambers after 10(9) Escherichia coli C-25 had been placed on the mucosal side of the ileal membranes (n = 6-7/group). In a second set of experiments, ileal membranes were exposed to 100 microg/mL lipopolysaccharide with or without the addition of the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine at a concentration of 10 mM (n = 7-8/group). Bacterial translocation of E. coli C-25 from the mucosal to the serosal side of the ileal membrane was measured every hour during the 3-hr experimental period, as were serial measurements of the potential difference and resistance values of the ileal membranes. At the conclusion of the 3-hr period, the ileal membranes were harvested and levels of inducible nitric oxide synthase and constitutive nitric oxide synthase activity were measured. The incidence of E. coli C-25 passage across the ileal membranes mounted in the Ussing chambers was significantly increased in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide (71% and 86%, respectively) vs. the control membranes (0%) or the membranes exposed to 1 microg/mL of lipopolysaccharide (0%) (p < .05). This increase in E. coli C-25 passage in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide was associated with a decrease in ileal membrane resistance and an increase in inducible nitric oxide synthase activity (p < .05). The addition of N(G)-monomethyl-L-arginine protected against lipopolysaccharide-induced bacterial translocation and prevented the lipopolysaccharide-induced increase in ileal membrane inducible nitric oxide synthase activity. These results indicate that lipopolysaccharide induction of increased ileal inducible nitric oxide synthase activity is necessary for lipopolysaccharide-induced E. coli C-25 translocation to occur in normal ileal mucosal membranes tested in the Ussing chamber system.

Intestinal cytokine response after gut ischemia: role of gut barrier failure.

To investigate the effect of intestinal ischemia with and without a reperfusion injury on intestinal cytokine production and gut permeability. In humans and in animal models, the gut has been implicated as a cytokine-producing organ after ischemia/reperfusion (I/R)-type injuries. Because of the limitations of in vivo models, it has been difficult to demonstrate directly that the gut releases cytokines after an I/R injury or whether there is a relation between the magnitude of the ischemic process and the cytokine response. Ileal mucosal membranes from rats subjected to sham or 45 or 75 min of superior mesenteric occlusion (SMAO) or 45 minutes of SMAO and 30 minutes of reperfusion (SMAO 45/30) were mounted in the Ussing chamber system. Levels of tumor necrosis factor-alpha and interleukin-6 were serially measured in the mucosal and serosal reservoirs of the Ussing system, as was mucosal permeability as reflected by the passage of bacteria or phenol red across the ileal membrane. In a second group of experiments, Escherichia coli C25 was added to the mucosal reservoir to determine if the cytokine response would be increased. Mucosal and serosal levels of tumor necrosis factor-alpha were equally increased after SMAO, with the highest levels in the 75-minute SMAO group. The highest levels of interleukin-6 were found in rats subjected to 75 minutes of SMAO or SMAO 45/30; the serosal levels of interleukin-6 were four to sixfold higher than the mucosal levels. The addition of E. coli C25 resulted in a significant increase in the amount of interleukin-6 or tumor necrosis factor-alpha recovered from the mucosal reservoir. Increased ileal membrane permeability was observed only in rats subjected to 75 minutes of SMAO or SMAO 45/30. These results directly document that the levels of tumor necrosis factor-alpha and interleukin-6 released from the gut increase after an ischemic or I/R injury, such as SMAO, and that there is a relation between the magnitude of the gut ischemic or I/R insult and the cytokine response.

CAMP-independent chloride secretion activated by a vasoactive intestinal peptide in a monolayer culture of human bronchial epithelial cells.

To investigate the effects of vasoactive intestinal peptide (VIP) on Cl- transport across normal human bronchial epithelial (NHBE) cells grown in a monolayer, changes in short-circuit current (Isc) were measured in Ussing chamber systems. In the presence of 10(-4) M amiloride, the addition of VIP to the serosal solution led to an increase in the Isc in a concentration-dependent manner, the 50% effective concentration (EC50) being 2.6 x 10(-11) M. However, the addition of 10(-5) M forskolin had little effect on the increase in Isc. On the other hand, in the intracellular cAMP measurement, 10(-5) M forskolin remarkably increased the cAMP levels, but 10(-7) M VIP did not. This result suggests that Cl- secretion by VIP is not related to the raised intracellular cAMP levels in NHBE cells.

Effect of t-butylhydroperoxide on chloride secretion in rat tracheal epithelia.

Oxidative stress has been known to play important roles in various inflammatory diseases of lung such as allergic bronchitis, dust particle-induced inflammatory diseases, or chronic bronchitis. However, the effects of oxidants on Cl- secretion in tracheal epithelia have not been determined. To examine the effects of oxidants on Cl- secretion of the airway epithelia rat tracheal epithelial cells were cultured on porous filters and short circuit current (Isc) was measured in an Ussing chamber system. t-Butylhydroperoxide, which was widely used as a model substance to study the mechanism of cell injury resulted from oxidative stress, induced a transient increase in Isc by dose-dependent manner. The response was not observed in Cl(-)-free medium, and inhibited by 100 microM bumetanide. N(-Diphenyl-1,4-phenylene-diamine (DPPD, 5 microM), an inhibitor of lipid peroxidation, blocked the t-butylhydroperoxide response. When t-butylhydroperoxide was added after the administration of forskolin or H-89, a protein kinase A inhibitor, the t-butylhydroperoxide-induce Isc increase was abolished. Pretreatment of indomethacin (10 microM) completely inhibited the t-butylhydroperoxide response, but pretreatment of thapsigargin (1 microM) did not, t-Butylhydroperoxide induced gradual increases in cytosolic Ca2+ level, and increased [3H]arachidonic acid release in the presence of thapsigargin. These results indicate that t-butylhydroperoxide stimulates Cl-secretion via activation of phospholipase A2 and subsequent production of cyclooxygenase metabolities by Ca(2+)-dependent and -independent mechanisms.

Hypoxia/Reoxygenation-Induced Intestinal Mucosal Injury is Prevented by Pretreatment with a Calcium Blocker it the Ussing Chamber System

Hypoxia/Reoxygenation-Induced Intestinal Mucosal Injury is Prevented by Pretreatment with a Calcium Blocker it the Ussing Chamber System

In vitro assessment of gastric mucosal transfer of anti-Helicobacter therapeutic agents.

A novel animal model for studying antibiotic transfer across gastric mucosa was developed by using adult rats. Gastric corpus mucosa was mounted in an Ussing chamber system and bathed in oxygenated Krebs solution. Metronidazole flux from serosa to mucosa (J(S-->M)) was measured over 60 min under basal conditions and compared with mucosa-to-serosa flux (J(M-->S)). The effects of varying the chamber cross-sectional diameter and of stimulation by histamine and carbachol were assessed. Metronidazole J(M-->S) was measured with the mucosal pH at 2.2, 2.7, 3.2, and 7.4. Amoxicillin J(S-->M) under basal conditions was also measured and compared with metronidazole J(S-->M). Metronidazole J(S-->M) was proportional to serosal concentration (P < 0.001) under basal conditions, being 3.98 nmol x h(-1) x cm(-2) with a serosal concentration of 0.2 mmol/liter. Amoxicillin J(S-->M) was significantly lower under similar conditions at 0.50 nmol x h(-1) x cm(-2) (P < 0.01). Metronidazole J(S-->M) was not significantly different from J(M-->S), between chambers of different sizes, or following stimulation. When the mucosal pH was changed, J(M-->S) was proportional to the un-ionized concentration on the mucosal side (P < 0.001). Therefore, this model shows properties analogous to those of human gastric mucosa in vivo, with partitioning of metronidazole on the mucosal side according to pH, diffusion of metronidazole across the mucosa in both directions, and selectivity for different antibiotics, and it will be useful for the study of other therapeutic agents in the treatment of Helicobacter pylori.

Characterization of K+ channels in the basolateral membrane of rat tracheal epithelia.

To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 microg/ml) in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristate 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of ISK channel. RT-PCR analysis revealed the presence of ISK cDNA in the rat trachea epithelia. Although 0.1 mM Ba2+ only had minimal affect on short-circuit current (Isc) induced by cAMP in intact epithelia, 0.1 mM clofilium strongly inhibited it. These results indicate that ISK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.

Methodological and Functional Aspects of the Isolated Bovine Rumen Epithelium in Ussing Chamber Flux Studies

Abstract The properties of isolated sheets of bovine rumen epithelium in the Ussing chamber system were studied. The viability and time-dependent change in performance, as expressed by electrical parameters, was acceptable and comparable to what has been obtained with other epithelia in vitro. The viability and time-dependent change in performance was not influenced by transport time (5 to 45 minutes) between the slaughterhouse and the laboratory. Flux rates of 22Na+ and 14C-propionate showed that an equilibration period of 45 minutes after addition of tracers will be sufficient to obtain steady-state conditions with this epithelium. Compared with the time-dependent changes in electrical parameters, this shows that rumen epithelium cannot truly be used as its own control over time. The varying papillation caused surface enlargement factors between one and five, estimated by the dry weight of stripped epithelium. However, the dry weight was not correlated to epithelial capacitance or flux rates. It is conc...

Elemental diet and IV-TPN-induced bacterial translocation is associated with loss of intestinal mucosal barrier function against bacteria.

The goal of the current study was to directly assess the role of loss of mucosal barrier function in nutritionally induced bacterial translocation. Parenteral and certain elemental enteral diets have been shown to promote bacterial translocation. The mechanisms underlying this observation, especially the question of whether nutritionally induced bacterial translocation is primarily related to loss of intestinal barrier function, versus an impaired immune system, remain to be fully elucidated. Bacterial translocation was measured in vivo, ileal mucosal membranes were harvested, and their electrophysiologic properties and barrier function were measured ex vivo in the Ussing chamber system 7 days after receiving total parenteral nutrition solution parenterally (IV-TPN) or enterally (elemental diet). Chow-fed rats served as control subjects. The incidence of bacterial translocation was significantly increased both to the mesenteric lymph nodes in vivo and across the in vitro Ussing chamber-mounted ileal mucosal membranes of the elemental diet-fed and IV-TPN-fed rats. The magnitude of Escherichia coli and phenol red transmucosal passage in the Ussing chamber was significantly higher in the IV-TPN-fed rats than in the elemental diet-fed or chow-fed animals. The potential differences across the ileal membrane were similar between the three groups at all time points. However, the specific resistances of the ileal membranes of the IV-TPN and elemental diet groups were significantly less than the chow-fed animals, indicating increased membrane permeability. Loss of intestinal barrier function plays a major role in nutritionally induced bacterial translocation, and the loss of mucosal barrier function to both E. coli and phenol red appeared greater in the IV-TPN than the elemental diet-fed rats.

EFFECTS OF VARIOUS FACTORS ON PULMONARY ABSORPTION OF PEPTIDE DRUGS USING &lt;I&gt;IN VITRO&lt;/I&gt; EXPERIMENTAL SYSTEMS

We examined the in vitro penetration characteristics of FITC-dextrans (FDs) with various molecular weights and peptides such as calcitonin and insulin across human lung carcinoma A-549 cell monolayer. The permeability coefficients of FDs through the A-549 monolayers (type-II like cells) were much larger than those in the rat alveolar epithelial cell monolayers (RAEMs: type-I like cells), which were established by Kim and coworkers. The high permeability characteristics of A-549 monolayers may be attributed to their morphological differences as compared with the RAEMs.On the other hand, we studied the degradation characteristics of peptide drugs such as calcitonin and insulin using rat lung and A-549 cell homogenates. Calcitonin was rapidly disappeared than insulin in both homogenates. The apparent degradation clearance (Km/Vmax) values of these peptides were higher in the cytosol fraction than membrane fraction. In both homogenates, chymotrypsin activity was the highest among the six protease activities such as trypsin, elastase, chymotrypsin, Leu-aminopeptidase, aminopeptidase B and aminopeptidase N examined in this study. These results confirmed our previous report that the degradation of insulin in the rat lung homogenate was suppressed by the addition of soybean trypsin inhibitor.Furthermore, the penetration characteristics of water soluble compounds, such as phenol red and FDs across the Xenopus isolated pulmonary membrane were examined with the modified Ussing chamber system. The permeability coefficients of FDs with various molecular weights through the Xenopus isolated pulmonary membrane were similarto those through the small and large intestine. A good correlation was observed between the penetrated amounts of drugs across the Xenopus isolated pulmonary membrane with the in vitro Ussing chamber system and their absorbed amounts calculated by deconvolution method in an in situ rat intrapulmonary absorption experiment. Three types of absorption enhancers, such as laurylmaltoside, mixed micelle and Na-caprate, increased the penetration of phenol red across the Xenopus isolated pulmonary membrane, while EDTA and Na-salicylate did not affect its penetration across the Xenopus isolated pulmonary membrane. These results were in agreement with our previous report of an in situ rat intrapulmonary experiment.

Evaluation of insulin permeation across the rabbit nasal mucosa using the Ussing chamber system

Permeation of insulin across the nasal mucosa, a desirable absorption site for peptide and protein drugs to avoid their first-pass effect and gastrointestinal degradation, excised from rabbits and the effect of bile salts as permeation enhancers on it were evaluted by using Ussing chamber system. Insulin was degraded in this system, and the degradation in the mucosal side was slightly sooner than that in the serosal side. Every bile salt (0.5%) used in this study reduced transmucosal electrical resistance (Rm) and enhanced insulin permeation. The degradation of insulin in the mucosal side was inhibited by addition of a bile salt, sodium taurodihydrofusidate. These results suggest that the Ussing chamber system becomes a useful tool to evaluate the enzymatic barrir function of nasal mucosa.

A simple approach to measurement of electrical parameters of cultured epithelial monolayers: Use in assessing neutrophil-epithelial interactions

Typically, electrophysiologic studies of epithelial monolayers are either performed in formalized Ussing chamber systems which yield highly accurate results or in simple setups using recording devices which have limitations in accuracy or the range of measurements which can be performed or both. Here we detail a simple method of interfacing traditionally accurate Ussing chambers systems with commercially available filter supports on which epithelial monolayers can be grown. We also detail simple methods for growing inverted monolayers, suitable for electrophysiologic assays, for use in studies where cells or particles must be layered by gravity on the undersurface (basolateral pole) of the filter. Both approaches allow experiments to be performed on large numbers of monolayers synchronously. As an example of the use of this system, we analyze the sequelae of neutrophil migration across monolayers of the intestinal cell line T84. Neutrophil migration across monolayers can occur in either direction, is dependent on neutrophil surface β2 integrins, and is paralleled by a decrease in epithelial barrier function as detected electrically. We have previously shown in formal Ussing chamber studies that neutrophil-epithelial interactions elicit a modest short-circuit current indicative of electrogenic ion transport. We show here that this short-circuit current response can be readily detected using the simple approach described.

Estimating tissue permeability and other bioelectrical parameters using membrane voltage and short-circuit current.

Evaluating the acute toxic effects of drugs or toxins is based mainly on studies which require the use of light microscopy. Recently, the effects of such substances on biological membranes, such as the nasal membrane, has been studied using the traditional Ussing chambers, which make it possible to study the transepithelial flux of drugs across membranes and to measure some bioelectrical parameters. A model is described, with which the changes in the membrane permeability, for sodium, potassium and chlorine, can be calculated directly, based on values obtained from the Ussing chamber system. Also, an experiment is described for evaluating the toxic effects of the cholera toxin B subunit, by measuring these changes in isolated rabbit nasal mucosa.

Morphological effects of aspirin and prostaglandin on the canine gastric mucosal surface

Abstract An iodoplatinate technique that selectively stains phospholipid was used to evaluate morphologically the effects of aspirin and 16,16-dimethyl prostaglandin E 2 on canine gastric mucosa in an in vitro Ussing chamber system. The association between these morphological alterations and the changes in mucosal surface hydrophobicity as determined by contact angle analysis was also investigated. Prostaglandin (1 μg/ml) did not alter aspirin-induced cell injury, although it reduced (45%) the detachment of damaged cells from the lamina propria. Mucus release appeared to be stimulated independently both by aspirin and prostaglandin treatment and by the blotting and drying procedure routinely performed before contact angle analysis. When an extracellular mucus gel coat was present, it contained numerous iodoplatinate-reactive vesicles and myelinated or lamellated structures. Iodoplatinate-reactive substances also appeared at the luminal surface of the mucus gel as a filamentous band. This band of reactivity was frequently seen after simultaneous treatment with prostaglandin and aspirin but only sporadically observed after aspirin treatment alone. The hydrophobic nature of the canine gastric mucosa under the above experimental conditions correlated well (r = 0.743, p

Morphological effects of aspirin and prostaglandin on the canine gastric mucosal surface: Analysis with a phospholipid-selective cytochemical stain

An iodoplatinate technique that selectively stains phospholipid was used to evaluate morphologically the effects of aspirin and 16,16-dimethyl prostaglandin E2 on canine gastric mucosa in an in vitro Ussing chamber system. The association between these morphological alterations and the changes in mucosal surface hydrophobicity as determined by contact angle analysis was also investigated. Prostaglandin (1 μg/ml) did not alter aspirin-induced cell injury, although it reduced (45%) the detachment of damaged cells from the lamina propria. Mucus release appeared to be stimulated independently both by aspirin and prostaglandin treatment and by the blotting and drying procedure routinely performed before contact angle analysis. When an extracellular mucus gel coat was present, it contained numerous iodoplatinate-reactive vesicles and myelinated or lamellated structures. Iodoplatinate-reactive substances also appeared at the luminal surface of the mucus gel as a filamentous band. This band of reactivity was frequently seen after simultaneous treatment with prostaglandin and aspirin but only sporadically observed after aspirin treatment alone. The hydrophobic nature of the canine gastric mucosa under the above experimental conditions correlated well (r = 0.743, p < 0.005) with the percentage of mucosal surface covered by this filamentous band, and less so with the density of iodoplatinate-reactive structures within the mucus gel. We conclude that phospholipid structures in the mucus gel may account for the hydrophobic nature of the gastric mucosal surface and the ability of damaging and “cytoprotective” agents to influence both surface wettability and barrier integrity.

Human amnion as a model for IgG transport.

The ability of fresh human amnion to bind and internalize horseradish peroxidase-labeled IgG (IgG-HRP) was examined in an in vitro Ussing chamber system. The amnion demonstrated unique cell membrane receptors for the Fc portion of IgG molecules (Fc gamma R). The Fc gamma R exhibit exquisite specificity and affinity for IgG monomers as demonstrated by staining with labeled IgG. Labeled IgA, IgM, F(ab')2 fragments of IgG, aggregated IgG, and antigen-antibody complexes all failed to bind to the amnionic epithelial cells. Binding was only minimally affected by changes in ionic strength or pH when viewed at the light microscopic level. The Fc gamma R are located on both the apical and basal cell membranes. The binding of IgG-HRP to the amnion cell membrane was detectable within 1 min, and internalization of the ligand occurred within 5 min. No binding of IgG-HRP was observed following treatment of the membrane with 0.25% trypsin for 30 min at room temperature. Incubation of the amnion at 4 degrees C or in the presence of colchicine or cytochalasin D prevented internalization of the IgG-HRP. These experiments demonstrate Fc gamma R on human amnionic epithelial cells that both bind and internalize IgG, thus allowing the amnion to be used as a model system for studying IgG transport.