Transepithelial Potential Difference Research Articles

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na(+) absorption, a defective cAMP-dependent Cl(-) conductance, higher concentrations of Na(+), Cl(-), K(+), and Ca(2+) in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca(2+) entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.

Intestinal Na+ Loss and Volume Depletion in JAK3-Deficient Mice

Background/Aims: The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na⁺ channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na⁺ exretion, blood pressure and extracellular fluid volume regulation. Methods: Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3^-/-) and in wild type mice (jak3^+/+). Colonic ENaC activity was estimated from amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na⁺ concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. Results: The amiloride (50 µM)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na⁺ excretion significantly higher in jak3^-/- mice than in jak3^+/+ mice. Moreover, systolic arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher in jak3^-/- mice than in jak3^+/+ mice. Both, absolute and fractional renal Na⁺ excretion were significantly lower in jak3^-/- mice than in jak3^+/+ mice. Conclusions: JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na⁺ loss, decrease of blood pressure, increased aldosterone release and subsequent stimulation of renal tubular Na⁺ reabsorption.

Kidney-specific WNK1 regulates sodium reabsorption and potassium secretion in mouse cortical collecting duct

Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is a kinase-deficient variant of WNK1 that is expressed exclusively in the kidney. It is abundantly expressed in the distal convoluted tubule (DCT) and to a lesser extent in the cortical thick ascending limb (cTAL), connecting tubule, and cortical collecting duct (CCD). KS-WNK1 inhibits Na(+)-K(+)-2Cl(-)- and sodium chloride cotransporter-mediated Na(+) reabsorption in cTAL and DCT, respectively. Here, we investigated the role of KS-WNK1 in regulating Na(+) and K(+) transport in CCD using in vitro microperfusion of tubules isolated from KS-WNK1 knockout mice and control wild-type littermates. Because baseline K(+) secretion and Na(+) reabsorption were negligible in mouse CCD, we studied tubules isolated from mice fed a high-K(+) diet for 2 wk. Compared with that in wild-type tubules, K(+) secretion was reduced in KS-WNK1 knockout CCD perfused at a low luminal fluid rate of ~1.5 nl/min. Na(+) reabsorption and the lumen-negative transepithelial potential difference were also lower in the KS-WNK1 knockout CCD compared with control CCD. Increasing the perfusion rate to ~5.5 nl/min stimulated K(+) secretion in the wild-type as well as knockout CCD. The magnitudes of flow-stimulated increase in K(+) secretion were similar in wild-type and knockout CCD. Maxi-K(+) channel inhibitor iberiotoxin had no effect on K(+) secretion when tubules were perfused at ~1.5 nl/min, but completely abrogated the flow-dependent increase in K(+) secretion at ~5.5 nl/min. These findings support the notion that KS-WNK1 stimulates ROMK-mediated K(+) secretion, but not flow-dependent K(+) secretion mediated by maxi-K(+) channels in CCD. In addition, KS-WNK1 plays a role in regulating Na(+) transport in the CCD.

Regional differences in rat conjunctival ion transport activities

Active ion transport and coupled osmotic water flow are essential to maintain ocular surface health. We investigated regional differences in the ion transport activities of the rat conjunctivas and compared these activities with those of cornea and lacrimal gland. The epithelial sodium channel (ENaC), sodium/glucose cotransporter 1 (Slc5a1), transmembrane protein 16 (Tmem16a, b, f, and g), cystic fibrosis transmembrane conductance regulator (Cftr), and mucin (Muc4, 5ac, and 5b) mRNA expression was characterized by RT-PCR. ENaC proteins were measured by Western blot. Prespecified regions (palpebral, fornical, and bulbar) of freshly isolated conjunctival tissues and cell cultures were studied electrophysiologically with Ussing chambers. The transepithelial electrical potential difference (PD) of the ocular surface was also measured in vivo. The effect of amiloride and UTP on the tear volume was evaluated in lacrimal gland excised rats. All selected genes were detected but with different expression patterns. We detected αENaC protein in all tissues, βENaC in palpebral and fornical conjunctiva, and γENaC in all tissues except lacrimal glands. Electrophysiological studies of conjunctival tissues and cell cultures identified functional ENaC, SLC5A1, CFTR, and TMEM16. Fornical conjunctiva exhibited the most active ion transport under basal conditions amongst conjunctival regions. PD measurements confirmed functional ENaC-mediated Na(+) transport on the ocular surface. Amiloride and UTP increased tear volume in lacrimal gland excised rats. This study demonstrated that the different regions of the conjunctiva exhibited a spectrum of ion transport activities. Understanding the specific functions of distinct regions of the conjunctiva may foster a better understanding of the physiology maintaining hydration of the ocular surface.

Suitability of Isolated Rat Jejunum Model for Demonstration of Complete Absorption in Humans for BCS-Based Biowaiver Request

The objective of this study was to evaluate the suitability and acceptance criteria of isolated rat jejunum model for Biopharmaceutics Classification System (BCS)-permeability classification. The evaluation followed recommended procedures including investigation of tissue integrity by applying damage triggers (nitrogen gassing, elevated temperature, azide addition), characterization of transporter functionality, and expression and development of correlation between rats' apparent permeability coefficient (P(app) ) versus humans' and versus oral fraction absorbed (f(a) ) in humans. Firstly, damage triggers caused a decrease in transepithelial resistance and potential difference and increase in lucifer yellow (LY) and fluorescein isothiocyanate (FITC)-dextran P(app) . However, only FITC-dextran P(app) 's increase was considered significant in all treatment groups, making FITC-dextran a better indicator of jejunum integrity than LY. Secondly, the expression level of selected intestinal transporters highly correlated between rat and human and functionality of P-glycoprotein was confirmed. P(app) of investigated drugs correlated with human f(a) (R(2) = 0.85, n = 20) and with human permeability coefficients (R(2) = 0.86, n = 13). Utility of described model for BCS classification was shown on levetiracetam, where significantly higher permeability of levetiracetam as compared with internal standard metoprolol was determined (t-test, p = 0.005). In conclusion, isolated rat jejunum model is a useful tool not only for mechanistic investigation, but also for permeability classification according to BCS and consequently for BCS-based biowaiver procedures.

Effects of diacetyl vapor exposure on human cultured airway epithelial cell ion transport

In August 2000, NIOSH investigated the development of bronchiolitis obliterans following inhalation of the butter flavoring, diacetyl, in employees at a popcorn manufacturing plant. Rats exposed to diacetyl vapor for 6 h developed diacetyl dose‐dependent epithelial damage of the upper airways 18 h after exposure. Nevertheless, diacetyl inhalation did not result in hyperreactivity to methacholine (MCh) both in vivo (inhaled MCh aerosol) and in vitro (isolated, perfused trachea), despite damage to the epithelium. In order to investigate the direct effects of diacetyl on airway epithelium, we examined ion transport of human air‐interface, cultured epithelial cells following diacetyl exposure. We hypothesized that altered epithelial ion transport may be among the earliest events associated with the onset of diacetyl toxicity. Using an exposure system designed for cultured cells, cells were exposed for 6 h to diacetyl vapors (100, 200, 300, 360 ppm). Eighteen hours after exposure, cells were placed into an Ussing chamber to measure transepithelial potential difference (Vt). The results demonstrated that, whereas inhalation of diacetyl had no effect in vivo or in vitro, all concentrations of diacetyl abolished Vt in cultured cells. Our results indicate that diacetyl causes substantial damage to airway epithelium in culture at exposure concentrations that do not alter reactivity of the airways in vivo or in vitro.

Fibroblast growth factor-23 abolishes 1,25-dihydroxyvitamin D3-enhanced duodenal calcium transport in male mice

Despite being widely recognized as the important bone-derived phosphaturic hormone, whether fibroblast growth factor (FGF)-23 modulated intestinal calcium absorption remained elusive. Since FGF-23 could reduce the circulating level of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], FGF-23 probably compromised the 1,25(OH)₂D₃-induced intestinal calcium absorption. FGF-23 may also exert an inhibitory action directly through FGF receptors (FGFR) in the intestinal cells. Herein, we demonstrated by Ussing chamber technique that male mice administered 1 μg/kg 1,25(OH)₂D₃ sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH)₂D₃-induced calcium absorption in the duodenal tissues taken from the 1,25(OH)₂D₃-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH)₂D₃ preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH)₂D₃-induced upregulation of TRPV5, TRPV6, and calbindin-D(9k), but not PMCA(1b) expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH)₂D₃-induced calcium absorption.

Effects of copper on osmoregulation in sheepshead minnow, Cyprinodon variegatus acclimated to different salinities

The sheepshead minnow, Cyprinodon variegatus is a euryhaline fish that inhabits estuaries and coastal marshes where it encounters a wide range of salinities. Many of these areas also have elevated levels of contaminants, creating the potential for toxic ions to interfere with the uptake of ions for osmoregulation. To determine whether the effect of copper on osmoregulatory activity is dependent on the osmotic conditions that individuals have been living at, fish were acclimated for 14 days to 2.5, 10.5 or 18.5ppt seawater and then exposed to a fixed free cupric ion level (14.6μM Cu2+) for 6h. Plasma Na, plasma Cl, wet/dry weight ratio, transepithelial potential difference (TEPD) and branchial Na+/K+-ATPase activity were determined before and after copper exposure. We also computed Na and Cl equilibrium potentials. Following the salinity acclimation (in fish not yet exposed to copper), fish from the low salinity group (2.5ppt) had lower TEPD, lower plasma Na levels and higher branchial Na+/K+-ATPase activity compared to the fish acclimated to higher salinities. No differences in plasma Cl and wet/dry weight ratio were detected. Copper exposure caused a significant decrease in plasma Na levels and Na+/K+-ATPase activity and an increase in wet/dry weight ratio, but these changes were limited to the 2.5ppt salinity group. No significant changes in plasma Cl were detected. Copper treatment resulted in a small decrease in TEPD for all except the lowest salinity acclimation group. A comparison of equilibrium potentials with TEPD showed evidence of active transport of both Na and Cl in 2.5ppt acclimated fish but not for the 10.5 or the 18.5ppt acclimated fish. Our results show that effects of copper on osmoregulation are dependent on the fish’ past salinity regime, and that these effects tend to be more pronounced for euryhaline fish that have been living under hyposmotic conditions.

An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ∼40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.

Requirement of Potassium for the Action of Anti-Diuretic Hormone (ADH) on Frog Skin

The aim of the present study is to study whether the presence of K(+) in bathing media is required for the action of ADH to the ionic transport across the skin in the frog species Rana hexadactyla. lonic transport was measured as transepithelial potential difference (TEPD) and short circuit current (SCC) by using an indigenously developed computer based voltage-clamp technique. Addition of ADH (40 nM) on the serosal side significantly increased the TEPD and SCC with Normal Ringer (NR) on both sides. ADH had no effect subsequent to amiloride (100 µM) pre-treatment, which confirmed the ADH-induced Na(+) transport. Chloride also has a significant role in the development of TEPD. To determine the role of K(+), Potassium-free Ringer (KFR) was placed on both sides; addition of ADH had no effect consequently. Further experiments were carried out to find out which side of K(+) was required for the action of ADH. There was a lack of ADH effect with apical NR and serosal KFR, demonstrating that serosal K(+) is essential to activate Na(+), K(+)- ATPase. Similarly, the ADH effect was lacking with apical KFR and serosal NR that was the novel finding of this study. Due to the concentration gradient, the K(+) was secreted from serosal side to apical side through barium (1 mM) blockable K(+) channel. This study provides evidence that serosal as well as apical K(+) are necessary for the action of ADH.

Feasibility of the Ussing chamber technique for the determination of in vitro jejunal permeability of passively absorbed compounds in different animal species

The aim of this study was to assess the feasibility of the Ussing chamber technique for the determination of the jejunal permeability of passively absorbed, high permeability model compounds (acetaminophen and ketoprofen) in different animal species. Additionally, electrophysiological measurements and histological examination of pre- and post-incubation tissue specimens were performed. Apparent permeability coefficients of turkey and dog jejunum were low and highly variable due to tissue fragility caused by differences in thickness of the remaining intestinal layers after stripping and resulting in severe damage. Pig and horse jejunum were markedly more suitable for permeability determinations and mild signs of deterioration were noticed after 120 min of incubation. Transepithelial electrical resistance and potential difference did not correlate well with the observed tissue damage. From these data, the Ussing chamber technique appears to allow for permeability measurements within a species, but seems unsuitable for interspecies permeability comparison. However, further validation of the method with low permeability compounds and actively transported compounds is needed.

Effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin and [D-Ala2,D-Leu5]enkephalin on ion and amino acid transport in rabbit ileum

The selective mu-opioid agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) (10 microM) reduced the short circuit (Isc) and the L-valine induced increase of the transepithelial potential difference and Isc(delta Vms and delta Isc) measured in-vitro in rabbit ileum, with a mechanism antagonized by naloxone (1 microM). [D-Ala2,D-Leu5]enkephalin (DADLE) (10 microM) had no significant effect on the transepithelial potential difference (Vms), Isc, delta Vms and delta Isc. In the ileum deprived of the serosa and muscolaris, DAGO reduced the delta Vms and delta Isc, but not the Vms and Isc, suggesting localization of the receptors responsible for this latter effect in the myenteric plexus and/or the muscularis mucosae. These preliminary results suggest that in the rabbit ileum opioids influence electrolyte and amino acid transport and these effects may be at least partly mediated by mu-receptors.

Ambroxol Inhibits Na+ Absorption by Canine Airway Epithelial Cells in Culture

To study the effect of ambroxol on ion transport functions of airway mucosa, we measured bioelectric properties of canine cultured tracheal epithelium under short-circuit conditions in-vitro. Addition of ambroxol to the submucosal but not to the mucosal solution in an Ussing chamber decreased short-circuit current, transepithelial potential difference and cell conductance. The ambroxol-induced decrease in short-circuit current was not affected by bumetanide or diphenylamine-2-carboxylate, but it was abolished by pretreatment of cells with amiloride. These results suggest that ambroxol may selectively inhibit Na+ absorption by airway epithelium, thereby increasing water composition in airway surface fluid and reducing mucus viscosity.

Salbutamol improves markers of epithelial function in mice with chronic allergic pulmonary inflammation

We investigated the effects of salbutamol on the markers of epithelial function in a murine model of chronic allergic pulmonary inflammation by recording the ciliary beat frequency (CBF) and the transepithelial potential difference (PD) in vivo. Mice were sensitized and received four challenges of ovalbumin (OVA group) or 0.9% saline (control group). Forty-eight hours after the 4th inhalation, we observed eosinophilia in the bronchoalveolar lavage and epithelium remodeling with stored acid mucus in the OVA group (P < 0.001). No difference in the baseline CBF was noticed between the groups; however, the OVA group had a significantly lower baseline PD (P = 0.013). Salbutamol increased the CBF in all groups studied, and the dose response curve to salbutamol increased the PD in the OVA group from 10(-4)M to 10(-2)M. We suggest that salbutamol affects the CBF and the depth of the periciliary layer, which, in great part, determines the ability of the cilia to propel the mucus layer. This effect may have a positive impact on airway mucociliary transport in asthma and may have clinical implications.

Glucocorticoid-induced Leucine Zipper 1 Stimulates the Epithelial Sodium Channel by Regulating Serum- and Glucocorticoid-induced Kinase 1 Stability and Subcellular Localization

Serum- and glucocorticoid-induced kinase 1 (SGK1) is a multifunctional protein kinase that markedly influences various cellular processes such as proliferation, apoptosis, glucose metabolism, and sodium (Na(+)) transport via the epithelial Na(+) channel, ENaC. SGK1 is a short-lived protein, which is predominantly targeted to the endoplasmic reticulum (ER) to undergo rapid proteasome-mediated degradation through the ER-associated degradation (ERAD) system. We show here that the aldosterone-induced chaperone, GILZ1 (glucocorticoid-induced leucine zipper protein-1) selectively decreases SGK1 localization to ER as well as its interaction with ER-associated E3 ubiquitin ligases, HRD1 and CHIP. GILZ1 inhibits SGK1 ubiquitinylation and subsequent proteasome-mediated degradation, thereby prolonging its half-life and increasing its steady-state expression. Furthermore, comparison of the effect of GILZ1 with that of proteasome inhibition (by MG-132) supports the idea that these effects of GILZ1 are secondary to physical interaction of GILZ1 with SGK1 and enhanced recruitment of SGK1 to targets within an "ENaC regulatory complex," thus making less SGK1 available to the ERAD machinery. Finally, effects of GILZ1 knockdown and overexpression strongly support the idea that these effects of GILZ1 are functionally important for ENaC regulation. These data provide new insight into how the manifold activities of SGK1 are selectively deployed and strengthened through modulation of its molecular interactions, subcellular localization, and stability.

An International Randomized Multicenter Comparison of Nasal Potential Difference Techniques

The transepithelial nasal potential difference (NPD) is used to assess cystic fibrosis transmembrane conductance regulator (CFTR) activity. Unreliability, excessive artifacts, and lack of standardization of current testing systems can compromise its use as a diagnostic test and outcome measure for clinical trials. To determine whether a nonperfusing (agar gel) nasal catheter for NPD measurement is more reliable and less susceptible to artifacts than a continuously perfusing nasal catheter, we performed a multicenter, randomized, crossover trial comparing a standardized NPD protocol using an agar nasal catheter with the same protocol using a continuously perfusing catheter. The data capture technique was identical in both protocols. A total of 26 normal adult subjects underwent NPD testing at six different centers. Artifact frequency was reduced by 75% (P < .001), and duration was less pronounced using the agar catheter. The measurement of sodium conductance was similar between the two catheter methods, but the agar catheter demonstrated significantly greater CFTR-dependent hyperpolarization, because Δ zero Cl- + isoproterenol measurements were significantly more hyperpolarized with the agar catheter (224.2 ± 12.9 mV with agar vs 18.2 ± 9.1 mV with perfusion, P < .05). The agar nasal catheter approach demonstrates superior reliability compared with the perfusion nasal catheter method for measurement of NPD. This nonperfusion catheter method should be considered for adoption as a standardized protocol to monitor CFTR activity in clinical trials.

Rat Parotid Gland Cell Differentiation in Three-Dimensional Culture

The use of polarized salivary gland cell monolayers has contributed to our understanding of salivary gland physiology. However, these cell models are not representative of glandular epithelium in vivo, and, therefore, are not ideal for investigating salivary epithelial functions. The current study has developed a three-dimensional (3D) cell culture model for rat Par-C10 parotid gland cells that forms differentiated acinar-like spheres on Matrigel. These 3D Par-C10 acinar-like spheres display characteristics similar to differentiated acini in salivary glands, including cell polarization, tight junction (TJ) formation required to maintain transepithelial potential difference, basolateral expression of aquaporin-3 and Na+/K+/2Cl- cotransporter-1, and responsiveness to the muscarinic receptor agonist carbachol that is decreased by the anion channel blocker diphenylamine-2-carboxylic acid or chloride replacement with gluconate. Incubation of the spheres in the hypertonic medium increased the expression level of the water channel aquaporin-5. Further, the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma induced alterations in TJ integrity in the acinar-like spheres without affecting individual cell viability, suggesting that cytokines may affect salivary gland function by altering TJ integrity. Thus, 3D Par-C10 acinar-like spheres represent a novel in vitro model to study physiological and pathophysiological functions of differentiated acini.

Hymenolepis diminuta: The effects of infection on transepithelial ion transport and tight junctions in rat intestines

In this study, we examine the effect of Hymenolepis diminuta on ion transport in the ileum and on tight junctions in the ileum and colon of rats. We also evaluate the effect of H. diminuta on C-fiber endings in the ileum, the direct habitat of H. diminuta, before and after mechanical stimulation and pharmacological modification by capsaicin (C-fiber irritant).Wistar rats were orally infected with five cysticercoids of H. diminuta. Using a modified Ussing chamber, electrophysiological parameters of the ileum were measured (transepithelial electrical potential difference and transepithelial electrical resistance) as well as the deposition of occludin (a tight junction protein) in the ileum and colon of the rats 8, 16, 25, 35, 40 and 60days post infection.We observed a significant reduction in transepithelial electrical potential difference in the ileum of rats infected with H. diminuta. In both the ileum and colon of rats infected with H. diminuta we also observed a decrease in occludin deposition, which indicates leakage of tight junctions, correlating with the decrease in transepithelial electrical resistance of these tissues. The application of capsaicin confirmed the hypothesis that H. diminuta in rats affects the C-fiber sensory receptors, causing changes in ion transport in the ileum.The results of the performed electrophysiological and immunohistochemical examinations indicate hymenolepidosis-related changes in the active transport of ions and the passive movement of ions.

P.3.c.039 Effects of neuroleptic treatment on oxidative stress parameters in schizophrenia patients

To clarify whether the epithelium of the human urinary tract possesses an active transepithelial sodium transport, the unidirectional sodium fluxes and the electrical parameters of the epithelium were determined on isolated pieces of renal pelvis. The epithelium was incubated in isotonic Ringer’s solution at 37.3C in Ussing-type chambers. The transepithelial potential difference was found to be 15.8 ± 3.0 mV and the transepithelial electrical resistance 760 ± 48Ω.-cm.−2. The transepithelial, unidirectional sodium fluxes were measured under short-circuit conditions. From both the unidirectional sodium influx from the luminal to the serosal side (7.01 ± 2.17) and from the efflux in the opposite direction (0.45 ± 0.07) a transepithelial sodium net flux of 6.56 ± 2.20 nmol. · minutes−1 · cm.−2 was calculated. Since there are no passive forces across the epithelium under short circuited conditions to explain the net flux, an active sodium transport has to be postulated. In all experiments, the simultaneously measured short circuit current (6.43 ± 2.18 nmol. · minutes−1·cm.−2) was not significantly different from the net sodium flux, and can therefore be taken as a measure of the net flux.

Lipopolysaccharide hyperpolarizes guinea pig airway epithelium by increasing the activities of the epithelial Na+ channel and the Na+-K+ pump

Earlier, we found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference (V(t)) of tracheal epithelium in the isolated, perfused trachea (IPT) of the guinea pig 18 h after injection. As well, LPS increased the hyperpolarization component of the response to basolateral methacholine, and potentiated the epithelium-derived relaxing factor-mediated relaxation responses to hyperosmolar solutions applied to the apical membrane. We hypothesized that LPS stimulates the transepithelial movement of Na(+) via the epithelial sodium channel (ENaC)/Na(+)-K(+) pump axis, leading to hyperpolarization of V(t). LPS increased the V(t)-depolarizing response to amiloride (10 μM), i.e., offset the effect of LPS, indicating that Na(+) transport activity was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the Na(+)-K(+) pump with basolateral amphotericin B (7.5 μM). LPS had no effect on the hyperpolarization response to apical trypsin (100 U/ml) in the Ussing chamber, indicating that channel-activating proteases are not involved in the LPS-induced activation of ENaC. To assess Na(+)-K(+) pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater V(t) in the presence of amphotericin B in tracheas from LPS-treated animals compared with controls revealed that LPS increased Na(+)-K(+) pump activity. This finding was confirmed in the Ussing chamber by inhibiting the Na(+)-K(+) pump via extracellular K(+) removal, loading the epithelium with Na(+), and observing a greater hyperpolarization response to K(+) restoration. Together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the Na(+)-K(+) pump.