Increase In Isc Research Articles

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichia coli enterotoxin STa across the colon of the gerbil ( Gerbillu cheesmani) in different dietary states

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa, 30 ng mL −1) on the basal short-circuit current (Isc in μA cm −2) across stripped and unstripped sheets of proximal, mid and distal colon were investigated. Samples were taken from fed, starved (4 days, water ad lib) and undernourished (50% of control food intake for 21 days) gerbils ( Gerbillus cheesmani). The effect of neurotoxin tetrodotoxin (TTX 10 μM) and the effect of replacing chloride by gluconate or the effect of removing bicarbonate from bathing buffer on the maximum increase in Isc induced by E. coli heat stable enterotoxin STa were also investigated. The results showed that there is a segmental difference both in the basal and STa-induced Isc. Also, STa is more effective in the proximal than distal colon in the three feeding conditions. Undernourishment raised the STa-induced Isc in the three regions of the colon. In fed and starved gerbils part of STa-induced Isc in the proximal colon was chloride-dependent, while the other was bicarbonate-dependent; in the mid colon, the STa-induced Isc was bicarbonate-dependent only. In the three regions of the colon taken from undernourished gerbil, the STa-induced Isc was both chloride-and bicarbonate-dependent. The increase in STa-induced Isc as a results of undernourishment in proximal and mid colon was chloride-dependent, while in the distal colon, it was both chloride- and bicarbonate-dependent.

NSP4 PLAYS A MAJOR ROLE IN THE EARLY SECRETORY DIARRHEA INDUCED BY ROTAVIRUS IN A CACO-2 EXPERIMENTAL MODEL

Aim: Among Rotavirus' (RV) multiple mechanisms of diarrhea a major role is played by viral enterotoxin NSP4. RV infection induces an early anion secretion and subsequent cell damage in a Caco-2 cell model. Aim of the study was to address the role of NSP4 in a model of RV infection in human-derived intestinal cell line. Methods: Caco-2 cells were infected with RV strain SA-11 (5PFU/cell) and mounted in Ussing chambers at different times after infection. Production of NSP4 was assessed by western blotting in immunoprecipitates of supernatants and cell lysates. Short circuit current (Isc) and trans-epithelial resistance (TEER), reflecting transepithelial ion transport and tissue integrity were recorded. In parallel experiments, non-specific human immunoglobulins (Ig) preparations for parenteral use and specific anti-NSP4 Ig were added to enterocyte monolayers. Results: RV induced a biphasic response with a significant increase in Isc after 1-3 hours of infection, consistent with active anion secretion. TEER started to fall progressively 24 hours after infection, indicating monolayer loss of integrity. Starting from 1 hour after infection, NSP4 was detected in the supernatant of infected cells at increasing concentrations up to 48 hours post-infection. Addition of anti-NSP4 Ig, as well as no-specific human Ig, blocked the RV-induced secretion but not TEER modifications. Discussion: RV induces a dual, chronologically distinct, pathogenic effect in human enterocytes: 1) an early anion secretion which is NSP4-dependent and inhibited by specific Ig and by non-specific human Ig; 2) a subsequent cytopathic damage which is not prevented by anti-NSP4 Ig but blocked by human Ig.

A pore-forming toxin produced by Aeromonas sobria activates Ca 2+ dependent Cl − secretion

Bacteria produce many types of hemolysin that induce diarrhea by mechanisms that are not completely understood. Aeromonas sobria hemolysin (ASH) is a major virulence factor produced by A. sobria, a human pathogen that causes diarrhea. Since epithelial cells in the intestine are the primary targets of hemolysin, we investigated the effects of ASH on ion transport in human colonic epithelial (Caco-2) cells. ASH increased short-circuit currents (Isc) in a dose-dependent manner, and it also activated a 125I efflux from Caco-2 cells. ASH-induced Isc increases and 125I efflux activations were both suppressed by low Ca 2+ levels in the extracellular solution or by pretreatment with the Ca 2+ chlelator BAPTA-AM. Intracellular Ca 2+ levels were increased by ASH in a biphasic fashion characterized by a rapid sharp increase (peak 1) followed by a sustained low plateau (peak 2). ASH-induced peak 1 was inhibited by pretreatment with pertussis toxin, indicating that Ca 2+ was mobilized from intracellular stores, and peak 2 was induced by an influx of extracellular Ca 2+. Peak 2 but not peak 1 was related to Cl − secretion. These results indicate that ASH activates Ca 2+-dependent Cl − secretion.

Sodium and anion transport across the avian uterine (shell gland)epithelium

The uterine (shell gland) epithelium from the domestic chicken was mounted in Ussing chambers, bathed in symmetric avian saline solution on both apical and basolateral aspects and voltage clamped at 0 mV. The epithelium exhibited a basal short circuit current (I(sc)) that was partially inhibited by the epithelial Na(+) channel (ENaC) blockers, amiloride and benzamil (IC(50) values of 0.8 and 0.12 micromol l(-1), respectively). Inhibition of basal Na(+) absorption by 10 micromol l(-1) amiloride was confirmed by measurements of transepithelial Na(+) and Cl(-) fluxes, where inhibition of the apical-to-basolateral and net Na(+) flux occurred, but no significant effects on Cl(-) fluxes were detected. The amiloride-insensitive portion of the basal I(sc) was both Cl(-) and HCO(3)(-) dependent and was inhibited by the Cl(-) channel blocker, diphenyl-2-carboxylate (DPC; 100 micromol l(-1)). Stimulation with 8-(4-chlorophenylthio)-cyclic 3'-5', adenosine monophosphate (8-cpt cAMP) produced a sustained increase in I(sc) that was dependent on both Cl(-) and HCO(3)(-). The magnitude of the amiloride-sensitive I(sc) was approximately twofold greater in birds where shell formation was complete, but oviposition had not yet occurred. In addition, the amiloride-sensitive I(sc) was greater in hens over the age of 55 weeks and in molting birds. The anion-dependent component of the basal I(sc) was reduced in older birds, and electrogenic HCO(3)(-) transport was nearly absent in molting birds. These results demonstrated that electrogenic Na(+) transport in avian shell gland was similar to the mammalian uterine epithelium and increased with age and during molting. Electrogenic Cl(-) and HCO(3)(-) transport were coupled under basal and cAMP stimulated conditions and basal anion transport decreased with age and during molting.

Interferon-γ Down-regulates Adenosine 2b Receptor-mediated Signaling and Short Circuit Current in the Intestinal Epithelia by Inhibiting the Expression of Adenylate Cyclase

Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.

Electrical parameters and water permeability properties of monolayers formed by T84 cells cultured on permeable supports

T84 is an established cell line expressing an enterocyte phenotype whose permeability properties have been widely explored. Osmotic permeability (POSM), hydraulic permeability (PHYDR) and transport-associated net water fluxes (JW-transp), as well as short-circuit current (ISC), transepithelial resistance (RT), and potential difference (deltaVT) were measured in T84 monolayers with the following results: POSM 1.3 +/- 0.1 cm.s-1 x 10-3; PHYDR 0.27 +/- 0.02 cm.s-1; RT 2426 +/- 109 omega.cm2, and deltaVT 1.31 +/- 0.38 mV. The effect of 50 microM 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a "net Cl- secretory agent", on T84 cells was also studied. We confirm the reported important increase in ISC induced by DCEBIO which was associated here with a modest secretory deltaJW-transp. The present results were compared with those reported using the same experimental approach applied to established cell lines originating from intestinal and renal epithelial cells (Caco-2, LLC-PK1 and RCCD-1). No clear association between PHYDR and RT could be demonstrated and high PHYDR values were observed in an electrically tight epithelium, supporting the view that a "water leaky" barrier is not necessarily an "electrically leaky" one. Furthermore, the modest secretory deltaJW-transp was not consistent with previous results obtained with RCCD-1 cells stimulated with vasopressin (absorptive fluxes) or with T84 cells secreting water under the action of Escherichia coli heat stable enterotoxin. We conclude that, while the presence of aquaporins is necessary to dissipate an external osmotic gradient, coupling between water and ion transport cannot be explained by a simple and common underlying mechanism.

Apical trypsin increases ion transport and resistance by a phospholipase C-dependent rise of Ca2+

We investigated the mechanisms by which serine proteases alter lung fluid clearance in rat lungs and vectorial ion transport in airway and alveolar epithelial cells. Inhibition of endogenous protease activity by intratracheal instillation of soybean trypsin inhibitor (SBTI) or alpha(1)-antitrypsin decreased amiloride-sensitive lung fluid clearance across rat fluid-filled lungs; instillation of trypsin partially restored this effect. Gelatin zymography demonstrated SBTI-inhibitable trypsin-like activity in rat lung lavage fluid. Apical trypsin and human neutrophil elastase, but not agonists of protease activated receptors, increased Na(+) and Cl(-) short-circuit currents (I(sc)) and transepithelial resistance (R(TE)) across human bronchial and nasal epithelial cells and rat alveolar type II cells, mounted in Ussing chambers, for at least 2 h. The increase in I(sc) was fully reversed by amiloride and glibenclamide. The increase in R(TE) was not prevented by ouabain, suggesting that trypsin decreased paracellular conductance. Apical trypsin also induced a transient increase in intracellular Ca(2+) in human airway cells; treatment of these cells with BAPTA-AM mitigated the trypsin-induced increases of intracellular Ca(2+) and of I(sc) and R(TE). Increasing intracellular Ca(2+) in airway cells with either ionomycin or thapsigargin reproduced the increase in I(sc), whereas inhibitors of phospholipase C (PLC) prevented the increases in both Ca(2+) and I(sc). These data indicate trypsin-like proteases and elastase, either present in lung cells or released by inflammatory cells into the alveolar space, play an important role in the clearance of alveolar fluid by increasing ion transport and paracellular resistance via a PLC-initiated rise of intracellular Ca(2+).

Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Stimulation of colonic anion secretion by monochloramine: action sites

During inflammatory bowel disease, reactive oxygen metabolites are released by phagocytes reacting with intraluminal NH3 to produce monochloramine (NH2Cl). NH2Cl is assumed to play role in the pathogenesis of inflammation-associated diarrhoea, as it is able to induce intestinal secretion. The aim of the present study was to determine the action sites of NH2Cl in rat colonic epithelium with Ussing chamber and fura-2 experiments. In intact mucosa, NH2Cl (5.10(-6)-10(-4) mol.l(-1)) evoked a concentration-dependent increase in short-circuit current (Isc), consistent with the induction of anion secretion, as demonstrated by anion substitution and transport blocker experiments. When the apical membrane was permeabilised by the ionophore nystatin, two basolateral action sites of NH2Cl (5.10(-5) mol.l(-1)) could be identified, i.e. an increase in the K+ conductance and a stimulation of the Na+-K+ pump. When tissues were basolaterally depolarised by a high K+ concentration, the stimulation of an apical Cl- conductance by NH2Cl was observed. In isolated colonic crypts loaded with the Ca2+-sensitive fluorescent dye fura-2, NH2Cl (5.10(-5) mol.l(-1)) evoked an increase in the intracellular Ca2+ concentration. This increase was independent from the presence of Ca2+ in the extracellular medium, but was inhibited by blockade of intracellular sarcoplasmatic, endoplasmatic Ca2+-ATPases with cyclopiazonic acid (10(-5) mol.l(-1)). The NH2Cl-evoked Ca2+ release was sensitive against inhibition of ryanodine receptors with ruthenium red (5.10(-5) mol.l(-1)) and against inhibition of inositol-1,4,5-trisphosphate (IP3) receptors with 2-aminoethoxydiphenylborate (10(-4) mol.l(-1)). Both blockers also inhibited the NH2Cl-induced increase in Isc. These results indicate that an intracellular Ca2+ release via ryanodine and/or IP3 receptors is involved in oxidant stimulation of anion secretion in rat colon.

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.

Na+/Ca2+ exchange regulates Ca2+-dependent duodenal mucosal ion transport and HCO3− secretion in mice

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca(2+), which regulates ion transport, including HCO(3)(-) secretion. However, the underlying Ca(2+) handling mechanisms are poorly understood. The aim of the present study was to determine whether Na(+)/Ca(2+) exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO(3)(-) secretion by controlling Ca(2+) homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (I(sc)), and HCO(3)(-) secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca(2+) in duodenocytes was measured by fura 2. Carbachol (100 muM) increased I(sc) in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO(3)(-) secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 muM) or LiCl (30 mM) significantly reduced the initial peak in I(sc) by 51 or 47%, respectively, and abolished the plateau phase of I(sc) without affecting HCO(3)(-) secretion induced by carbachol. Ryanodine (100 muM), caffeine (10 mM), and nifedipine (10 muM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10-30 muM) significantly inhibited carbachol-induced increases in duodenal mucosal I(sc) and HCO(3)(-) secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca(2+) imaging experiments where Na(+) depletion elicited Ca(2+) entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion that results from stimulation of muscarinic receptors.

ClC-2 chloride secretion mediates prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum

Ischemia results in the breakdown of the intestinal barrier, predisposing patients to sepsis and multiple organ failure. Prostaglandins play a critical role in mediating recovery of barrier function in ischemia-injured intestine through a mechanism involving stimulation of Cl - secretion. In the present study, we investigated the contributory role of individual Cl - channels in the recovery of barrier function in ischemia-injured porcine ileum. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short-circuit current (Isc) and transepithelial resistance (TER) were measured in response to prostaglandin E 2 (PGE 2 ) and pharmacologic inhibitors of epithelial Cl - channels. Immunoassays were used to assess the expression and localization of ion channels. Application of PGE 2 to ischemia-injured ileal mucosa stimulated increases in Isc, an indicator of Cl - secretion, that was followed by marked increases in TER, an indicator of barrier function recovery. In vitro studies revealed that although PGE 2 induced Cl - secretion via at least 3 distinct secretory pathways, recovery of barrier function was initiated by Cl - secretion via ClC-2 Cl - channels co-expressed with occludin and localized to tight junctions within restituting epithelium. Intravenous administration of furosemide to pigs subjected to 1 hour of ileal ischemia impaired recovery of barrier function, as evidenced by decreased TER and increased mucosal-to-serosal 3 H-mannitol flux after a 2-hour reperfusion/recovery period, confirming an important role for Cl - secretory pathways in vivo. ClC-2-mediated intestinal Cl - secretion restores TER in ischemia-injured intestine. These data may provide the basis for targeted pharmacologic therapy for diseases associated with impaired barrier function.

Weaning Induces Both Transient and Long-Lasting Modifications of Absorptive, Secretory, and Barrier Properties of Piglet Intestine

This study investigated intestinal physiology of piglets at weaning. Piglets (n = 60) weaned at 21 d were food deprived for 2 d and then tube-fed using 2 different diets (a conventional diet vs. a wheat-enriched diet). They were slaughtered at d 0, 2, 5, 8, or 15 postweaning. Jejunum, ileum, and colon were mounted in Ussing chambers. In addition, segments of the proximal jejunum of 4 growing pigs were studied 35 d after weaning. Secretory function was assessed by basal short-circuit current (Isc) and secretagogue-stimulated Isc. Glucose absorption was measured by the increase in Isc after the addition of glucose. Epithelial barrier function was measured by transmucosal resistance (R) and horseradish peroxidase (HRP) fluxes across the epithelium. There were no significant differences between the pigs fed the 2 diets for any of the parameters studied. As already reported, a transient villous atrophy was observed. At the same time, we observed an increased basal Isc in jejunum and colon, increased glucose absorption and a dramatic drop of R in jejunum. These parameters had returned to preweaning values by d 5. Weaning was also followed by long-lasting modifications. In jejunum, responses to the secretagogues and glucose absorption were decreased at wk 2 after weaning and were not different between d 15 and 35. Ileal transmucosal resistance increased on d 5 and was stable thereafter. HRP flux in jejunum declined on d 2 and stayed at this low level throughout the experiment. We conclude that weaning induces transient dramatic changes in intestinal physiology but is also a period of maturation of the intestine.

Role of anions in nitric oxide-induced short-circuit current increase in isolated porcine ciliary processes.

To investigate how nitric oxide (NO) modulates short-circuit current (Isc) in isolated porcine ciliary processes. Isc changes (Ussing-type chamber) induced either by the NO donors SNP or SIN-1, or by the cGMP analogue 8-pCPT-cGMP were assessed. The effect of inhibitors of guanylate cyclase (10 microM ODQ, 100 microM LY83583), protein kinase G (30 microM Rp-8-pCPT-cGMP, 3 microM KT 5823), protein kinase A (1 microM KT 5720), or protein kinase C (1 microM Go6983) on SNP- or 8-pCPT-cGMP-induced Isc changes were investigated. The effect of inhibitors of anion channel (100 microM niflumic acid, 1 mM DIDS, and 1 mM 9-AC), K+-channel (10 mM TEA, 10 mM BaCl2), Na+-channel blockers (1 mM amiloride), Na+-K+-2Cl- cotransporter inhibitor (0.5 mM bumetanide), or carbonic anhydrase inhibitor (1 mM acetazolamide) was studied. In Cl(-)- or HCO3(-)-free Krebs-Ringer solution, the effect of SNP- or 8-pCPT-cGMP-induced Isc changes was accessed. SNP, SIN-1, or 8-pCPT-cGMP increased Isc with a change in the potential difference that became more negative toward the nonpigmented epithelium (aqueous) side. The Isc increase induced by SNP or SIN-1, but not by 8-pCPT-cGMP, was prevented by ODQ and LY83583. SNP- and 8-pCPT-cGMP-induced Isc increases were prevented by Rp-8-pCPT-cGMP or KT5823 (but not by KT5720 or Go6983), or by niflumic acid, DIDS, 9-AC, or acetazolamide (but not by TEA, BaCl2, amiloride, or bumetanide). The effect of SNP and 8-pCPT-cGMP was abolished in Cl(-)- and reduced in HCO3(-)-free solutions. NO activates a guanylate cyclase-cGMP-protein kinase G pathway that appears to stimulate stroma-to-aqueous anionic transport, possibly Cl-, in porcine ciliary epithelium.

PGE2 stimulates Cl- secretion in murine M-1 cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is thought to be an important modulator of renal ion and water transport, but its effects remain complex and incompletely understood. Here we examined the effects of PGE2 on transepithelial ion transport of M-1 mouse cortical collecting duct cells using short-circuit current (ISC) measurements. Basolateral addition of PGE2 (1 microM) produced a transient peak increase in ISC of 6.3+/-0.8 microA cm(-2) (n=11), followed by a sustained plateau. The PGE2-evoked response was preserved in the presence of 100 micro M apical amiloride with an average peak increase of 10.6+/-1.0 microA cm(-2) (n=23). However, it was greatly diminished in both the presence of apical diphenylamine-2-carboxylic acid (DPC, 1 mM) and the absence of extracellular Cl-, indicating that Cl- secretion had been stimulated. Basolateral PGE2 induced a concentration dependent response, with an EC50 of about 8 nM. Apical addition of PGE2 elicited an ISC response similar to that observed with basolateral PGE2. Furthermore, apical exposure to arachidonic acid (AA) produced a similar increase in ISC, which could be prevented by the cyclooxygenase inhibitor indomethacin, while AA failed to exert an additional effect in the presence of PGE2. Using RT-PCR, we confirmed the expression of the PGE2 (EP) receptor subtypes EP1, EP3 and EP4 but not of EP2 in cultured M-1 CCD cells. We conclude that M-1 cells express functional cyclooxygenase activity and can generate PGE2 which acts in an autocrine manner, causing Cl- secretion.

Electrophysiological Properties of the Airway: Epithelium in the Murine, Ovalbumin Model of Allergic Airway Disease

The electrophysiological properties of cultured tracheal cells (CTCs) were examined in a murine (C57BL/6J), ovalbumin (OVA)-induced model of allergic airway disease (AAD) at early (3-day OVA-aerosol) and peak (10-day OVA-aerosol) periods of inflammation. Transepithelial potential difference, short-circuit current (Isc), and resistance (RT) were lower in CTCs from 10-day OVA-aerosol animals compared to CTCs from naïve mice. In cells cultured for 5 weeks, RT was greater in naive CTCs than in 10-day OVA-aerosol CTCs at all times (P < 0.01). The Isc response to mucosal amiloride (10(-4) mol/L) was increased in CTCs from 10-day OVA-aerosol mice compared to naïve mice (6.0 +/- 0.37 microA/cm2 versus 1.8 +/- 0.56 microA/cm2; P < 0.001) with intermediate values for CTCs from 3-day OVA-aerosol mice. The cAMP-induced increase in Isc was blunted in 10-day OVA-aerosol animals compared to CTCs from naïve mice (9 +/- 12% versus 39 +/- 7%; P < 0.01) with intermediate values for CTCs from 3-day OVA-aerosol mice. There was no difference in mannitol flux in naïve compared to 10-day OVA-aerosol CTCs. Similar results were found using intact tracheas mounted in a perfusion chamber. These data demonstrate changes in airway epithelial cell function in the OVA-induced model of AAD that may contribute to the pathogenesis of airway inflammation.

A regulated apical Na(+) conductance in dexamethasone-treated H441 airway epithelial cells.

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na(+) channel alpha- and beta-subunits and increased transepithelial ion transport (measured as short-circuit current, I(sc)) from <4 microA.cm(-2) to 10-20 microA.cm(-2). This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil >or= amiloride > EIPA and can thus be attributed to active Na(+) absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na(+) pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na(+) conductance (G(Na)) from a negligible value to 100-200 microS.cm(-2). Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na(+) and Li(+) and that the permeability to these cations was approximately fourfold greater than to K(+). There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an approximately 60% increase in I(sc), and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in G(Na). This cAMP-dependent control over G(Na) was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na(+) transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.

Effects of TAK-637 on NK 1 receptor-mediated mechanisms regulating colonic secretion

This study investigates the effect of a selective NK 1 receptor antagonist TAK-637 on enteric mechanisms involved in regulation of epithelial secretion in the colon. Mucosal sheets isolated from guinea-pig colon were placed in modified Ussing chambers and the net active transport of electrolytes was measured as short-circuit current (Isc). GR-73632, a selective NK 1 receptor agonist, induced an increase in basal Isc, which was inhibited by TAK-637 (IC 50 of 21 nM). The increase in Isc induced by GR-73632 was significantly attenuated by tetrodotoxin (TTX, 1 μM), indicating that TAK-637 inhibits neuronal NK 1 receptors. Moreover, TAK-637 reduced the TTX-resistant component of the response to GR-73632 suggesting that NK 1 receptors expressed by epithelial cells are inhibited by TAK-637. In separate experiments, TAK-637 partially inhibited the submaximal Isc induced by electrical field stimulation (EFS, 0.5 ms, 15 Hz) of enteric nerves or by activation of primary afferent fibers using capsaicin (50 μM). TAK-637 had no significant effect on the basal Isc or on responses induced by neurokinin A (NKA), senktide, or forskolin. The results imply that inhibition of peripheral NK 1 receptors may reduce autonomic epithelial secretion in response to activation of autonomic secretomotor pathways, while having no significant effect on basal epithelial transport.

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichia coli heat stable enterotoxin across small intestine of the gerbil (Gerbillus cheesmani).

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa 30 ng ml(-1)) on the basal short-circuit current (Isc in microA cm(-2)) across stripped and unstripped sheets of jejuna and ilea taken from fed, starved (4 days, water ad lib) and undernourished (50% control food intake for 21 days) gerbil (Gerbillus cheesmani) were investigated. The effect of neurotoxin tetrodotoxin (TTX 10 microM) and the effects of replacing chloride by gluconate or the effects of removing bicarbonate from bathing buffers on the maximum increase in Isc induced by STa were also investigated. The maximum increase in Isc which resulted from the addition of STa were significantly higher in jejuna and ilea taken from starved and undernourished gerbils when compared with the fed control both using stripped and unstripped sheets. In the two regions of the small intestine taken from fed and starved animals TTX reduced the maximum increase in Isc induced by STa across unstripped sheets only. Moreover in jejuna and ilea taken from undernourished gerbils TTX reduced significantly the maximum increase in Isc induced by STa across stripped and unstripped sheets. Replacing chloride by gluconate decreased the maximum increase in Isc induced by STa across jejuna and ilea taken from undernourished gerbils only. Removing bicarbonates from bathing buffer decreased the maximum increase in Isc across the jejuna and ilea taken from starved and undernourished gerbils.

Evidence for epidermal growth factor receptor as negative-feedback control in aldosterone-induced Na+ reabsorption.

Aldosterone enhances Na(+) reabsorption via epithelial Na(+) channels (ENaC). Aldosterone also stimulates the protein kinase ERK1/2- and the epidermal growth factor (EGF) receptor (EGFR)-signaling pathway. Yet EGF and ERK1/2 are known inhibitors of ENaC-mediated Na(+) reabsorption. In the present study, using the well-established Madin-Darby canine kidney C7 cell line, we tested the hypothesis that EGFR represents a negative-feedback control for chronic aldosterone-induced Na(+) reabsorption [amiloride-inhibitable short-circuit current (I(sc))]. Mineralocorticoid receptor expression was confirmed by RT-PCR and Western blot analysis. Aldosterone enhanced ERK1/2 phosphorylation in an EGFR-dependent way. Furthermore, aldosterone stimulated EGFR expression. Aldosterone (10 nmol/l) induced a small transient increase in I(sc) under control conditions. Inhibition of ERK1/2 phosphorylation with U-0126 (10 micromol/l) stimulated I(sc), indicating constitutive ENaC inhibition. Aldosterone exerted a significantly larger effect in the presence of U-0126 than without U-0126. EGF (10 microg/l) inhibited I(sc), whereas inhibition of EGFR kinase by tyrphostin AG-1478 (100 nmol/l) enhanced I(sc). Aldosterone was more effective in the presence of AG-1478 than without AG-1478. In summary, we propose that the EGFR-signaling cascade can serve as a negative-feedback control to limit the effect of aldosterone-induced Na(+) reabsorption.