Changes In Short Circuit Current Research Articles

198 Bitter Stimuli Induce ERK Activation in Mouse and Human Cell Lines

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS) to release secretagogues. We have previously demonstrated that extracellular calcium-sensing receptor (CaSR) agonists can abrogate toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Based on our observation that CaSR is expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, we hypothesized that CaSR agonists may also function through neuronal pathways. To examine this possibility, rat colon segments containing intact ENS were isolated, mounted on Ussing chambers and perfused. Basal and cyclic nucleotide-stimulated anion secretions were monitored by measuring changes in short circuit current (Isc). CaSR was activated by R-568 either before or after cyclic nucleotide stimulation to mimic diarrhea prevention or treatment, respectively, and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of Isc in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. Activation of CaSR with R-568 significantly reduced basal and forskolin-induced Isc. A similar inhibitor effect of R-568 on Isc was achieved following the forskolin stimulation, suggesting that this agonist might be useful both for diarrhea prevention and treatment. Interestingly, when colon tissues were pretreated with TTX to inhibit the ENS activity, the inhibitory effects of R-568 were either significantly attenuated or abolished, indicating that R-568 exerts its antisecretory effect on the intestine primarily by inhibiting the ENS. The antisecretory effect of R-568 appears to be largely mediated by the submucosal Meissner's plexus because removal of the muscular layers and the myenteric Auerbach's plexus from the colon did not significantly reduce the inhibitory effect of R568. Our results suggest a new model in which the neuronal component of intestinal fluid transport can be modulated by the inhibitory effects of CaSR on the ENS. The ability of CaSR agonist to reduce secretagogue-stimulated anion secretion suggests that modulation of CaSR activity may provide a new therapy for secretory diarrheas and other ENS-mediated conditions.

Epidermal growth factor chronically upregulates Ca2+‐dependent Cl− conductance and TMEM16A expression in intestinal epithelial cells

Dysregulated epithelial fluid and electrolyte transport is a common feature of many intestinal disorders. However, molecular mechanisms that regulate epithelial transport processes are still poorly understood, thereby limiting development of new therapeutics. Previously, we showed that epidermal growth factor (EGF) chronically enhances intestinal epithelial secretory function. Here, we investigated a potential role for altered expression or activity of apical Cl(−) channels in mediating the effects of EGF. Cl(−) secretion across monolayers of T(84) colonic epithelia was measured as changes in short-circuit current. Protein expression/phosphorylation was measured by RT-PCR and Western blotting. Under conditions that specifically isolate apical Ca(2+)-activated Cl(−) channel (CaCC) currents, EGF pretreatment (100 ng ml(−1) for 15 min) potentiated carbachol (CCh)-induced responses to 173 ± 25% of those in control cells, when measured 24 h later (n = 26; P < 0.01). EGF-induced increases in CaCC currents were abolished by the transmembrane protein 16A (TMEM16A) inhibitor, T16A(inh)-A01 (10 μm). Furthermore, TMEM16A mRNA and protein expression was increased by EGF to 256 ± 38% (n = 7; P < 0.01) and 297 ± 46% (n = 9, P < 0.001) of control levels, respectively. In contrast, EGF did not alter CFTR expression or activity. EGF-induced increases in Cl(−) secretion, CaCC currents and TMEM16A expression were attenuated by a PKCδ inhibitor, rottlerin (20 μm), and a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042 (25 μm). Finally, LY290042 inhibited EGF-induced phosphorylation of PKCδ. We conclude that EGF chronically upregulates Ca(2+)-dependent Cl(−) conductances and TMEM16A expression in intestinal epithelia by a mechanism involving sequential activation of PI3K and PKCδ. Therapeutic targeting of EGF receptor-dependent signalling pathways may provide new approaches for treatment of epithelial transport disorders.

TMEM16A and NHERF1 Regulate Ca2+ and cAMP Stimulated Cl‐ Secretion in Murine Colon

TMEM16A is a calcium activated chloride channel (CaCC). However, its role in colonic Cl secretion is unknown. By qPCR, we identified TMEM16A as the predominant CaCC in mouse colon and hypothesized that it was involved in basal and Ca2+‐ stimulated colonic Cl secretion. Because the NHERF family of PDZ domain containing proteins has been shown to regulate intestinal ion transporters, we tested whether NHERF1 and NHERF2 are involved in TMEM16A regulation. Colonic Cl secretion was measured as the change in short circuit current (ΔIsc) by Ussing chamber. Digallic acid, a CaCC inhibitor inhibited basal Isc by 50% (p&lt;0.05) and completely abolished carbachol (CCH) stimulated Isc in the wild type (WT) mouse colon. In TMEM16A+/− colon, basal, CCH and forskolin (FSK) stimulated Isc were reduced by 47% (p&lt;0.04), 67% (p&lt;0.001) and 75% (p&lt;0.001), respectively. In NHERF1−/− mice, basal and CCH stimulated Isc were reduced by 60% (P&lt;0.01) and 92% (P&lt;0.001), while there was no significant change in the FSK stimulated Isc. Basal, CCH and FSK stimulated Isc in NHERF2−/− colonic tissues were not different from WT. Our results suggest that in mouse colon (1) TMEM16A is a major contributor of basal, CCH and unexpectedly also cAMP stimulated Cl secretion; (2) NHERF1, but not NHERF2 plays an important role in basal and CCH stimulated Cl secretion. In conclusion, NHERF1 affects colonic Cl secretion via effects on TMEM16A.

Allergic sensitization enhances anion current responsiveness of murine trachea to PAR-2 activation

Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor possibly involved in the pathogenesis of asthma. PAR-2 also modulates ion transport in cultured epithelial cells, but these effects in native airways are controversial. The influence of allergic inflammation on PAR-2-induced changes in ion transport has received little attention. Here, we studied immediate changes in transepithelial short circuit current (I (sc)) induced by PAR-2 activation in the tracheas of naive and allergic mice. Activation of PAR-2 with an apically added activation peptide (AP) induced a small increase in I (sc), while a much larger increase was observed following basolateral AP addition. In ovalbumin-sensitized and -challenged animals used as a model of allergic airway inflammation, the effect of basolateral AP addition was enhanced. Responses to basolateral AP in both naive and allergic mice were not decreased by blocking sodium absorption with amiloride or CFTR function with CFTR(inh)172 but were reduced by the cyclooxygenase inhibitor indomethacin and largely blocked (>80%) by niflumic acid, a calcium-activated chloride channels' (CaCC) blocker. Allergic mice also showed an enhanced response to ATP and thapsigargin. There was no change in mRNA expression of Par-2 or of the chloride channels Ano1 (Tmem16a) and Bestrophin 2 in tracheas from allergic mice, while mRNA levels of Bestrophin 1 were increased. In conclusion, basolateral PAR-2 activation in the mouse airways led to increased anion secretion through apical CaCC, which was more pronounced in allergic animals. This could be a protective mechanism aimed at clearing allergens and defending against mucus plugging.

Quercetin Increases Cystic Fibrosis Transmembrane Conductance Regulator–Mediated Chloride Transport and Ciliary Beat Frequency: Therapeutic Implications for Chronic Rhinosinusitis

Increasing epithelial chloride (Cl(-)) secretion in the upper airways represents a putative method for promoting mucociliary clearance through augmentation of airway surface liquid depth. Several naturally occurring flavonoid compounds, including quercetin, have shown the capacity to increase transepithelial Cl(-) transport. Quercetin exhibits well-known antioxidant and anti-inflammatory activity and is now recognized as a potent activator of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel activity in a fashion largely independent of cyclic adenosine monophosphate signaling. The present study investigates whether this compound activates Cl(-) secretion and ciliary beat frequency (CBF) in well-characterized culture models of sinonasal epithelium. Cystic fibrosis and non-cystic fibrosis primary human sinonasal epithelial (HSNE) and murine nasal septal epithelial (MNSE) cultures were studied for transepithelial ion transport in Ussing chambers under voltage clamp conditions and CBF was performed using pharmacologic manipulation. Change in short circuit current (DeltaI(SC), expressed as microamperes per squared centimeter) in response to quercetin were significantly greater than controls in both MNSE (23.23 ± 5.44 versus 2.47 ± 1.62; p < 0.0001) and HSNE (-8.72 ± 1.88 versus -1.88 ± 0.66; p < 0.01) cultures. CBF was significantly increased in quercetin-treated cells (expressed as fold change over baseline) in wild type (1.65 ± 0.13 versus 1.23 ± 0.05 [control]; p < 0.01), but not CFTR(-/-) (1.65 ± 0.29 versus 1.48 ± 0.38; p = 0.23). Quercetin significantly increased transepithelial Cl(-) transport and CBF in MNSE and HSNE cultures. Future studies investigating quercetin as a means to promote mucociliary transport in individuals with rhinosinusitis are warranted.

The role of rectal chloride secretion in childhood constipation

Disturbance in fluid secretion, driven by chloride secretion, might play a role in constipation. However, disturbed chloride secretion in those patients has yet to be evaluated. Therefore, the aim of this study was to compare chloride secretion in rectal biopsies of children with functional constipation (FC) to those without constipation. To measure changes in short circuit current (I(sc) in μA cm(-2)) reflecting chloride secretion, intestinal biopsies from children with constipation, to either exclude or diagnose Hirschsprung's disease, and from children without constipation (controls) undergoing colonoscopy for screening of familial adenomatous polyposis, juvenile polyps or inflammatory bowel disease (IBD), were compared and studied in Ussing chambers. Following electrogenic sodium absorption blockade by amiloride, chloride secretory responses to calcium-linked (histamine, carbachol) and cAMP-linked (IBMX/forskolin) secretagogues were assessed. Ninety-six patients (46 FC) participated; nine FC patients (n = 1 congenital syndrome and n = 8 technical problems) and 13 controls (n = 6 IBD; n = 7 technical problems) were excluded. No significant difference was found in mean (±SE) basal chloride currents between children with FC and controls (9.6 ± 1.1 vs 9.2 ± 0.8; P = 0.75, respectively). Responses to calcium-linked chloride secretagogues (histamine and carbachol) were significantly higher in controls (33.0 ± 3.0 vs 24.5 ± 2.3; P = 0.03 and 33.6 ± 3.4 vs 26.4 ± 2.7; P = 0.05 following histamine and carbachol, respectively). Calcium-linked chloride secretion is disturbed in children with FC. Whether this defect occurs at the level of histamine receptors, components of receptor-linked signal transduction pathways or basolateral Ca(2+) -sensitive K(+) channels enhancing the electrical driving force for apical chloride secretion, remains to be explored.

Myosin 5 is involved in cAMP‐induced ENaC trafficking in a mCCD cell line

The molecular motor which traffics epithelial sodium channels (ENaC) to and from the apical membrane is still unknown. We hypothesized that myosins, specifically Myosin 5 (Myo5), is involved in ENaC exocytosis. To determine the role of Myo5 in ENaC trafficking, we knocked down Myo5a and Myo5c in mCCD cells using shRNAs. We then measured the short-circuit current (Isc) and membrane capacitance change (ΔCT) of cells in Ussing chambers before and following cAMP activation. The Isc and CT change following cAMP-induced trafficking in knockdowns was significantly smaller than mCCD controls. Cells with Myo5c knocked down had a ΔIsc of 2.9 μA ± 1.03 μA compared to controls ΔIsc of 4.7 μA ± 0.68μA. There was a significant reduction in ΔCT (21% for controls vs. 14% in Myo5c) following cAMP stimulation. No significant change in Isc or CT response was observed for Myo5a knockdowns. There was no significant difference in basal (unstimulated) Isc between controls and Myo5c knockdowns. These results suggest that Myo5c is specifically involved in cAMP-induced ENaC trafficking to the apical membrane in response to cAMP stimulation and not constitutive ENaC protein turnover.

Evaluation of the secretory response to endogenous and exogenous prostaglandins in mucosa from the proximal and distal portions of the colon of dogs

To compare secretory responses to prostaglandin (PG) E(2) in mucosa obtained from the proximal and distal portions of the colon of dogs. Colonic mucosa from cadavers of 18 clinically normal adult dogs. Short-circuit current (I(SC)) and maximum change in I(SC) (ΔI(sc)) in response to administration of 1μM PGE(2) were measured across mucosa obtained from the proximal and distal portions of the colon. Responses were evaluated in mucosa (n = 6 dogs) incubated in Ussing chambers with or without 1 mM amiloride or without chloride in the Ringer's bathing solution. Responses were also evaluated in mucosa (n = 9 dogs) incubated with or without pretreatment with 1 μM indomethacin, with or without amiloride in the subsequent bathing solution. Histologic changes in mucosa from 3 dogs were assessed over time. I(SC) and ΔI(SC) were significantly reduced when chloride was removed from, but not when amiloride was added to, the bathing solution and were significantly reduced after pretreatment with indomethacin. The ΔI(SC) was significantly greater in mucosa from the distal portion of the colon than in the proximal portion of the colon. Histologic changes after incubation for 3 hours were minimal. I(SC) and ΔI(SC) resulted from electrogenic chloride secretion. Chloride secretion was reduced when release of PGs was prevented by indomethacin and was induced by administration of PGE(2). Chloride secretion in response to PGE(2) was greater in mucosa from the distal portion of the colon than in mucosa from the proximal portion of the colon.

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium. Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²(+) concentration ([Ca²(+)](i)) and Ca²(+) in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123. NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na(+) -K(+)-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²(+)-dependent) basolateral K(+) channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²(+)](i) , an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²(+). Initial falls in [Ca²(+)](i) were not mediated by a sequestration of Ca²(+) in intracellular Ca²(+) storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²(+)](i) were inhibited by 2',4'-dichlorobenzamil, an inhibitor of the Na(+)-Ca²(+)-exchanger, and attenuated in Na(+)-free buffer, suggesting a transient stimulation of Ca²(+) outflow by this transporter, directly demonstrated by Mn²(+) quenching experiments. ATP-sensitive and Ca²(+)-dependent basolateral K(+) conductances, the basolateral Na(+)-K(+)-pump as well as Ca²(+) transporters were involved in the action of H₂S in regulating colonic ion secretion.

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl−/1HCO3− exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.

THE RATE OF CURRENT CHANGE DURING A SHORT CIRCUIT IN THE POWER CIRCUITS OF THE ELECTRIC ROLLING STOCK WITH REGARD TO EDDY CURRENTS

In the article the issue of influence of vortical currents on rate of change of short circuit current is considered, a mathematical model for the calculation of short circuit currents in the traction mode in the power circuits of DC electric rolling stock is presented, and the research results are given.

Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea

Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

T2054 Platelet Activating Factor (PAF) Enhances Secretion and Motility in Human Small Intestine

G A A b st ra ct s effects on intestinal permeability. Methods: BALB/c (WT) and IL-13Rα1KO mice were infected with Heligmosomoides polygyrus and studied 12 days after a secondary infection (Hp2) to evoke a memory response. Muscle-free sections of jejunum were taken from WT or IL-13Rα1KO mice and mounted in microsnapwells to determine transepithelial electrical resistance (TEER), an index of mucosal permeability. Segments of jejunum were placed in Ussing chambers to assess changes in basal short circuit current (Isc). mRNA expression was determined by quantitative real-time PCR. Results: The Hp2-induced increase in Th2 cytokine expression was associated with decreased TEER (↑permeability) and basal Isc, consistent with enhanced paracellular permeability (Table). This was associated with an increase in mucosal mast cell protease-1 (mMCP-1) and claudin-2 expression (3.0 ± 0.7 fold). In contrast, Hp2 infection did not alter TEER or basal Isc in IL-13Rα1KO mice despite upregulation of Th2 cytokines, mMCP-1 and claudin-2 (3.5 ± 0.6 fold). Hp2 increased PAR2 expression in WT, but not in IL-13Rα1KO mice. Conclusions: These data question the perceived redundancy of the biological effects of IL-4 and IL-13. The effects of IL-4 on intestinal barrier function appear to be mediated by mastocytosis and increased expression of PAR-2 and claudin-2. In contrast, IL-13 working through IL-13Rα1 is critical for the Hp2induced changes in permeability, which appears to be due, in part, to increased expression of PAR-2, but independent of mastocytosis and claudin-2.

New developments in the fabrication of amorphous silicon photovoltaic modules on very large 2.60 m × 2.20 m glass

AbstractThe fabrication of photovoltaic thin film modules based on hydrogenated amorphous silicon (a‐Si:H) technology on very large float glass is presented. In high volume manufacture of about 900 modules/day production present an average power of about 105 W ±5 W (initial state, 1.43 m2 modules). Light‐induced degradation (LID) of modules results in an average power of about 85±2 W after about 500 kWh of degradation. Degradation of the fill factor (FF) of current voltage curves under illumination is the main contributor to degradation reducing from about 70% to 60% (artificial light). The dependency of the power in the initial and in the degraded state on the intrinsic layer thickness of the pin‐structure is investigated. Thicker intrinsic layer results in higher initial power, however also the degradation rate is higher resulting in very similar power of all modules in the degradation state. LID is performed in in‐door conditions under metal‐halogen lamp light and in outdoor conditions with sun‐light. Under outdoor conditions the degraded state of the modules is not stable, but depends on the season, resulting in lower power in winter months and higher power in summer months. Main contributor to this effect is the change of short circuit current. (© 2010 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Bicarbonate Transport by the Human Pancreatic Ductal Cell Line HPAF

The human pancreatic duct cell line, HPAF, has been shown previously to secrete Cl(-) in response to Ca(2+)-mobilizing stimuli. Our aim was to assess the capacity of HPAF cells to transport and secrete HCO3(-). HPAF cells were grown as confluent monolayers on permeable supports. Short-circuit current was measured by voltage clamp. Intracellular pH (pHi) was measured by microfluorometry in cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In HCO3(-)-free solutions, ATP-evoked changes in short-circuit current were inhibited by bumetanide, and the recovery of pHi from acid loading was abolished by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). In the presence of HCO3(-), ATP-evoked secretion was no longer inhibited by bumetanide, and there was a strong EIPA-insensitive recovery from acid loading, which was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS). ATP, but not forskolin, stimulated HCO3(-) efflux from the cells. In the absence of HCO3(-), ATP-evoked Cl(-) secretion is driven by a basolateral Na(+)-K(+)-2Cl(-) cotransporter, and pH(i) is regulated by apical and basolateral Na(+)/H(+) exchangers. In the presence of HCO3(-), ATP-evoked secretion is sustained in the absence of Na(+)-K(+)-2Cl(-) cotransporter activity and is probably driven by basolateral Na(+)-HCO3(-) cotransport.

Electrophysiological characterization of chloride secretion across the jejunum and colon of pigs as affected by age and weaning

Hypersecretion of chloride can cause diarrhea, a disease frequently occurring in young pigs, particularly around weaning. We investigated the contribution of different channels to intestinal Cl(-) secretion as influenced by age and weaning. Jejunal and colonic epithelia from 4-month-old pigs and 4-week-old piglets were incubated in Ussing chambers and stimulated by carbachol and forskolin. Changes in short-circuit currents were taken as measure of electrogenic net Cl(-) secretion. DIDS or NPPB served to inhibit Ca-activated Cl(-)-channels and outwardly rectifying Cl(-)-channels (ORCC) or cystic fibrosis transmembrane regulator (CFTR), respectively. Depolarizing the basolateral membrane allowed to examine the influence of K(+)-channels on Cl(-) secretion. Forskolin-stimulated Cl(-) secretion was mediated by CFTR. ORCC were not involved. Carbachol-induced Cl(-) secretion could be ascribed to an enhanced driving force due to the opening of K(+)-channels, whereas Ca-dependent Cl(-) channels seemed not to be involved. In jejunum, piglets showed higher Cl(-) secretion than pigs. Two days after weaning forskolin induced an I (sc) overshoot and a faster increase in G (t). In colon, Cl(-) secretion was neither influenced by age nor by weaning. The data suggest a disposition of porcine jejunum for a higher Cl(-) secretion in young and freshly weaned piglets, which might be a natural defense mechanism as well as a predisposing factor for diarrhea.

The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

It has recently been shown that the epithelial Na(+) channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the beta- and gamma-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.

Black walnut extract inhibits secretory pathways in equine cecum and colon via mechanisms that overlap with cyclooxygenase signaling

Disturbances in the hind‐gut have been linked to the induction of equine laminitis, presumably by initiating enteric signaling pathways that result in release of mediators that affect distant fragile vascular beds such as that in the hoof. Laminitis has been experimentally induced by gavaging with black walnut extract (BWE) toxin, leading to changes in both the gut and laminae. Cecal and colonic tissue were mounted on Ussing chambers and treated with escalating doses of BWE (0.6, 1.25, 2.5 and 5%). Changes in short circuit current (Isc), transepithelial electrical resistance, and 3H‐mannitol fluxes were measured. BWE treatment did not affect gut barrier function as there were no significant changes in resistance or mannitol flux. However, BWE caused significant decreases in Isc when compared with controls, most likely due to inhibition of basal Cl− secretion. Pre‐treatment with the cyclooxygenase inhibitor flunixin (2.7μM) resulted in a similar reduction in Isc, which has previously been shown to inhibit Cl− secretion. Treatment with both BWE and flunixin had no additive effect, further suggesting that BWE and flunixin have overlapping signaling pathways. The precise mechanism by which BWE reduces Isc in the equine gut will require further study, but early evidence is suggestive of an inhibitory effect on Cl− secretion. This in turn may affect colonic luminal contents, with systemic effects such as laminitis.

Prostaglandin E2Derived from Cyclooxygenases 1 and 2 Mediates Intestinal Epithelial Ion Transport Stimulated by the Activation of Protease-Activated Receptor 2

Proteinase-activated receptor (PAR)(2) is activated by trypsin-like serine proteinases and has been implicated in intestinal inflammation. However, its role in the regulation of intestinal mucosal function remains unclear. Using the intestinal epithelial cell line, SCBN, we have studied the stimulus-secretion coupling mechanisms of PAR(2)-induced epithelial chloride transport, focusing on cyclooxygenase (COX)-1 and COX-2 activities and prostaglandin (PG) E(2) secretion. SCBN monolayers were grown on Snapwell supports, mounted in modified Ussing chambers, and exposed to the activating peptide, SLIGRL-NH(2) (50 microM), to activate PAR(2). Pretreatment with inhibitors of cytosolic PLA(2) (cPLA(2)) (AACOCF3, arachidonyltrifluoromethyl ketone), COX-1 [SC560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2 (celecoxib) resulted in a significant concentration-dependent attenuation of PAR(2)-induced changes in short-circuit current. Immunoblot analysis showed a PAR(2)-induced increase in cPLA(2) phosphorylation that was blocked by the mitogen-activated protein kinase kinase inhibitor, PD98059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C(16)H(13)NO(3)], and the pan-protein kinase C inhibitor, GFX (bisindolylmaleimide). PAR(2) stimulation also resulted in a large increase in the production of PGE(2) as determined by enzyme-linked immunosorbent assay and was also blocked by PD98059 and GFX. Immunofluorescence and immunoblot analysis determined that EP2 and EP4 are expressed at the basolateral membrane of SCBN cells. Through the use of selective inhibitors (EP2, AH6809 [6-isopropoxy-9-oxoxanthene-2-carboxylic acid]; EP4, GW627368X [N-[2[4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl] acetyl]benzene sulphonamide]), it was found that both EP2 and EP4 were involved in mediating the PAR(2)-induced chloride secretory response. We conclude that basolateral PAR(2) activation induces epithelial chloride secretion that is mediated by cPLA(2), COX-1, COX-2, and the subsequent release of PGE(2). The production of PGE(2) results in an autocrine secretory response that is dependent on basolateral EP2 and EP4 receptors.

Enterocyte chloride and water secretion into the small intestine after enterotoxin challenge: Unifying hypothesis or intellectual dead end?

Many forms of diarrhoeal disease, particularly so called "secretory" diarrhoeal disease are thought to arise by the active secretion of chloride ion from the enterocytes, creating an osmotic gradient for fluid movement into the small intestinal lumen. This model implies that normally occurring intestinal secretion is catastrophically enhanced by bacterial enterotoxins. This review advocates that neither normal nor abnormal intestinal secretion from the enterocytes occurs and that no competent proof for chloride secretion exists. Prior to 1970, the physiological evidence failed to support the concept of the formation of intestinal juice as a normal intestinal event. The concept was later revived to explain the high rate of fluid entry into the lumen after exposure to cholera toxin. Much evidence has been advanced for the chloride secretion hypothesis, the dominant secretory paradigm after 1974, but is the evidence sufficiently compelling for it to be regarded as proving the chloride secretory model? The evidence falls into four categories and a fifth conjectural argument that proposes that an abnormal chloride ion channel in cystic fibrotic sufferers confers a natural selective advantage by preventing diarrhoeal disease. Secretion is putatively demonstrated by 1) showing that mass transfer of fluid is into the lumen (secretion) and not merely a failure to transport out of the lumen (failed absorption). Support is offered by 2) chloride ion flux measurements in vitro in Ussing chambers and by 3) short-circuit current measurements that are consistent with and purport to show chloride ion movement into the lumen. In addition, 4) pharmacological agents are identified that affect short-circuit current and these are assumed to be anti-secretory, consistent with the biochemical mechanism for secretion, confirmed wherever possible by mouse knock-out models. Finally, the proxy methods used to study water movement such as elevated short-circuit current measurements show these to be absent in cystic fibrotic patients. The enterocyte secretion hypothesis is challenged here on the basis of an examination of the methods used to show secretion, particularly after exposing the small intestine to heat stable enterotoxin (STa) from E. coli. STa is thought to be secretory because fluid entry into the lumen is claimed, enhanced isotopic flux of chloride ion towards the lumen occurs, an increase in short-circuit current is found, preventable by various drugs that are deemed likely to be anti-secretory and also because the short-circuit current changes after STa are not seen in cystic fibrotic patients. Using volume recovery in vivo, STa is found not to be secretory but only anti-absorptive. Hence, other techniques used to show secretion are not fit for that purpose. If STa is identified as secretory and yet no secretion occurs, how reliable is the evidence for other toxins being secretory when these methods are used? This review concludes that chloride ion secretion is unproven. A review of the literature indicates that secretion occurs not because epithelial cells actively pump water but by interdiction of fluid absorption, increased conductivity through tight junctions and an increased hydrostatic driving force through elevated capillary pressure. The exclusive focus on chloride secretion may explain the failure to develop antisecretory drugs over the last three decades.