Increase In Isc Research Articles

Actions of hydrogen sulphide on ion transport across rat distal colon

The aim of this study was to identify the actions of H(2)S on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by the H(2)S-donor, NaHS, were measured in Ussing chambers. Cytosolic Ca(2+) concentration was evaluated using fura-2. NaHS concentration-dependently induced a change in Isc, that was only partially inhibited by the neurotoxin, tetrodotoxin. Lower concentrations (< or =10(-3) mol.L(-1)) of NaHS induced a monophasic increase in Isc, whereas higher concentrations induced an additional, secondary fall of Isc, before a third phase when Isc rose again. Blockers of H(2)S-producing enzymes (expression demonstrated immunohistochemically) decreased basal Isc, suggesting that endogenous production of H(2)S contributes to spontaneous anion secretion. The positive Isc phases induced by NaHS were due to Cl(-) secretion as shown by anion substitution and transport inhibitor experiments, whereas the transient negative Isc induced by higher concentrations of the H(2)S-donor was inhibited by mucosal tetrapentylammonium suggesting a transient K(+) secretion. When applied from the serosal side, glibenclamide, an inhibitor of ATP-sensitive K(+) channels, and tetrapentylammonium, a blocker of Ca(2+)-dependent K(+) channels, suppressed NaHS-induced Cl(-) secretion suggesting different types of K(+) channels are stimulated by the H(2)S-donor. NaHS-induced increase in cytosolic Ca(2+) concentration was confirmed in isolated, fura-2-loaded colonic crypts. This response was not dependent on extracellular Ca(2+), but was inhibited by blockers of intracellular Ca(2+) channels present on Ca(2+) storage organelles. H(2)S induces colonic ion secretion by stimulation of apical as well as basolateral epithelial K(+) channels.

Unravelling a role for KCNQ1 in K+ recycling and gastric acid secretion

Unravelling a role for KCNQ1 in K⁺ recycling and gastric acid secretion

Inhibition of anion fluxes in Calu‐3 cells by polystyrene nanoparticles: role of lipid rafts

IntroductionThere is growing concern about the potential toxicity of non‐engineered and engineered nanoparticles. Lungs are particularly susceptible to the injury brought about by inhalation of gases and aerosols containing nanoparticles. Aims: We investigated the effects of apical exposure (up to 60 min) of cells to polystyrene nanoparticles (PNPs) (positive, negative, uncharged; 20, 50, 100nm) on transepithelial anion fluxes.MethodsCalu‐3 cells grown on inserts were mounted into modified Ussing chambers. Anion fluxes (measured as short‐circuit current, Isc) were stimulated with forskolin (10µM) in the presence or absence of PNPs (88‐104 µg/ml). In some experiments lipid rafts were disrupted by removing cell surface cholesterol with cyclodextrin (CD).ResultsForskolin resulted in mean peak increase in Isc from 29.4 to 108.4µA/cm2. All charged PNPs caused a significant (p&lt;0.05, n.≥3) reduction of forskolin‐stimulated anion fluxes (from 24 to 40‰), an effect undetectable with uncharged particles. The CD treatment led to a significant potentiation of the inhibitory effects of 20 and 50nm PNPs. In contrast, the treatment abolished the inhibitory effects of 100nm PNPs.ConclusionOur results suggest that charge, size and interactions with lipid rafts are essential factors in determining the extent of PNPs‐mediated inhibition of forskolin‐stimulated anion fluxes in Calu‐3 cells.

Epithelia derived from human mammary tissue exhibits unique ion transport characteristics

Ion transport and barrier characteristics of mammary epithelium are poorly defined for most species, including humans. MCF10a cells, derived from non‐neoplastic human mammary tissue (Cancer Res 50: 6075, 1990), were cultured on permeable supports in typical medium (DMEM/F12) for 9‐17 days in the absence and presence of cholera toxin (Ct). At all time points tested, MCF10a cells generated an electrically tight epithelial barrier (1010±110 O cm2) that exhibited a positive short circuit current (Isc; 6.1±0.3 μA cm−2) indicative of net cation absorption or anion secretion. Cells cultured in Ct exhibited greater basal Isc and greater amiloride‐sensitive Isc (4.3 vs. 2.5 μA cm−2), indicating enhanced sodium absorption. Culturing the cells in the presence of forskolin (0.2 μM) did not replicate the Ct effect. Additionally, it was shown that the effect of Ct‐enhanced amiloride‐sensitive Isc was greatest at the shortest exposure period tested, 1 day. Regardless of culture conditions, acute exposure to forskolin (2 μM) induced a modest, slowly developing increase in Isc. Bumetanide and glibenclamide (300 μM) were associated with modest reductions in Isc (&lt;0.7 μA cm−2). These results demonstrate that MCF10a cells provide a valuable model to identify transport mechanisms and regulatory pathways that can account for the unique composition of human milk. [NIH (P20‐RR017686), KS Ag Exp Stn, and KSU SUROP support]

Apical Methyl-β-cyclodextrin (mβCd) Treatment In A6 Renal Cells Does Not Affect Basolateral PGE2-induced Cl- Secretion , But Stimulates An Early Cl- Peak Current, Activated By Hypotonic Shock

We explore in this study whether cholesterol content of the apical membrane of distal kidney cells from Xenopus laevis (A6) affects the behavior of two types of anion channels, recently described by Bao et al. (AJP,2008,295,G234). Voltage clamp technique in an Ussing-type chamber, enabling the recording of short-circuit current, was applied on polarized monolayers of A6 cells, grown on permeable filter supports for 13 - 31 days. To create a gradient for Cl- secretion, the apical solution was Cl- and Na+ free, whereas the basolateral solution contained normal NaCl. Twice basolateral addition of PGE2 (1 μM) during 15 minutes with an interval of 60 minutes produced a first transient peak increase in Isc of 7.25 ± 0.98 μAcm-2 (N=6), followed by a plateau of 2.77 ± 0.50 μAcm-2. The second PGE2 stimulation elicited a peak current of 6.63 ± 1.20 μAcm-2. When the apical membrane was treated for at least 1h with 10 mM mβCD, PGE2 evoked Cl- secretion was not different from control. A hypotonic shock was induced twice with an interval of 60 minutes on the basolateral side by a sudden reduction of solution osmolality from 260 to 140 mOsm/kg H2O. In control a transient increase in Isc of 0.27 ±0.11 μAcm-2 (N=3) was observed during the second stimulation only. After 60 minutes treatment with 10 mM mβCD the hypotonic shock evoked an augmented transient Isc of 1.82 ± 0.08 μAcm-2 (N=5). These results suggest that apical Cl- channels in A6 cells, activated either by PGE2 or by cell swelling, are regulated differently by the membrane cholesterol content. This work was supported by the grants FWO Flanders G.0270.07 and BOF 03B0BF.

Stimulation of mucosal secretion by lubiprostone (SPI-0211) in guinea pig small intestine and colon

Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1-3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1-3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1-3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.

Intestinal Structure and Function of Broiler Chickens on Diets Supplemented with a Synbiotic Containing Enterococcus faecium and Oligosaccharides

A feeding trial was conducted on broiler chickens to study the effects of the synbiotic BIOMIN IMBO [a combination of Enterococcus faecium, a prebiotic (derived from chicory) and immune modulating substances (derived from sea algae)], with a dose of 1 kg/ton of the starter diets and 0.5 kg/ton of the grower diets on the intestinal morphometry and nutrient absorption. The general performance was improved (P < 0.05) by the dietary inclusion of synbiotic compared with the controls. Furthermore, the addition of synbiotic increased (P < 0.001) the villus height/crypt depth ratio and villus height in ileum. However, the ileal crypt depth was decreased by dietary supplementation of synbiotic compared with control. The addition of glucose in Ussing chamber produced a significant increase (P ≤ 0.001) in short-circuit current (Isc) in jejunum and colon relative to the basal values in both synbiotic and control groups. However, in jejunum the percentage of Isc increase after glucose addition was higher for synbiotic group (333 %) than control group (45 %). In conclusion, dietary inclusion of synbiotic BIOMIN IMBO increased the growth performance and improved intestinal morphology and nutrient absorption.

The Cystic Fibrosis Transmembrane Conductance Regulator and Chloride-Dependent Ion Fluxes of Ovine Vocal Fold Epithelium

Ion-driven transepithelial water fluxes participate in maintaining superficial vocal fold hydration, which is necessary for normal voice production. The authors hypothesized that Cl(-) channels are present in vocal fold epithelial cells and that transepithelial Cl(-) fluxes can be manipulated pharmacologically. Immunohistochemical assays were used to identify cystic fibrosis transmembrane regulator Cl(-) channels in ovine vocal fold mucosae (n = 2). Electrophysiological responses of vocal fold mucosae (n = 80) to Cl(-) channel inhibitors and secretagogues were evaluated in an ovine model using a randomized controlled experimental design. Cystic fibrosis transmembrane regulator channels were localized to the plasma membranes of epithelial cells. The Cl(-) transport inhibitor, diphenylamine-2-carboxylate, elicited a 30% decrease in mean short-circuit current (I(sc); n = 10). The secretagogue, isobutylmethylxanthine, yielded a 31.7% increase in mean I(sc) (n = 10). Another secretagogue, uridine triphosphate, elicited a 48.8% immediate and 17.3% sustained increase in mean I(sc) (n = 10). No sustained increases occurred following application of secretagogues to mucosae bathed in a low Cl(-) environment (n = 10), suggesting that responses were Cl(-) dependent. The authors provide structural and functional evidence for the presence of a transepithelial pathway for Cl(-) fluxes. Pharmacological manipulation of this pathway may offer a mechanism for maintaining superficial vocal fold hydration.

Regulation of electrolyte transport across cultured endometrial epithelial cells by prolactin

The effect of prolactin (PRL) on ion transport across the porcine glandular endometrial epithelial cells was studied in primary cell culture using the short-circuit current technique. Addition of 1 microg/ml PRL either to the apical solution or to the basolateral solution produced a peak followed by a sustained increase in Isc, but with a lesser response when PRL was added apically. Basolateral addition of PRL increased the Isc in a concentration-dependent manner with a maximum effect at 1 microg/ml and an effective concentration value of 120 ng/ml. The PRL-stimulated Isc was significantly reduced by pretreatment with an apical addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM), diphenylamine-2-carboxylic acid (1 mM) or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (200 microM), Cl(-) channel blockers, but not by amiloride (10 microM), a Na(+) channel blocker. In addition, pretreatment with bumetanide (200 microM), a Na(+)-K(+)-2Cl(-) cotransporter inhibitor, in the basolateral solution significantly reduced the PRL-stimulated Isc. Replacement of Cl(-) or in the bathing solutions also decreased the Isc response to PRL. Pretreatment of the monolayer with AG490 (50 microM), an inhibitor of JAK2 activity significantly inhibited the PRL-induced increase in Isc. Western blot analysis of the porcine endometrial epithelial cells revealed the presence of short isoform of PRL receptor (PRLR-S) that could be regulated by 17beta-estradiol. The results of this investigation showed that PRL acutely stimulated anion secretion across the porcine endometrial epithelial cells possibly through PRLR-S present in both apical and basolateral membranes. The PRL response appeared to be mediated by the JAK2-dependent pathway.

W1374 Excitation of Neurons in the Enteric Nervous System (ENS), Degranulation of Enteric Mast Cells and Direct Action On Enterocytes Underlie Stimulatory Action of Bradykinin (Bk) On Mucosal Secretion in Guinea-Pig Small Intestine

Neurons in the g-pig small intestinal ENS express the BK-B2 receptor. Stimulation of the BK-B2 receptor elevates neuronal excitability (J Pharmacol Exp Ther 2004; 309:310-9; & Br J Pharmacol 2003; 138:1221-32). BK-2 stimulation releases prostaglandins, which feedback in autocoid manner to excite the same neurons that release the prostaglandins. Full thickness and muscle-stripped segments of g-pig ileum were mounted in Ussing flux chambers for recording short-circuit current (Isc), an indicator of Clsecretion. Application of BK in the serosal compartment of the chambers evoked increases in Isc in a concentration-dependent manner with an EC50 8.0±0.1nM. Higher concentrations of 1-5μMwere required for stimulation of Isc with application in the mucosal compartment. Pretreatment with 0.5-μM tetrodotoxin (TTX) partially suppressed the BK-evoked increases in Isc. Transmural electrical field stimulation evoked neurally-mediated increases in Isc. These responses were abolished by TTX in each of 18 preps. Application of the mast cell degranulating agent, compound 48/ 80 (60μg/ml), increased Isc (P 0.05). The BK-B2 antagonist, WIN64388 (1-10μM), partially suppressed BK-evoked increases in Isc. WIN64388 at 10μM suppressed BK-evoked Isc by 62.8% in 12 preps. Pre-incubation for 30 min with the selective cyclooxygenase-1 (COX-1) inhibitor, SC560 (10μM), abolished increases in Isc evoked by 10-nM BK in each of 10 preps. The COX-2 inhibitor, NS398 (2μM) reduced BK-evoked increases in Isc by 28.9 percent in 6 preps. Pre-treatment with a prostanoid receptor antagonist, AH6809 (5μM), significantly suppressed BK-evoked stimulation of Isc (P<0.05) for all of 6 preps. BK-evoked increases in Isc were suppressed by pre-treatment with the mast cell stabilizing agents: cromolyn (10-30μM; P<0.05); doxantrazole (30-50μM; P<0.05) or ketotifen (10μM; P<0.05) with each agent tested on 10 preps. Carbachol acted directly on the epithelium to increase Isc. This well-known action of the muscarinic agonist was suppressed by ketotifen, but not by cromolyn or doxantrazole. The results suggest a role for enteric mast cells in BK-stimulation of mucosal secretion, with a prostaglandin involved as a mediator. Once released from mast cells prostaglandins stimulate secretomotor neuronal activity; as well as, act in direct concert with BK on the enterocytes. All of which might underlie the involvement of BK in inflammation-related secretory diarrhea. Supported by: NIH RO1DK57075 & KO8 DK060468

Epithelial Barrier Modulation by a Channel Forming Peptide

NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (Isc) was <3 microA cm(-2) for T-84, IPEC-J2 and PVD9902 cell monolayers and <8 microA cm(-2) for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in Isc to >15 microA cm(-2), indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm(-2): T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells

We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl(-) secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl(-) transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca(2+). Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH(2)-induced increases in I(sc). Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I(sc). We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl(-) secretion via cAMP- and Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.

Hydrogen peroxide stimulates chloride secretion in primary inner medullary collecting duct cells via mPGES-1-derived PGE2

We investigated the role and mechanism of H2O2 in regulation of NaCl transport in primary inner medullary collecting duct (IMCD) cells. IMCD cells were isolated from wild-type mice and grown onto semipermeable membranes, and short-circuit current (Isc) was determined by Ussing chamber. Exposure of IMCD cells to H2O2 at a range of 100-300 microM caused a rapid increase in Isc in a time- and dose-dependent manner. This increase was almost abolished by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel inhibitors diphenylamine-2-carboxylic acid (DPC) and CFTR inhibitor-172. In contrast, the magnitude of stimulation was unaffected by the epithelial Na+ channel (ENaC) inhibitor amiloride. The H2O2-induced Cl(-) secretion was significantly inhibited by indomethacin, as well as by microsomal PGE synthase-1 (mPGES-1) deficiency. Like H2O2, PGE2 treatment induced a twofold increase in Isc that was reduced by the protein kinase A (PKA) inhibitors H-89 and KT5720. These data suggest that H2O2 stimulates CFTR Cl(-) channel-mediated Cl(-) secretion through cyclooxygenase- and mPGES-1-dependent release of PGE2 and subsequent activation of PKA.

Orexins Control Intestinal Glucose Transport by Distinct Neuronal, Endocrine, and Direct Epithelial Pathways

Orexins are neuropeptides involved in energy homeostasis. We investigated the effect of orexin A (OxA) and orexin B (OxB) on intestinal glucose transport in the rat. Injection of orexins led to a decrease in the blood glucose level in oral glucose tolerance tests (OGTTs). Effects of orexins on glucose entry were analyzed in Ussing chambers using the Na(+)-dependent increase in short-circuit current (Isc) to quantify jejunal glucose transport. The rapid and marked increase in Isc induced by luminal glucose was inhibited by 10 nmol/l OxA or OxB (53 and 59%, respectively). Response curves to OxA and OxB were not significantly different with half-maximal inhibitory concentrations at 0.9 and 0.4 nmol/l, respectively. On the one hand, OxA-induced inhibition of Isc was reduced by the neuronal blocker tetrodotoxin (TTX) and by a cholecystokinin (CCK) 2R antagonist, indicating involvement of neuronal and endocrine CCK-releasing cells. The OX(1)R antagonist SB334867 had no effect on OxA-induced inhibition, which is likely to occur via a neuronal and/or endocrine OX(2)R. On the other hand, SB334867 induced a significant right shift of the concentration-effect curve for OxB. This OxB-preferring OX(1)R pathway was not sensitive to TTX or to CCKR antagonists, suggesting that OxB may act directly on enterocytic OX(1)R. These distinct effects of OxA and OxB are consistent with the expression of OX(1)R and OX(2)R mRNA in the epithelial and nonepithelial tissues, respectively. Our data delineate a new function for orexins as inhibitors of intestinal glucose absorption and provide a new basis for orexin-induced short-term control of energy homeostasis.

Effects of cardiogenic edema fluid on ion and fluid transport in the adult lung

We have previously shown that cardiogenic pulmonary edema fluid (EF) increases Na(+) and fluid transport by fetal distal lung epithelia (FDLE) (Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM and O'Brodovich H. J Physiol 544: 537-548, 2002). We now report the effect of EF on Na(+) and fluid transport by the adult lung. We first studied primary cultures of adult type II (ATII) epithelium and found that overnight exposure to EF increased Na(+) transport, and this effect was mainly due to factors other than catecholamines. Plasma did not stimulate Na(+) transport in ATII. Purification of EF demonstrated that at least some agent(s) responsible for the amiloride-insensitive component resided within the globulin fraction. ATII exposed to globulins demonstrated a conversion of amiloride-sensitive short-circuit current (I(sc)) to amiloride-insensitive I(sc) with no increase in total I(sc). Patch-clamp studies showed that ATII exposed to EF for 18 h had increased the number of highly selective Na(+) channels in their apical membrane. In situ acute exposure to EF increased the open probability of Na(+)-permeant ion channels in ATII within rat lung slices. EF did increase, by amiloride-sensitive pathways, the alveolar fluid clearance from the lungs of adult rats. We conclude that cardiogenic EF increases Na(+) transport by adult lung epithelia in primary cell culture, in situ and in vivo.

Acute inflammation alters bicarbonate transport in mouse ileum

T-cell mediated acute inflammation of the ileum may occur during Crohn's disease exacerbations. During ileal inflammation, absorption of nutrients and electrolytes by villus cells is decreased with a concomitant increase in crypt and/or villus fluid secretion. These alterations lead to fluid accumulation and the subsequent diarrhoea. Net intestinal fluid secretion consists of HCO3--rich plasma-like fluid. However, the regulation and mechanisms of HCO3- secretion in normal and acutely inflamed ileum are not clearly understood. To study this phenomenon, anti-CD3 monoclonal antibody (mAb)- induced in vivo ileal inflammatory mouse models was used for in vitro functional studies with Ussing chamber and pH stat techniques. Three hours after anti-CD3 mAb injection, ileal mucosa stripped of muscular and serosal layers showed a significant increase in short circuit current (Isc) (0.58+/-0.07 microEq h(-1) cm2 versus 1.63+/-0.14 microEq h(-1) cm2). The cAMP-stimulated Isc component was sensitive to glibenclamide but not to DIDS, suggesting that a cystic fibrosis transmembrane conductance regulator (Cftr)-mediated anion conductance was responsible. Basal Cl--dependent HCO3- secretion, measured using a pH stat technique, was decreased significantly in anti-CD3-injected mice, with a simultaneous increase in Cl--independent HCO3- secretion that was also inhibited by glibenclamide. Experiments using Cftr-/- mice showed neither an increase in Isc nor an increase in HCO3- secretion, confirming the role for Cftr protein in stimulating anion secretion following anti-CD3 treatment. Western blot analysis indicated that Cftr protein levels were unaltered by anti-CD3 treatment, at least acutely. Finally, an immunoassay for cAMP showed significant increases in intracellular cAMP in villus cells, but not in crypt cells. These studies therefore suggest a shift from a predominantly electroneutral Cl-HCO3- exchange in normal mice, to a predominantly electrogenic anion secretion including HCO3- that occurs via functional Cftr during anti-CD3-mediated acute inflammation.

A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic agonists

The causal factors of equine anhidrosis have not yet been elucidated but defective electrolyte transport mechanisms in the gland are likely to be involved. To investigate this possibility, experiments were performed on cultured equine sweat gland epithelia from five free-sweating UK horses (3 intact males, 2 mares, aged 2-4 years) and from three free-sweating Singapore horses (1 intact male, 2 mares, aged 3-5 years) and three anhidrotic (Singapore) horses (1 intact male, 1 gelding, 1 mare, aged 3-6 years). Cultured cells from each animal were grown on permeable supports and loaded into Ussing chambers to quantify transepithelial resistance and agonist-induced electrolyte transport by the short circuit current (Isc) technique. Transepithelial resistances across the layers of cultured cells were not significantly different between cells from UK and Singapore free-sweating horses, but were significantly reduced in anhidrotic animals. Purinergic agonists added to the apical and basolateral aspects of the cultured cells caused similar increases in Isc between the two populations of unaffected cells, but Isc increases were significantly reduced in anhidrotic animals. Beta-adrenergic agonist stimulation of the anhidrotic cell layers failed to elicit any change in Isc. These pilot results not only confirm earlier conclusions from anatomical findings that failure in the secretory process occurs in anhidrosis but also indicate that both of the known ion transport mechanisms are involved. The trigger for these failures warrants further investigation.

5-Hydroxytryptamine Induces Electrogenic Secretion in the Duodenum of Gerbil (Gerbillus cheesmani)

The effect of serosally added 5-hydroxytryptamine (5-HT 100 I¼M) on the short circuitcurrent (Isc) across duodenum taken from fed, starved (4 days, water ad lib) and undernourished (50% control food intake for 21 days) gerbils (Gerbillus cheesmani) were investigated. The effect of the neurotoxin, tetrodotoxin (TTX 10 I¼M) and atropine (100I¼M) on the maximum increase in Isc induced by 5-HT were also studied. The 5-HT-induced Isc were higher in unstripped than in the stripped sheets in the three feeding conditions. TTX reduced the maximum increase in Isc induced by 5-HT across stripped and unstripped sheets taken from fed, starved and undernourished gerbils. Atropine decreased the 5-HT-induced Isc of stripped sheets in the three feeding conditions and it also decreased the 5-HTinduced Isc in unstripped sheets in fed duodenum. Therefore, the duodenal response to 5-HT occur partly by activation of a nonneural pathway and partly by activating electrogenic ion transport via muscarinic neural mechanism. It also showed that the 5-HT-induced Isc was chloride-dependent in fed duodenum and were chloride and bicarbonate dependent in the duodenum taken from starved and undernourished gerbil The results also showed that the increase in 5-HT-induced Isc as a results of starvation and undernourishment were TTX-sensitive and both chloride and bicarbonate dependent.

Effects of 5-hydroxytryptamine on the short-circuit current across the small intestine of the gerbil (Gerbillus cheesmani) in different dietary states

The effects of serosally added 5-hydroxytryptamine (5-HT, 100 microM) on the short circuit-current (Isc) across jejunum and ileum taken from fed, starved and undernourished (Gerbillus cheesmani) were investigated. The effects of the neurotoxin, tetrodotoxin (TTX, 10 microM) on the basal Isc as well as on the maximum increase in Isc induced by 5-HT were also studied. There were regional variations in the basal Isc as well as in the way by which the small intestine responds to 5-HT. The basal Isc was greater in jejunum than in ileum and such differences were TTX-sensitive. The maximum increase in Isc, which results from addition of 5-HT, was higher in jejunum than in ileum under all three feeding conditions. TTX reduced the maximum increase in Isc induced by 5-HT across stripped and intact intestine of the two regions in the three nutritional states. The 5-HT-induced Isc in the jejunum of both starved and undernourished gerbils and in the ileum of starved animals was the function of both submucosal and myenteric plexus. In jejunum and ileum taken from starved and undernourished gerbils the 5-HT-induced Isc was both chloride- and bicarbonate-dependent. Thus the results indicated that both starvation and undernourishment increase that response and such increases were TTX-sensitive and both chloride- and bicarbonate-dependent.