Increase In Isc Research Articles

Choline acetyltransferase and organic cation transporters are responsible for synthesis and propionate-induced release of acetylcholine in colon epithelium

Acetylcholine is not only a neurotransmitter, but is found in a variety of non-neuronal cells. For example, the enzyme choline acetyltransferase (ChAT), catalyzing acetylcholine synthesis, is expressed by the colonic epithelium of different species. These cells release acetylcholine across the basolateral membrane after luminal exposure to propionate, a short-chain fatty acid. The functional consequence is the induction of chloride secretion, measurable as increase in short-circuit current (Isc) in Ussing chamber experiments. It is unclear how acetylcholine is produced and released by colonic epithelium. Therefore, the aim of the present study was the identification (on mRNA and protein level) and functional characterization (in Ussing chamber experiments combined with HPLC detection of acetylcholine) of transporters/enzymes in the cholinergic system of rat colonic epithelium. Immunohistochemical staining as well as RT-PCR revealed the expression of high-affinity choline transporter, ChAT, carnitine acetyltransferase (CarAT), vesicular acetylcholine transporter (VAChT), and organic cation transporters (OCT 1, 2, 3) in colonic epithelium. In contrast to blockade of ChAT with bromoacetylcholine, inhibition of CarAT with mildronate did not inhibit the propionate-induced increase in Isc, suggesting a predominant synthesis of epithelial acetylcholine by ChAT. Although being expressed, blockade of VAChT with vesamicol was ineffective, whereas inhibition of OCTs with omeprazole and corticosterone inhibited propionate-induced Isc and the release of acetylcholine into the basolateral compartment. In summary, OCTs seem to be involved in regulated acetylcholine release by colonic epithelium, which is assumed to be involved in chemosensing of luminal short-chain fatty acids by the intestinal epithelium.

Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells.

Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19‐day gestational age rat fetuses. Equivalent short‐circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The I SC showed a fast dose‐dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5‐(N‐Ethyl‐N‐isopropyl)amiloride inhibition of ISC was not affected, however, benzamil‐sensitive ISC was increased in insulin‐stimulated monolayers. The application of LY‐294002 and Akti1/2 both completely blocked the stimulating effect of insulin on I SC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF‐1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF‐1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.

Evidence for expression and function of angiotensin II receptor type 1 in pulmonary epithelial cells

There is a growing evidence that the peptide hormone angiotensin II (ANGII) can act as an auto-/paracrine mediator to regulate epithelial ion transport processes. The present study focused on the impact of ANGII on transepithelial ion transport in pulmonary epithelia. Transcripts for the ANGII receptor type 1 (ATR1) were detected in lungs of Xenopus laevis and H441 cells (human pulmonary epithelial cell line). Native Xenopus lung preparations were used for Ussing chamber recordings and apically applied ANGII (10μM) induced a significant increase of short-circuit current (ISC: 8±2%, n=13). Pre-incubation with losartan (LOS), an antagonist of ATR1 prevented the effect of ANGII on ISC.Transcripts for ATR1 in Xenopus lungs and H441 cells were detected and an increase of ISC was observed by ANGII in native Xenopus lung epithelia. This indicates that ANGII is a potential auto-/paracrine mediator for ion transport regulation in pulmonary epithelia.

Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins

Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

Endogenous PYY and GLP‐1 mediate l‐glutamine responses in intestinal mucosa

l-glutamine (Gln) is an energy source for gastrointestinal (GI) epithelia and can stimulate glucagon-like peptide 1 (GLP-1) release from isolated enteroendocrine L-cells. GLP-1 and peptide YY (PYY) are co-secreted postprandially and both peptides have functional roles in glucose homeostasis and energy balance. The primary aim of this project was to establish the endogenous mechanisms underpinning Gln responses within intact GI mucosae using selective receptor antagonists. Mouse mucosae from different GI regions were voltage-clamped and short-circuit current (Isc) was recorded to Gln added to either surface in the absence or presence of antagonists, using wild-type (WT) or PYY-/- tissues. The glucose sensitivity of Gln responses was also investigated by replacement with mannitol. Colonic apical and basolateral Gln responses (at 0.1 and 1 mM) were biphasic; initial increases in Isc were predominantly GLP-1 mediated. GLP-1 receptor antagonism significantly reduced the initial Gln response in the PYY-/- colon. The slower reductions in Isc to Gln were PYY-Y1 mediated as they were absent from the PYY-/- colon and were blocked selectively in WT tissue by a Y1 receptor antagonist. In jejunum mucosa, Gln stimulated monophasic Isc reductions that were PYY-Y1 receptor mediated. Gln effects were partially glucose sensitive, and Calhex 231 inhibition indicated that the calcium-sensing receptor (CaSR) was involved. Gln stimulates the co-release of endogenous GLP-1 and PYY from mucosal L-cells resulting in paracrine GLP-1 and Y1 receptor-mediated electrogenic epithelial responses. This glucose-sensitive mechanism appears to be CaSR mediated and could provide a significant therapeutic strategy releasing two endogenous peptides better known for their glucose-lowering and satiating effects.

Actions of Angeli's salt, a nitroxyl (HNO) donor, on ion transport across mucosa—submucosa preparations from rat distal colon

The aim of this study was to investigate whether nitroxyl (HNO), a redox variant of the radical gasotransmitter nitric oxide (NO) with therapeutically promising properties, affects colonic ion transport. Changes in short-circuit current (Isc) induced by the HNO donor Angeli's salt were recorded in Ussing chambers. Cytosolic Ca2+ concentration was measured with fura-2. The nitroxyl donor induced a concentration-dependent increase in Isc across rat distal colon which was due to a stimulation of chloride secretion. The secretion induced by Angeli's salt (5×10−4mol/l) was not altered by the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), but was abolished by the HNO scavenger l-cysteine. The response was not dependent on the activity of soluble guanylate cyclase or enteric neurons, but was inhibited by indomethacin. Experiments with apically permeabilized epithelia revealed the activation of basolateral K+ channels and a stimulation of the current carried by the basolateral Na+—K+-pump by Angeli's salt. The secretion induced by Angeli's salt was reduced in the absence of extracellular Ca2+. A prominent increase in the cytosolic Ca2+ concentration was evoked by Angeli's salt predominantly in subepithelial cells within the submucosa, which had the same dependence on extracellular Ca2+ as the Angeli's salt-induced Cl− secretion. Consequently, Angeli's salt induces a soluble guanylate cyclase-independent, Ca2+-dependent Cl− secretion via activation of the Na+—K+-ATPase and of basolateral K+ channels. Cyclooxygenase metabolites produced within the submucosa seem to be involved in this response.

Ursodeoxycholic acid attenuates colonic epithelial secretory function

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

Basolateral transport of bicarbonate by avian renal proximal tubule cells in culture

Bicarbonate (HCO3−) is an important component of acid/base regulation. Therefore, it is crucial to understand the mechanism of bicarbonate reabsorption in the kidney proximal tubule (PT). In mammals HCO3−reabsorption is well understood, but evidence suggests that birds may use a different mechanism. We hypothesize that in the avian PT HCO3−crosses the luminal membrane in ionic form, rather than as CO2, and that HCO3− ions then leave the basolateral side via an NBC1‐like transporter, as in the mammalian PT. Experiments used primary cell cultures of chick PT and electrophysiological studies to measure currents (ISC) associated with ion transport. Monolayers were first stimulated with 1μM forskolin, which activated a chloride secretory current in the avian PT. This was followed by basolateral application of 100 μM DIDS, an inhibitor of NBC1. With bicarbonate in the bathing solution DIDS caused an increase in ISC of 6.25 ± 1.55 μAmps/cm2 (n = 6), but only 2.30 ± 0.58 μAmps/cm2 (n = 5) in the absence of bicarbonate. The increased ISC is consistent with inhibition of electrogenic transport of HCO3− ions via the NBC1 transporter, and the decreased effect of DIDS in the nominal absence of bicarbonate further supports this. Additional studies support both mRNA expression and NBC1 protein expression (western blotting) in chick PT cultures and native tissue. Experiments are underway to test for an alternative apical transporter.

Fusion‐activated cation entry (FACE)viaP2X4couples surfactant secretion and alveolar fluid transport

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X₄ receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X₄ abrogated this effect by ∼50%, whereas potentiating P2X4 lead to ∼80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.

Galanin is a modulator of eccrine sweat gland secretion

The neuropeptide galanin has been ascribed different roles in modulating physiological functions in the skin. The present study examined the function of galanin in eccrine sweat gland physiology. We demonstrated secretion of galanin by sweat glands in vivo by radioimmunoassay of human sweat (20-192 fmol galanin/ml). Furthermore, human sweat glands expressed galanin receptors GalR2 and GalR3. Using chamber short-circuit current (Isc) measurements showed that application of galanin to human NCL-SG3 cells led to a significant increase in Isc, which was inhibited by the presence of chloride channel blockers and in chloride-free Krebs solution. Additionally, application of SNAP 37889, a non-peptidergic selective antagonist of GalR3, abolished the effect of galanin on Isc. In summary, our results show that galanin can regulate transepithelial chloride ion transport and fluid secretion by stimulating GalR3 in NCL-SG3 cells and demonstrate a possible important extraneural function of galanin in sweat gland physiology.

Reliable Metallization Process for Ultra Fine Line Printing

In this paper we present an approach to achieve both 100mg paste deposit on a c-Si solar cell and efficiency gains with no impact on peel strength at extremely narrow finger widths. Using the double print process with suitable pastes and screens we demonstrate an 0.18% absolute efficiency gain and 20% paste saving over the standard process. This benefit is correlated to the finger width reduction from 63μm to 47μm, leading to an increase in Isc and Voc, due to the reduced recombination area under the metal contacts. The peel strength is also shown to increase from 1.5N to 3N for the same busbar thickness, when different paste compositions for the DP process are used. We also show screen lifetimes above 30k prints in production, with a stable process in terms of efficiency gain and paste saving.

Effects of leukotriene C4 on the bioelectric properties and ion transport of equine tracheal epithelium

To determine effects of leukotriene (LT) C(4) on ion transport across equine tracheal epithelium. Sample-Tracheal epithelium from cadavers of 24 horses considered free of respiratory tract disease. Mucosae were mounted into Ussing chambers, and short-circuit current (I(sc)) was monitored over time. Effects of LTC(4) were examined for various conditions, including addition of amiloride (10μM) to the mucosal bath solution, addition of bumetanide (10μM) to the serosal bath solution, addition of barium (1mM) to the serosal bath solution, and substitution of gluconate for chloride and HEPES for bicarbonate in bath solutions. Electrolyte transport was assessed via (22)Na and (36)Cl isotope fluxes. Addition of LTC(4) (50nM) to the serosal bath solution caused an increase in I(sc) for basal conditions and a larger increase after pretreatment with amiloride. The increase was negated in part by the addition of bumetanide to the serosal bath solution and further reduced by substitution of HEPES for bicarbonate in bath solutions. Remaining current was reduced to values less than those before treatment with LTC(4) by the addition of barium to the serosal solution. There was a small increase in I(sc) after the addition of amiloride and substitution of gluconate for chloride. Radioisotope flux indicated that addition of LTC(4) to the serosal bath solution increased chloride secretion and reduced sodium absorption. LTC(4) stimulated chloride secretion through a predominately bumetanide-sensitive pathway, with a smaller contribution from a bicarbonate-dependent pathway. Thus, LTC(4) appears to be a potential mediator of airway hypersecretion in horses.

Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD(c14) cortical collecting duct cell line to determine effects of ATP on sodium (Na(+)) and chloride (Cl(-)) transport with an Ussing chamber system. ATP, at a concentration of 10(-6) M or less, did not inhibit ENaC-mediated short-circuit current (I(sc)) but instead stimulated a transient increase in I(sc). The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V(te)) is clamped to less than -10 mV. We hypothesized that this negative V(te) might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD(c14) cells with aldosterone (10(-6) M) and then clamped the V(te) to -50 mV, the V(te) of aldosterone-stimulated cells under open-circuit conditions. ATP (10(-6) M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl(-) absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V(te), may dictate the direction of ATP-stimulated Cl(-) transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.

Can morphology tailoring improve the open circuit voltage of organic solar cells?

While the effect of interfacial morphology on the short circuit current (ISC) of organic photovoltaic devices (OPVs) is well known, its impact on open circuit voltage (VOC) and fill-factor (FF) are less clear. Since the output power of a solar cell Pout=ISCVOCFF, such understanding is critical for designing high-performance, morphology-engineered OPVs. In this letter, we provide an explicit analytical proof that any effort to radically improve VOC by tailoring bulk heterojunction morphology is futile, because any increase in ISC due to larger interface area is counterbalanced by corresponding increase in recombination current, so that the upper limit of VOCBHJ cannot exceed that of the corresponding planar heterojunction devices, i.e., VOCBHJ≤VOCPHJ. We discuss the implication of this VOC-constraint on the efficiency optimization of organic solar cells.

Modulation of ion transport across rat distal colon by cysteine

The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionine-β-synthase and cystathionine-γ-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and β-cyano-L-alanine, i.e., inhibitors of H2S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e., an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl− and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl− secretion, whereas Na cysteinate – after a transient inhibitory phase – activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K+ channels. Indeed, after preinhibition of basolateral K+ channels with tetrapentylammonium or Ba2+, the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H2S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H2S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H2S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter.

Activation of P2Y1 and P2Y2 receptors induces chloride secretion via calcium-activated chloride channels in kidney inner medullary collecting duct cells

Dysregulation of urinary sodium chloride (NaCl) excretion can result in extracellular fluid (ECF) volume expansion and hypertension. Recent studies demonstrated that urinary nucleotide excretion increases in mice ingesting a high-salt diet and that these increases in extracellular nucleotides can signal through P2Y(2) receptors in the kidney collecting duct to inhibit epithelial Na(+) channels (ENaC). However, under conditions of ECF volume expansion brought about by high-dietary salt intake, ENaC activity should already be suppressed. We hypothesized that alternative pathways exist by which extracellular nucleotides control renal NaCl excretion. We used an inner medullary collecting duct (mIMCD-K2) cell line in an Ussing chamber system as a model to study additional ion transport pathways that are regulated by extracellular nucleotides. When ENaC was inhibited, the addition of adenosine triphosphate (ATP) to the basal side of cell sheets activated both P2Y(1) and P2Y(2) receptors, inducing a transient increase in short-circuit current (I(sc)); addition of ATP to the apical side activated only P2Y(2) receptors, inducing first a transient and then a sustained increase in I(sc). The ATP-induced increases in I(sc) were blocked by pretreatment with a phospholipase C (PLC) inhibitor, a calcium (Ca(2+)) chelator, or Ca(2+)-activated Cl(-) channel (CACC) inhibitors, suggesting that ATP signals through both PLC and intracellular Ca(2+) to activate CACC. We propose that P2Y(1) and P2Y(2) receptors operate in tandem in IMCD cells to provide an adaptive mechanism for enhancing urinary NaCl excretion in the setting of high-dietary NaCl intake.

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

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

Normal Mucus Release Requires Both Ca2+‐mediated Exocytosis and cAMP Mediated HCO3‐ Transport

Mounting evidence indicates that the formation of normal mucus depends critically on the environment into which it is discharged. We applied histology, Ussing chamber pH-stat assays, and mucus collection methods to examine the links between mucin release and ion transport. In wild type mouse (C57BL/6) small intestine, the Ca2+ mediated agonist (carbachol) did not stimulate sustained HCO3− secretion, but induced mucin exocytosis (histologically) and a short circuit current (Isc) increase (possibly due to Cl− secretion). In contrast, cAMP mediated agonists (vasoactive intestinal peptide, isoproterenol) induced both HCO3− secretion and Isc increase, but little, if any, exocytosis. cAMP mediated agonists combined with carbachol discharged more mucus into the luminal perfusate than carbachol alone, but the cAMP enhancing effect was absent without HCO3−. In CF (ΔF508/ΔF508) mouse small intestine, neither Ca2+ nor cAMP mediated agonists induced significant HCO3− secretion, Isc increase or significant mucus discharge. These results indicate that normal mucus release requires Ca2+ mediated exocytosis, possibly Cl− secretion, and apparently post-exocytotic cAMP-mediated HCO3− secretion to affect normal mucin discharge. Concept: Mucus discharge requires Ca2+ mediated exocytosis + cAMP mediated HCO3− secretion. We thank the CFRI, CF Foundation, NIH-RO1 HL084042 and Dr. Hui Dong.

P2Y 2 receptor stimulation increases K secretion by activating K Ca 3.1 potassium channels in human mammary epithelial cells

Human mammary epithelial (HME) cells express several P2Y receptor subtypes located in both apical and basolateral membranes. Apical UTP (10 μM) or ATP-γ-S (10 μM) stimulation of monolayers mounted in Ussing chambers evoked a rapid, but transient decrease in short circuit current (Isc), consistent with activation of an apical K conductance. In contrast, basolateral stimulation with UTP (10 μM) produced a longer duration increase in Isc that was blocked by 5 μM benzamil. Inhibition of Ca2+ mobilization using BAPTA-AM abolished these changes in Isc. Moreover, apical pretreatment with charybdotoxin (200 nM), blocked the effect on Isc by >80% and a similar degree of inhibition was observed with clotrimazole (10 μM) and TRAM-34 (0.5 μM). Silencing expression of KCa3.1 by transfection of HME cells with an shRNA containing lentivirus construct targeting the channel produced ~80% inhibition of mRNA expression and 71% inhibition of the apical Isc response. In addition, silencing the P2Y2 receptor by expression of an shRNA containing lentivirus construct reduced the level of P2Y2 mRNA by 75% and inhibited the apical effects of 10 μM ATP-γ-S by 65%. These results suggest that P2Y2 receptors mediate the effects of purinoceptor agonists on K secretion by regulating the activity of KCa3.1 channels expressed in the apical membrane of HME cells.