Epithelial Cl Research Articles

All-trans Retinoic Acid Counteracts Diarrhea and Inhibition of Downregulated in Adenoma Expression in Gut Inflammation.

Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. All-trans retinoic acid (10 μM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1β and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.

Dexamethasone reduces airway epithelial Cl− secretion by rapid non-genomic inhibition of KCNQ1, KCNN4 and KATP K+ channels

Basolateral membrane K+ channels play a key role in basal and agonist stimulated Cl− transport across airway epithelial cells by generating a favourable electrical driving force for Cl− efflux. The K+ channel sub-types and molecular mechanisms of regulation by hormones and secretagoues are still poorly understood. Here we have identified the type of K+ channels involved in cAMP and Ca2+ stimulated Cl− secretion and uncovered a novel anti-secretory effect of dexamethasone mediated by inhibition of basolateral membrane K+ channels in a human airway cell model of 16HBE14o− cells commonly used for ion transport studies.Dexamethasone produced a rapid inhibition of transepithelial chloride ion secretion under steady state conditions and after stimulation with cAMP agonist (forskolin) or a Ca2+ mobilizing agonist (ATP). Our results show three different types of K+ channels are targeted by dexamethasone to reduce airway secretion, namely Ca2+-activated secretion via KCNN4 (KCa3.1) channels and cAMP-activated secretion via KCNQ1 (Kv7.1) and KATP (Kir6.1,6.2) channels. The down-regulation of KCNN4 and KCNQ1 channel activities by dexamethasone involves rapid non-genomic activation of PKCα and PKA signalling pathways, respectively. Dexamethasone signal transduction for PKC and PKA activation was demonstrated to occur through a rapid non-genomic pathway that did not implicate the classical nuclear receptors for glucocorticoids or mineralocorticoids but occurred via a novel signalling cascade involving sequentially a Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation.The rapid, non-genomic, effects of dexamethasone on airway epithelial ion transport and cell signalling introduces a new paradigm for glucocorticoid actions in lung epithelia which may serve to augment the anti-inflammatory activity of the steroid and enhance its therapeutic potential in treating airway hypersecretion in asthma and COPD.

MiR-125a-5p: a novel regulator of SLC26A6 expression in intestinal epithelial cells.

Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl-/ exchanger, also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed, Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore, we investigated the regulation of PAT-1 expression by microRNAs (miRNA), as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3'-untranslated region (UTR) revealed potential binding sites for several miRNAs, suggesting the role of miRNAs in modulating PAT1 expression. miRNAs showing highest context scores (125a-5p, 339-5p, 423-5p, 485-5p, and 501-3p) were selected as candidates for their effects on the activity of a 263-bp PAT-1 3'-untranslated region (UTR) fragment cloned into pmirGLO vector upstream of luciferase. The 3'-UTR activity was measured by dual luciferase reporter assay in Caco-2, T-84, HT-29, and SK-CO15 cells. Transient transfection of PAT-1 3'-UTR significantly decreased the relative luciferase activity compared with the empty vector suggesting binding of potential miRNA(s) to the PAT-1 3'-UTR. Among all the selected candidates, cotransfection with miRNA mimics 125a-5p and 423-5p further decreased PAT-1 3'-UTR activity. Furthermore, increasing miR-125a-5p abundance via mimic transfection in Caco-2 cells decreased both mRNA and protein levels of PAT-1. Our results demonstrate a novel regulatory mechanism of intestinal PAT-1 expression via miR-125a-5p that could be of therapeutic importance in disorders associated with decreased PAT-1 expression and function.

Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR.

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl− channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl− channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl− channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl− channels.

125Iodide as a Surrogate for 36Chloride in Tracing Transepithelial Intestinal Chloride Transport

Chloride (Cl−) transport by the large intestine is critically important, driving epithelial fluid secretion and absorption. Dysregulation of Cl− transport by the intestine results in diarrhea‐associated pathologies. Measurement of epithelial Cl− transport has long been achieved using the radioisotope 36Cl−. However, the price of 36Cl has sky‐rocketed, now costing over $100 per μCi compared to just $1.50 per μCi 15 years ago, making it prohibitively expensive and a barrier to future research. 125Iodide (125I−) has been used as an alternative to 36Cl− in some transport assays, including Cl− efflux and influx by cultured cells, but has never before been validated in parallel with 36Cl as an alternative for tracing transepithelial Cl− transport. The goal of this study was to examine whether 125I− can serve as a reliable surrogate for measuring Cl− transport by the intestinal epithelium. Under symmetrical short‐circuited conditions in the Ussing chamber, we simultaneously measured 36Cl− and 125I− fluxes across the cecum and distal colon from mice lacking the main Cl−/HCO3− exchanger, DRA (Slc26a3), relative to wild‐type controls. In addition, we used a cocktail of forskolin and 3‐isobutyl‐1‐methylxanthine (IBMX) to stimulate cAMP production and net Cl− secretion by these tissues. We found the unidirectional fluxes of 125I− correlated well with 36Cl− in all circumstances, even in the absence of DRA, with a significant R2 value of 0.86 across all fluxes, or higher (up to 0.93) when data was separated by genotype and intestinal segment. Unexpectedly, cAMP induced net Cl− secretion predominantly through a reduction in the absorptive, mucosal‐to‐serosal, Cl− flux rather than stimulating the secretory serosal‐to‐mucosal Cl− flux, which is noteworthy considering antidiarrheal drugs often target secretory processes. Also, rates of Cl− and I− absorption in DRA‐knockout mice were decreased by 79% and 50%, respectively, in the ceca, and 65% and 34% in the distal colon, suggesting this Cl− transporter may have a previously unrecognized role in I− uptake. In summary, we demonstrated 125I− is an adequate, cost‐effective alternative for tracing unidirectional Clfluxes across the intestinal epithelium. For example, the amount of 36Cl− used in this study cost $536 compared to a mere $4 worth of 125I−, thus making future research into epithelial Cltransport accessible. In addition, this study also provides insight into mechanisms of net Clsecretion and suggests that DRA may have a role in intestinal I− absorption.Support or Funding InformationThis study was supported by grants DK088892 and DK108755 to M. Hatch from the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

125 Iodide as a surrogate tracer for epithelial chloride transport by the mouse large intestine in vitro.

What is the central question of this study? The tracer 36 Cl- , currently used to measure transepithelial Cl- fluxes, has become prohibitively expensive, threatening its future use. 125 Iodide, previously validated alongside 36 Cl- as a tracer of Cl- efflux by cells, has not been tested as a surrogate for 36 Cl- across epithelia. What is the main finding and its importance? We demonstrate that 125 I- can serve as an inexpensive replacement for measuring Cl- transport across mouse large intestine, tracking Cl- transport in response to cAMP stimulation (inducing Cl- secretion) in the presence and absence of the main gastrointestinal Cl- -HCO3- exchanger, DRA. Chloride transport is important for driving fluid secretion and absorption by the large intestine, with dysregulation resulting in diarrhoea-associated pathologies. The radioisotope 36 Cl- has long been used as a tracer to measure epithelial Cl- transport but is prohibitively expensive. 125 Iodide has been used as an alternative to 36 Cl- in some transport assays but has never been validated as an alternative for tracing bidirectional transepithelial Cl- fluxes. The goal of this study was to validate 125 I- as an alternative to 36 Cl- for measurement of Cl- transport by the intestine. Simultaneous fluxes of 36 Cl- and 125 I- were measured across the mouse caecum and distal colon. Net Cl- secretion was induced by the stimulation of cAMP with a cocktail of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX). Unidirectional fluxes of 125 I- correlated well with 36 Cl- fluxes after cAMP-induced net Cl- secretion, occurring predominantly through a reduction in the absorptive mucosal-to-serosal Cl- flux rather than by stimulation of the secretory serosal-to-mucosal Cl- flux. Correlations between 125 I- fluxes and 36 Cl- fluxes were maintained in epithelia from mice lacking DRA (Slc26a3), the main Cl- -HCO3- exchanger responsible for Cl- absorption by the large intestine. Lower rates of Cl- and I- absorption in the DRA knockout intestine suggest that DRA might have a previously unrecognized role in iodide uptake. This study validates that 125 I- traces transepithelial Cl- fluxes across the mouse large intestine, provides insights into the mechanism of net Cl- secretion and suggests that DRA might be involved in intestinal iodide absorption.

Herbal dry extract BNO 1011 improves clinical and mucociliary parameters in a rabbit model of chronic rhinosinusitis.

Enhancing chloride (Cl- ) secretion in sinus epithelia represents a novel therapeutic approach to chronic rhinosinusitis (CRS). Herbal dry extract BNO 1011 enhances mucociliary clearance (MCC) via upregulation of Cl- secretion in sinonasal cultures in vitro and murine epithelium in vivo. The objective of this study is to evaluate whether the BNO 1011 improves MCC and clinical parameters in a rabbit model of CRS. After the development of CRS in 30 New Zealand white rabbits, animals were randomly assigned to receive oral placebo (n = 10), BNO 1011 (low dose [LD], 25 mg/kg/daily) (n = 10), or BNO1011 (high dose [HD], 125 mg/kg/daily) (n = 10) for 4 weeks. Outcomes included sinus opacification (Kerschner's rabbit sinus CT grade), maxillary epithelial Cl- secretion (sinus potential difference [PD] assay), airway surface liquid (ASL) depth using micro-optical coherence tomography (μOCT), and submucosal gland density (SMD) on histopathology. Outcome parameters were analyzed by 2 blinded investigators. BNO 1011 significantly cleared sinus opacification (HD = 1.21 ± 0.63, LD = 1.26 ± 0.37,) compared to placebo (4.02 ± 0.92) (p = 0.009). BNO 1011 resulted in markedly greater mean sinus PD polarization (HD = -12.23 ± 1.4 mV, LD = -12.0 ± 3.0 mV) when compared to rabbits treated with placebo (-4.1 ± 1.1 mV) (p = 0.03). ASL depth was significantly improved when treated with HD (4.08 ± 0.06 μm) and LD (4.05 ± 0.06 μm) compared to placebo (3.5 ± 0.05 μm) (post hoc analysis, p < 0.0001). Histologically, epithelial thickness (HD = 10.0 ± 0.7 μm; LD = 13.7 ± 0.9 μm; placebo = 21.1 ± 2.3 μm; p < 0.005), subepithelial thickness (HD = 63.1 ± 6.6 μm; LD = 103.2 ± 6.7 μm; placebo = 113.3 ± 6.0 μm; p < 0.001), and SMD (HD = 22.2 ± 2.9%; LD = 31.8 ± 1.1%; placebo = 43.8 ± 1.7%; p < 0.0001) were noticeably better with the HD. Herbal dry extract BNO 1011 improves radiographic, histologic, and MCC parameters in a rabbit model of CRS.

Quercetin is a Useful Medicinal Compound Showing Various Actions Including Control of Blood Pressure, Neurite Elongation and Epithelial Ion Transport.

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.

ADPKD cell proliferation and Cl--dependent fluid secretion.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by bilateral fluid-filled cysts, renal inflammation and extensive fibrosis, leading to the progressive decline in kidney function. Renal cyst formation begins in utero from aberrant proliferation of tubule epithelial cells; however, the mechanisms for cystogenesis remain unclear. Cell proliferation and Cl--dependent fluid secretion, which drives the accumulation of cyst fluid, are responsible for inexorable growth of cysts and the remarkable appearance of massively enlarged ADPKD kidneys. Investigators have used in vitro assays to explore cellular and molecular mechanisms involved in ADPKD cyst epithelial cell proliferation and Cl--dependent fluid secretion in experimentally controlled environments. These assays have been used to evaluate potential therapeutic approaches to inhibit cellular pathways involved in cyst growth. This chapter discusses methods for measuring ADPKD cell proliferation, transepithelial Cl- secretion, and net fluid transport across cyst epithelial cell monolayers.

Glutathione S-Transferase π-Activatable O2-(Sulfonylethyl Derived) Diazeniumdiolates Potently Suppress Melanoma in Vitro and in Vivo.

A group of glutathione S-transferase π (GSTπ) activatable O2-(sulfonylethyl derived) diazeniumdiolates 5-12 were designed and synthesized. These compounds could be activated by GSTπ to initiate the β-elimination reaction, liberating an active vinyl sulfone-based GSH derivative and a diazeniumdiolate anion which subsequently releases NO in situ. The most active compound 6 released relatively high levels of NO and inhibited proliferation of melanoma B16 cells, superior to a diazeniumdiolate-based anticancer agent JS-K (1). Importantly, 6 had 8-fold less inhibitory activity against normal epithelial JB6 Cl 30-7b cells. The inhibitory activity of 6 could be diminished by an NO scavenger or GSTπ inhibitor. Furthermore, 6 induced nitration of mitochondrial tyrosine in B16 cells and inoculated B16 tumors in mice, which might be responsible for oxidation of protein leading to tumor suppression. Finally, 6 significantly retarded the B16 cells growth in a mouse xenograft model. These findings suggest that 6 may have a potential to treat melanoma.

Mucosal glucagon‐like peptide 1 (GLP‐1) responses are mediated by calcitonin gene‐related peptide (CGRP) in the mouse colon and both peptide responses are area‐specific

Glucagon-like peptide (GLP)-1 is an incretin hormone and its mimetics are proven antidiabetic and antiobesity drugs. GLP-1 exerts antimotility and mucosal proliferative activities but its epithelial ion transport effects are uncharacterized and these may contribute to the gastrointestinal (GI) disturbance, i.e., diarrhea experienced with some GLP-1 mimetics. Our aim was to establish GLP-1 agonist mechanisms and identify potential mucosal mediator(s) in the colonic tissue from C57BL/6J mice. A tissue survey of GLP-1 responses (using exendin 4, Ex4) and α-calcitonin gene-related peptide (αCGRP) was undertaken, dividing the mouse colon into eight adjacent mucosal-submucosal preparations. Each preparation was voltage-clamped and changes in short-circuit current (Isc) measured. The involvement of submucosal neurons in GLP-1 agonism was tested using Ex(9-39) and tetrodotoxin (TTX), and CGRP receptors were blocked with BIBN4094. Ex4 responses along the length of the colon were inhibited by the GLP-1 antagonist, Ex(9-39) or TTX, indicating neural mediation in all colonic regions. In the ascending colon, Ex4 increased Isc levels that were abolished by 10nM BIBN4096, while in the descending colon it reduced Isc levels that were again BIBN4096-sensitive, but at 1μM. The latter αCGRP response was dependent on epithelial Cl- conductance and Na+ /K+ -ATPase, and was partially (~25%) peptide YY-mediated, but was not nitrergic, somatostatin sst2 , or α2 -adrenoceptor-mediated. GLP-1 modulates epithelial ion transport indirectly by activating CGRP-containing submucosal enteric neurons in the mouse colon. This GLP-1-CGRP response was area-specific and could potentially contribute to the diarrheal side effect of certain GLP-1R therapeutics.

Actions of quercetin, a flavonoid, on ion transporters: its physiological roles.

Flavonoids keep us healthy by controlling various body and cellular functions. It is well known that cations, such as Na+ , K+ , and Ca2+ , play important roles in the regulation of body and cellular functions, including generation of action potentials and the resting membrane potential of neural and muscle cells and signal transduction as intracellular second messengers. However, we have little information on the physiological roles of anions, particularly Cl- , in body and cellular functions. Quercetin, a flavonoid, stimulates Na+ -K+ -2Cl- cotransporter 1 (NKCC1), which is one of the most important ion transporters regulating the cytosolic Cl- concentration ([Cl- ]c ). Here, we introduce the molecular mechanism by which flavonoids, specifically quercetin, act through elevation of [Cl- ]c via activation of NKCC1 on important factors controlling various body and cellular functions, such as (1) antihypertensive actions controlling blood volume dependent on the amounts of renal Na+ reabsorption via expression of the epithelial Na+ channel, (2) neurite-elongating actions via polymerization of tubulin by inhibiting GTPase activity, and (3) antibacterial and antiviral infective actions through stimulation of epithelial Cl- secretion by increasing the driving force for epithelial Cl- secretion.

Stimulation of Na+ -K+ -pump currents by epithelial nicotinic receptors in rat colon.

Acetylcholine-induced epithelial Cl- secretion is generally thought to be mediated by epithelial muscarinic receptors and nicotinic receptors on secretomotor neurons. However, recent data have shown expression of nicotinic receptors by intestinal epithelium and the stimulation of Cl- secretion by nicotine, in the presence of the neurotoxin, tetrodotoxin. Here, we aimed to identify the transporters activated by epithelial nicotinic receptors and to clarify their role in cholinergic regulation of intestinal ion transport. Ussing chamber experiments were performed, using rat distal colon with intact epithelia. Epithelia were basolaterally depolarized to measure currents across the apical membrane. Apically permeabilized tissue was also used to measure currents across the basolateral membrane in the presence of tetrodotoxin. Nicotine had no effect on currents through Cl- channels in the apical membrane or on currents through K+ channels in the apical or the basolateral membrane. Instead, nicotine stimulated the Na+ -K+ -pump as indicated by Na+ -dependency and sensitivity of the nicotine-induced current across the basolateral membrane to cardiac steroids. Effects of nicotine were inhibited by nicotinic receptor antagonists such as hexamethonium and mimicked by dimethyl-4-phenylpiperazinium, a chemically different nicotinic agonist. Simultaneous stimulation of epithelial muscarinic and nicotinic receptors led to a strong potentiation of transepithelial Cl- secretion. These results suggest a novel concept for the cholinergic regulation of transepithelial ion transport by costimulation of muscarinic and nicotinic epithelial receptors and a unique role of nicotinic receptors controlling the activity of the Na+ -K+ -ATPase.

Chenodeoxycholic acid requires activation of EGFR, EPAC, and Ca2+ to stimulate CFTR-dependent Cl- secretion in human colonic T84 cells.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl- secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+ In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+ were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.

WS06.2 Altered epithelial Cl− secretion in primary human nasal epithelial cells of patients with chronic rhinosinusitis

WS06.2 Altered epithelial Cl− secretion in primary human nasal epithelial cells of patients with chronic rhinosinusitis

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Desacetyl bisacodyl-induced epithelial Cl(-) secretion in rat colon and rectum.

The purpose of this study was to clarify the mode of desacetyl bisacodyl (DAB)-induced secretory action in intestinal tissues using an Ussing chamber assay. DAB is the active metabolite of the laxative bisacodyl. In mucosal-submucosal preparations, mucosal application of DAB induced a transient decrease followed by subsequent increases in short-circuit current and tissue conductance in a concentration-dependent manner. DAB-induced responses occurred from the middle colon to the rectal segment but not in the proximal colon. Moreover, these responses were not observed under chloride (Cl(-))-free conditions or in the presence of DAB on the serosal side of the mucosalsubmucosal specimens. Treatment with tetrodotoxin had no effect on the DAB-induced responses; however, mucosal treatment with a COX inhibitor piroxicam resulted in the elimination of responses. These results suggest that DAB may contribute to the laxative action by inducing Cl(-) secretion which is associated with the COX signaling pathway. This study also demonstrated that the DAB target molecule is present on the mucosal side from the middle colon to the rectal segment.

Prevention of secretory diarrhea by ethanol extract of &lt;i&gt;Bistortae rhizoma&lt;/i&gt; through inhibition of chloride channel

&lt;p&gt;Inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca&lt;sup&gt;2+&lt;/sup&gt;-activated Cl&lt;sup&gt;-&lt;/sup&gt; channel (CaCC) represents an attractive approach for the treatment of secretory diarrhea. The aim of the study is to investigate the molecular basis of the anti-diarrheal effect of traditional Chinese herbal anti-diarrheal medicine &lt;em&gt;Bistortae rhizoma&lt;/em&gt;. Fluorescence quenching assay indicated that the 40% methanol /water fraction (D5) dose-dependently inhibited both CFTR and CaCC function in transfected Fischer rat thyroid (FRT) cells. Ex vivo studies indicated that D5 inhibited both forskolin (FSK)-activated CFTR current and CCh-induced CaCC current in rat colonic mucosa. In the mouse closed-loop model, intraluminal application of D5 (200 µg/mL) significantly reduced cholera toxin-stimulated fluid secretion. In the intestinal motility model, D5 significantly delayed intestinal peristalsis in mice. Our research suggests that CFTR and CaCC-mediated intestinal epithelial Cl&lt;sup&gt;-&lt;/sup&gt; secretion inhibiting and gastrointestinal motility delaying may account for the anti-diarrheal activity of &lt;em&gt;B. rhizoma&lt;/em&gt;. &lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Alternatively activated macrophages attenuate influenza-induced acute lung injury in mice heterozygous for the F508del mutation in CFTR. (INC6P.314)

Abstract Infection with influenza A virus can result in acute lung injury (ALI), characterized by pulmonary edema and hypoxemia. We found that influenza-induced ALI in C57BL/6 (WT) mice was associated with increased bronchoalveolar epithelial cell Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Influenza-induced ALI was highly attenuated in C57BL/6-congenic mice that are heterozygous for the F508del CFTR mutation (HETs), which exhibit a 50% reduction in CFTR expression and CFTR-mediated Cl- transport. This beneficial phenotype was alveolar macrophage (AM)-dependent and was not due to differences in viral replication between mouse strains. The aim of this study was to evaluate the role of HET AMs in the attenuation of influenza-induced ALI. We hypothesized that ALI attenuation in influenza-infected HET mice results from “alternative” activation (M2 polarization) of HET AMs in response to infection. Compared to WT AMs, Arginase 1 (Arg1) protein expression was higher while inducible nitric oxide synthase (iNOS) protein was lower in HET AMs following infection. Arg1 enzymatic activity was also higher in HET AM lysates. BALF from HET mice contained higher levels of urea and lower levels of nitrate/nitrite (end-products of Arg1 and iNOS, respectively) than WT. These findings indicate that reduced CFTR expression/activity promotes an anti-inflammatory, M2 AM polarization response to influenza infection, which contributes to ALI attenuation.

Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating.

Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. Cross-species comparative studies are a powerful approach to understanding the epithelial Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl(-) channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxineB failed to potentiate ovine CFTR Cl(-) currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl(-) channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.