Cholera Toxin-induced Intestinal Fluid Secretion Research Articles

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,β-dehydrolovastatin (DHLV), α,β-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 μM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 μM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 μM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

Identification of Fungus‐derived Compounds as a Novel CFTR inhibitor: Mechanism and Potential Anti‐diarrheal Application

Secretory diarrhea, which has been considered as a health burden worldwide, could be alleviated by inhibiting fluid secretion. CFTR is considered to be a drug target for development of anti‐secretory agent. However, there is no currently FDA‐approval drug targeting CFTR. This study aimed to identify CFTR inhibitors from a library of fungal bioactive metabolites found in Thailand. Electrophysiological analyses were used for identifying the inhibitory effect of 40 compounds. Three active compounds with the best one being DHLV, which effectively inhibited CFTR‐mediated Clˉ secretion with low micromolar potency (IC50 ~1.8 μM) in T84 cells without effect on Na+‐K+ ATPase activity. In addition, DHLV inhibited Ca2+‐activated Clˉ channels stimulated by ATP. In vivo experiments showed that administration of DHLV by intraperitoneal route (2 mg/kg) and intraluminal (22 µg/kg or 20 µM) injections reduced cholera toxin‐induced intestinal fluid secretion in mice by ∼46.7% and ∼42.8%, respectively without affecting intestinal fluid absorption. This study indicates that the novel CFTR inhibitor, DHLV, is of potential utility in the treatment of secretory diarrhea.

Activation of AMPK by chitosan oligosaccharide in intestinal epithelial cells: Mechanism of action and potential applications in intestinal disorders

Chitosan oligosaccharide (COS), a biomaterial derived from chitin, is absorbed by intestinal epithelia without degradation and has diverse biological activities including intestinal epithelial function. However, the mode of action is still unclear. This study aimed to investigate the effect of COS on AMP-activated protein kinase (AMPK) in intestinal epithelial cells (IEC) and its potential applications in the intestinal diseases benefited from AMPK activation. COS with molecular weights (MW) from 5,000Da to 14,000Da induced AMPK activation in T84 cells. That with MW of 5,000-Da was the most potent polymer and was used in the subsequent experiments. COS also activated AMPK in other IEC including HT-29 and Caco-2 cells. Mechanism of COS-induced AMPK activation in T84 cells involves calcium-sensing receptor (CaSR)-phospholipase C (PLC)-IP3 receptor channel-mediated calcium release from endoplasmic reticulum (ER). In addition, COS promoted tight junction assembly in T84 cells in an AMPK-dependent manner. COS also inhibited NF-κB transcriptional activity and NF-κB-mediated inflammatory response and barrier disruption via AMPK-independent mechanisms. Interestingly, luminal exposure to COS suppressed cholera toxin-induced intestinal fluid secretion by ∼30% concurrent with AMPK activation in a mouse closed loop model. Importantly, oral administration of COS prevented the development of aberrant crypt foci in a mouse model of colitis-associated colorectal cancer (CRC) via a mechanism involving AMPK activation-induced β-catenin suppression and caspase-3 activation. Collectively, this study reveals a novel action of COS in activating AMPK via CaSR-PLC-IP3 receptor channel-mediated calcium release from ER. COS may be beneficial in the treatment of secretory diarrheas and CRC chemoprevention.

Bioactivity-guided fractionation of an antidiarrheal Chinese herb Rhodiola kirilowii (Regel) Maxim reveals (-)-epicatechin-3-gallate and (-)-epigallocatechin-3-gallate as inhibitors of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the principal apical route for transepithelial fluid transport induced by enterotoxin. Inhibition of CFTR has been confirmed as a pharmaceutical approach for the treatment of secretory diarrhea. Many traditional Chinese herbal medicines, like Rhodiola kirilowii (Regel) Maxim, have long been used for the treatment of secretory diarrhea. However, the active ingredients responsible for their therapeutic effectiveness remain unknown. The purpose of this study is to identify CFTR inhibitors from Rhodiola kirilowii (Regel) Maxim via bioactivity-directed isolation strategy. We first identified fractions of Rhodiola kirilowii (Regel) Maxim that inhibited CFTR Cl- channel activity. Further bioactivity-directed fractionation led to the identification of (-)–epigallocatechin-3-gallate (EGCG) as CFTR Cl- channel inhibitor. Analysis of 5 commercially available EGCG analogs including (+)–catechins (C), (-)–epicatechin (EC), (-)–epigallocatechin (EGC), (-)–epicatechin-3-gallate (ECG) and EGCG revealed that ECG also had CFTR inhibitory activity. EGCG dose-dependently and reversibly inhibited CFTR Cl- channel activity in transfected FRT cells with an IC50 value around 100 μM. In ex vivo studies, EGCG and ECG inhibited CFTR-mediated short-circuit currents in isolated rat colonic mucosa in a dose-dependent manner. In an intestinal closed-loop model in mice, intraluminal application of EGCG (10 μg) and ECG (10 μg) significantly reduced cholera toxin-induced intestinal fluid secretion. CFTR Cl- channel is a molecular target of natural compounds EGCG and ECG. CFTR inhibition may account, at least in part, for the antidiarrheal activity of Rhodiola kirilowii (Regel) Maxim. EGCG and ECG could be new lead compounds for development of CFTR-related diseases such as secretory diarrhea.

Identification of Resveratrol Oligomers as Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator by High-Throughput Screening of Natural Products from Chinese Medicinal Plants

Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) have been widely used for characterizing CFTR function in epithelial fluid transport and in diseases such as secretory diarrhea, polycystic kidney disease and cystic fibrosis. Few small molecule CFTR inhibitors have been discovered so far from combinatorial compound library. In the present study, we used a high throughput screening (HTS)-based natural product discovery strategy to identify new CFTR inhibitors from Chinese medicinal herbs. By screening 40,000 small molecule fractions from 500 herbal plants, we identified 42 positive fractions from 5 herbs and isolated two compounds that inhibited CFTR conductance from Chinese wild grapevine (Vitis amurensis Rupr). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) studies determined the two active compounds as trans-ε-viniferin (TV) and r-2-viniferin (RV), respectively. Both compounds dose-dependently blocked CFTR-mediated iodide influx with IC50 around 20 μM. Further analysis by excised inside-out patch-clamp indicated strong inhibition of protein kinase A (PKA)-activated CFTR chloride currents by TV and RV. In ex vivo studies, TV and RV inhibited CFTR-mediated short-circuit Cl− currents in isolated rat colonic mucosa in a dose-dependent manner. In a closed-loop mouse model, intraluminal applications of TV (2.5 μg) and RV (4.5 μg) significantly reduced cholera toxin–induced intestinal fluid secretion. The present study identified two resveratrol oligomers as new CFTR inhibitors and validates our high-throughput screening method for discovery of bioactive compounds from natural products with complex chemical ingredients such as herbal plants.

Antidiarrheal Efficacy and Cellular Mechanisms of a Thai Herbal Remedy

Screening of herbal remedies for Cl− channel inhibition identified Krisanaklan, a herbal extract used in Thailand for treatment of diarrhea, as an effective antidiarrheal in mouse models of secretory diarrheas with inhibition activity against three Cl− channel targets. Krisanaklan fully inhibited cholera toxin-induced intestinal fluid secretion in a closed-loop mouse model with ∼50% inhibition at a 1∶50 dilution of the extract. Orally administered Krisanaklan (5 µL/g) prevented rotavirus-induced diarrhea in neonatal mice. Short-circuit current measurements showed full inhibition of cAMP and Ca2+ agonist-induced Cl− conductance in human colonic epithelial T84 cells, with ∼50% inhibition at a 1∶5,000 dilution of the extract. Krisanaklan also strongly inhibited intestinal smooth muscle contraction in an ex vivo preparation. Together with measurements using specific inhibitors, we conclude that the antidiarrheal actions of Krisanaklan include inhibition of luminal CFTR and Ca2+-activated Cl− channels in enterocytes. HPLC fractionation indicated that the three Cl− inhibition actions of Krisanaklan are produced by different components in the herbal extract. Testing of individual herbs comprising Krisanaklan indicated that agarwood and clove extracts as primarily responsible for Cl− channel inhibition. The low cost, broad antidiarrheal efficacy, and defined cellular mechanisms of Krisanaklan suggests its potential application for antisecretory therapy of cholera and other enterotoxin-mediated secretory diarrheas in developing countries.

Activation of AMP-Activated Protein Kinase by a Plant-Derived Dihydroisosteviol in Human Intestinal Epithelial Cell

Our previous study has shown that dihydroisosteviol (DHIS), a derivative of stevioside isolated from Stevia rebaudiana (Bertoni), inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial chloride secretion across monolayers of human intestinal epithelial (T84) cells and prevents cholera toxin-induced intestinal fluid secretion in mouse closed loop models. In this study, we aimed to investigate a mechanism by which DHIS inhibits CFTR activity. Apical chloride current measurements in Fisher rat thyroid cells stably transfected with wild-type human CFTR (FRT-CFTR cells) and T84 cells were used to investigate mechanism of CFTR inhibition by DHIS. In addition, effect of DHIS on AMP-activated protein kinase (AMPK) activation was investigated using Western blot analysis. Surprisingly, it was found that DHIS failed to inhibit CFTR-mediated apical chloride current in FRT-CFTR cells. In contrast, DHIS effectively inhibited CFTR-mediated apical chloride current induced by a cell permeable cAMP analog CPT-cAMP and a direct CFTR activator genistein in T84 cell monolayers. Interestingly, this inhibitory effect of DHIS on CFTR was significantly (p<0.05) reduced by pretreatment with compound C, an AMPK inhibitor. AICAR, a known AMPK activator, was able to inhibit CFTR activity in both FRT-CFTR and T84 cells. Western blot analysis showed that DHIS induced AMPK activation in T84 cells, but not in FRT-CFTR cells. Our results indicate that DHIS inhibits CFTR-mediated chloride secretion in T84 cells, in part, by activation of AMPK activity. DHIS therefore represents a novel candidate of AMPK activators.

Hydroxyxanthone as an inhibitor of cAMP-activated apical chloride channel in human intestinal epithelial cell

AimsPrevious investigation showed that polyphenols abundantly found in many plants could inhibit Cl− secretion. The present study was aimed to investigate the effect of phenol containing xanthone derivatives on cAMP-activated intestinal Cl− secretion and evaluate potential benefits of these compounds in the treatment of cholera. Main methodsFour hydroxy xanthones were synthesized via oxidative coupling reaction of the corresponding ortho-hydroxybenzoic acids and hydroxyphenols. Short-circuit current and apical Cl− current measurements across monolayers of human intestinal epithelial (T84) cell and Fisher rat thyroid cells transfected with human CFTR (FRT-hCFTR cell) were performed to determine the effect of hydroxyxanthones on cAMP-activated Cl− secretion. Intracellular cAMP was measured by immunoassay methods. Anti-diarrheal efficacy was evaluated using closed loop model of cholera. Key findingsAmong the tested xanthones, 1,3,6-trihydroxyxanthone (THX-001) was found to be the most potent derivative in the inhibition of cAMP-activated Cl− secretion across T84 cell monolayers (IC50~100μM). Electrophysiological analysis of T84 cells and FRT-hCFTR cells revealed that THX-001 targeted two distinct cAMP-activated Cl− channels in the apical membrane of T84 cells, namely, CFTR and inward rectifying Cl− channel (IRC). In contrast, THX-001 had no effect on intracellular cAMP levels in these cells. Importantly, THX-001 completely abolished cholera toxin-induced Cl− secretion across T84 cell monolayers and significantly inhibited cholera toxin-induced intestinal fluid secretion in mouse closed loop models. SignificanceThis study revealed that hydroxyxanthone represents another chemical class of polyphenolic compounds that may hold promise as anti-secretory therapy for cholera.

Novel Action of the Chalcone Isoliquiritigenin as a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibitor: Potential Therapy for Cholera and Polycystic Kidney Disease

Overstimulation of cAMP-activated Cl− secretion can cause secretory diarrhea. Isoliquiritigenin (ISLQ) is a plant-derived chalcone that has a wide range of biological activities. The present study thus aimed to investigate the effect of ISLQ on cAMP-activated Cl− secretion in human intestinal epithelium, especially the underlying mechanism and therapeutic application. Short-circuit current analysis of human intestinal epithelial (T84) cell monolayers revealed that ISLQ dose-dependently inhibited cAMP-activated Cl− secretion with an IC50 of approximately 20 μM. ISLQ had no effect on either basal short-circuit current or Ca2+-activated Cl− secretion. Apical Cl− current analysis of T84 cell monolayers indicated that ISLQ blocked mainly the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, but not other unidentified cAMP-dependent Cl− channels. ISLQ did not affect intracellular cAMP levels or cell viability. ISLQ completely abolished the cholera toxin-induced transepithelial Cl− secretion in T84 cells and reduced the cholera toxin-induced intestinal fluid secretion in mouse closed loop models by 90%. Similarly, ISLQ completely inhibited the cAMP-activated apical Cl− current across monolayers of Madin-Darby Canine Kidney (MDCK) cells and retarded cyst growth in MDCK cyst models by 90%. This study reveals a novel action of ISLQ as a potent CFTR inhibitor with therapeutic potential for treatment of cholera and polycystic kidney disease.

A Tannic Acid-Based Medical Food, Cesinex®, Exhibits Broad-Spectrum Antidiarrheal Properties: A Mechanistic and Clinical Study

The purpose of this investigation was to evaluate the efficacy and tolerability of a tannic acid-based medical food, Cesinex(®), in the treatment of diarrhea and to investigate the mechanisms underlying its antidiarrheal effect. Cesinex(®) was prescribed to six children and four adults with diarrhea. Patient records were retrospectively reviewed for the primary outcome. Cesinex(®) and its major component, tannic acid, were tested for their effects on cholera toxin-induced intestinal fluid secretion in mice. Polarized human gut epithelial cells (HT29-CL19A cells) were used to investigate the effects of tannic acid on epithelial barrier properties, transepithelial chloride secretion, and cell viability. Successful resolution of diarrheal symptoms was reported in nine of ten patients receiving Cesinex(®). The treatment of HT29-CL19A cells with clinically relevant concentrations of tannic acid (0.01-1 mg/ml) significantly increased transepithelial resistance (TER) and inhibited the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent or the calcium-activated Cl(-) secretion. Tannic acid could also improve the impaired epithelial barrier function induced by tumor necrosis factor alpha (TNFα) and inhibited the disrupting effect of TNFα on the epithelial barrier function in these cells. Cholera toxin (CTX)-induced mouse intestinal fluid secretion was significantly reduced by the administration of Cesinex(®) or tannic acid. Cesinex(®) has high antioxidant capacity. Cesinex(®) demonstrates efficacy and a good safety profile in the treatment of diarrhea. The broad-spectrum antidiarrheal effect of Cesinex(®) can be attributed to a combination of factors: its ability to improve the epithelial barrier properties, to inhibit intestinal fluid secretion, and the high antioxidant capacity.

A Plant-Derived Hydrolysable Tannin Inhibits CFTR Chloride Channel: A Potential Treatment of Diarrhea

The present study examined the effects and mechanisms of actions of penta-m-digalloyl-glucose (PDG), a hydrolysable tannin extracted from Chinese gallnut, on cystic fibrosis transmembrane conductance regulator protein (CFTR). Fisher rat thyroid cells stably expressing human CFTR (FRT cells) and human intestinal T84 cells were used as cell models to investigate the effects of PDG on chloride secretion using short-circuit current analysis. The mechanisms by which PDG affected chloride secretion were also examined. Finally, in vivo antidiarrheal efficacy and effects of PDG on intestinal fluid absorption were evaluated in mouse closed-loop models. In FRT cells, apical chloride current induced by forskolin, CPT-cAMP and apigenin were reversibly inhibited by PDG (IC50 approximately 10microM) without effects on intracellular cAMP content and cell viability. Similarly, in T84 cells, PDG effectively inhibited chloride secretion induced by forskolin and cholera toxin. However, it had no effect on calcium-induced chloride secretion. In mice, a single intraluminal injection of PDG (0.6 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 75% with no effect on intestinal fluid absorption. PDG represents a new class of CFTR inhibitors. Further development of this class of compounds may provide a new therapeutic intervention for diarrhea.

Thiazolidinone CFTR inhibitors with improved water solubility identified by structure–activity analysis

The thiazolidinone 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR inh-172) inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel conductance with submicromolar affinity and blocks cholera toxin-induced intestinal fluid secretion. Fifty-eight CFTR inh-172 analogs were synthesized to identify CFTR inhibitors with improved water solubility, exploring modifications in its two phenyl rings, thiazolidinone core, and core-phenyl connectors. Greatest CFTR inhibition potency was found for 3-CF 3 and polar group-substituted-phenyl rings, and a thiazolidinone core. Two compounds with ∼1 μM CFTR inhibition potency and solubility >180 μM (>10-fold more than CFTR inh-172) were identified: Tetrazolo-172, containing 4-tetrazolophenyl in place of 4-carboxyphenyl, and Oxo-172, containing thiazolidinedione in place of the thiazolidinone core. These water soluble thiazolidinone analogs had low cellular toxicity. The improved water solubility of Tetrazolo- and Oxo-172 make them potential lead candidates for therapy of secretory diarrheas and polycystic kidney disease.

A Natural Plant-Derived Dihydroisosteviol Prevents Cholera Toxin-Induced Intestinal Fluid Secretion

Stevioside and its major metabolite, steviol, have been reported to affect ion transport in many types of tissues, such as the kidney, pancreas, and intestine. The effect of stevioside, steviol, and its analogs on intestinal Cl(-) secretion was investigated in a human T84 epithelial cell line. Short-circuit current measurements showed that steviol and analogs isosteviol, dihydroisosteviol, and isosteviol 16-oxime inhibited in a dose-dependent manner forskolin-induced Cl(-) secretion with IC(50) values of 101, 100, 9.6, and 50 microM, respectively, whereas the parent compound stevioside had no effect. Apical Cl(-) current measurement indicated that dihydroisosteviol targeted the cystic fibrosis transmembrane regulator (CFTR). The inhibitory action of dihydroisosteviol was reversible and was not associated with changes in the intracellular cAMP level. In addition, dihydroisosteviol did not affect calcium-activated chloride secretion and T84 cell viability. In vivo studies using a mouse closed-loop model of cholera toxin-induced intestinal fluid secretion showed that intraluminal injection of 50 microM dihydroisosteviol reduced intestinal fluid secretion by 88.2% without altering fluid absorption. These results indicate that dihydroisosteviol and similar compounds could be a new class of CFTR inhibitors that may be useful for further development as antidiarrheal agents.

Lectin Conjugates as Potent, Nonabsorbable CFTR Inhibitors for Reducing Intestinal Fluid Secretion in Cholera

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to prevent intestinal fluid secretion in cholera. We previously discovered low- affinity glycine hydrazide (GlyH) CFTR inhibitors that block CFTR at its external pore. The goal of this study was to develop potent CFTR inhibitors that are minimally absorbed and washed out of the intestinal lumen for application as antisecretory agents in cholera. GlyH analogs (malonic hydrazides, MalH) were chemically conjugated to various lectins ("MalH-lectin") and purified. CFTR inhibition potency was measured by short-circuit current analysis, mechanism of action by patch-clamp, and antidiarrheal efficacy in closed-loop and suckling mouse models. By lectin conjugation, we improved CFTR inhibitory potency by approximately 100-fold (to 50 nmol/L) and retarded washout. High-affinity CFTR inhibition was abolished by MalH-lectin heat denaturation, protease digestion, or competition by mannose or unconjugated lectin. Patch-clamp analysis indicated CFTR inhibition by an external pore occlusion mechanism. Fluorescently labeled MalH-lectin remained membrane bound for >6 hours after washout, whereas washout occurred in a few minutes without the lectin. MalH-ConA and MalH-wheat (IC50 50-100 pmol) blocked cholera toxin-induced intestinal fluid secretion in closed intestinal loops in mice and greatly reduced mortality in a suckling mouse model of cholera. The high potency of MalH-lectin conjugates results from "anchoring" the CFTR-blocking MalH to cell surface carbohydrates by the lectin. The high-affinity, slow washout, and external site of action of the MalH-lectin conjugates support their further development as antisecretory drugs for enterotoxin-mediated secretory diarrheas.

Identification of New Small Molecule Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Protein: In Vitro and in Vivo Studies

Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated chloride channel that has been proposed as a pharmacological target to reduce intestinal fluid loss in cholera. The aim of this study was to identify new CFTR inhibitors by high-throughput screening. Screening of 50,000 drug-like small molecules was performed using a cell-based assay of iodide influx in Fisher rat thyroid (FRT) cells co-expressing human CFTR and halide-sensitive yellow fluorescent protein (YFP-H148Q). Two new CFTR inhibitors, 2-[N-(3-hydroxy-4-carboxyphenyl) amino]-4-(4-methylphenyl)-thiazole (INH 1) and 1-acetyl-5-bromo-2,3-dihydro-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1H-Indole-7-sulfonamide (INH 2), were identified. They were then determined for potency, reversibility and specificity by electrophysiological methods, and for in vivo efficacy in mouse model of cholera toxin-induced intestinal fluid secretion. INH 1 and INH 2 reversibly inhibited cAMP-activated apical chloride current in FRT cells with Kis of 15 and 20 microM, respectively. Similarly, in short-circuit current analysis in human colonic epithelial cell lines (T84 cells), cAMP-activated chloride secretion was inhibited by INH 1 and INH 2 with Kis of 24.5 and 25.3 microM, respectively. Calcium-activated chloride secretion in the T84 cells was markedly inhibited by 100 microM of INH 1, but was unaffected by 100 microM of INH 2. In vivo studies in mice showed that a single intraperitoneal injection of INH 1 (3 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 40%, whereas INH 2 produced no effect. Our results indicate that INH 1 could be a new class candidate for a blocker of cholera toxin-induced intestinal fluid secretion as well as a CFTR inhibitor.

Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor

Background & Aims: The cystic fibrosis transmembrane conductance regulator (CFTR) provides an important apical route for Cl − secretion across intestinal epithelia. A thiazolidinone-type CFTR blocker (CFTR inh-172) reduced cholera toxin-induced fluid accumulation in mouse intestinal loops. Here, we characterize the efficacy and pharmacodynamics of CFTR inh-172 in blocking cAMP and cGMP induced Cl −/fluid secretion in rodent and human intestine. Methods & Results: CFTR inh-172 inhibited cAMP and cGMP agonist induced short-circuit current by >95% in T84 colonic epithelial cells (K I ∼ 3 μmol/L) and in mouse and human intestinal sheets (K I ∼ 9 μmol/L). A single intraperitoneal injection of CFTR inh-172 (200 μg) blocked intestinal fluid secretion in a rat closed-loop model by >90% for cholera toxin and >70% for STa Escherichia coli toxin. In mice, CFTR inh-172 (20 μg) inhibited cholera toxin-induced intestinal fluid secretion by 90% (persistence t 1/2 ∼10 hours, K I ∼ 5 μg) and STa toxin by 75% (K I ∼ 10 μg). Tissue distribution and pharmacokinetic studies indicated intestinal CFTR inh-172 accumulation facilitated by enterohepatic circulation. An oral CFTR inh-172 preparation reduced fluid secretion by >90% in a mouse open-loop cholera model. Conclusions: A small molecule CFTR blocker markedly reduced intestinal ion and fluid secretion caused by cAMP/cGMP-dependent bacterial enterotoxins. CFTR inhibition may thus reduce fluid secretion in infectious secretory diarrheas.

Effects of Hange-shashin-to on cholera toxin-induced fluid secretion in the small intestine of rats.

The effects of Hange-shashin-to (TJ-14) on cholera toxin-induced intestinal fluid secretion were studied to elucidate the mechanism by which this kampo medicine manifests antidiarrheal effects. TJ-14 suppressed the intestinal fluid secretion induced by cholera toxin (1 microg/rat) in a dose-dependent manner at doses between 125 and 1000 mg/kg. It also inhibited the luminal prostaglandin E2 (PGE2) level. On the other hand, serotonin (5-HT) release was not affected by TJ-14. Subcutaneous injection of indomethacin at 10 mg/kg or ondansetron at 100 microg/kg significantly suppressed intestinal secretion. The luminal PGE2 level was also inhibited by indomethacin (10 mg/kg, s.c.). TJ-14, even at 10(-4) g/ml, had little effect on the phasic contraction of isolated guinea pig ileum induced by 5-HT (2 x 10(-6) g/ml), while ondansetron suppressed the phasic contraction caused by 5-HT. These results indicate that TJ-14 is useful in suppressing cholera toxin-stimulated intestinal fluid secretion, and that this effect is partially due to its suppressive action on the PGE2 level.

5-HT2 and 5-HT3 receptor subtypes mediate cholera toxin-induced intestinal fluid secretion in the rat

The mechanisms of diarrhea in Asiatic cholera have been studied extensively. Cyclic adenosine monophosphate, 5-hydroxytryptamine (5-HT), prostaglandins, and the function of neuronal structures have been implicated in the pathogenesis of cholera. To elucidate the action of 5-HT in mediating cholera secretion, in vivo experiments were performed in the rat jejunum. The inhibitory effects of the 5-HT2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist ICS 205–930 were studied in cholera toxin-and 5-HT-induced fluid secretion. Both ketanserin and ICS 205–930 dose-dependently but only partially reduced the secretory effect of cholera toxin. The combination of the two blockers totally abolished cholera toxin-induced secretion without any influence on cholera toxin-induced increase in cyclic adenosine monophosphate. Prostaglandin E2-and bisacodyl-induced secretion was not affected by the combined administration of 5-HT2 and 5-HT3 antagonists. The present results provide evidence for an important role of 5-HT in cholera toxin-induced secretion. The data suggest a model in which cholera toxin may initiate the release of 5-HT from enterochromaffin cells. 5-Hydroxytryptamine may then cause prostaglandin E2 formation via 5-HT2 receptors and activation of neuronal structures via 5-HT3 receptors. These two effects may finally lead to the profuse fluid secretion which can be totally blocked by the combination of a 5-HT2 blocker and a 5-HT3 blocker.

Reversal of cyclic AMP-mediated intestinal secretion by ethacrynic acid.

Ethacrynic acid (EA) has been reported to reduce cholera toxin-induced intestinal fluid secretion in the intact animal. We explored the nature of this inhibition in vitro by measuring unidirectional, transmural fluxes of (22)Na and (36)Cl across isolated rabbit ileal mucosa. Under control conditions (short-circuited mucosa bathed in bicarbonate-Ringer), there was net absorption of Na and Cl. Theophylline (10 mM), cyclic AMP (5 mM), and cholera toxin (added in vivo) abolished net Na flux and produced net Cl secretion. In the presence of either theophylline or cAMP, addition of 0.1 mM EA to the serosal bathing solution abolished net Cl secretion and restored net Na absorption. Cholera toxin-treated mucosa was exposed to 0.05 and 1.0 mM EA. The lower concentration restored net Na absorption but did not significantly reduce Cl secretion. The higher concentration abolished net transport of both Na and Cl. Short-circuit current and Na flux measurements in the presence and absence of glucose indicated that 0.1 mM EA does not inhibit glucose-coupled Na transport. Short-circuit current measurements in the presence of 1.0 mM EA suggested that even this concentration of EA does not inhibit glucose-coupled Na transport. Thus EA appears to specifically inhibit Cl (or NaCl) secretion without inhibiting the absorptive Na "pump." The anti-secretory effect of 0.1 mM EA does not appear to result from inhibition of adenylate cyclase since secretion stimulated by addition of 5 mM cAMP was abolished. Furthermore, 0.1 mM EA did not significantly reduce theophylline-augmented and cholera toxin-augmented cAMP levels in ileal mucosa. We conclude that EA interacts specifically with the active Cl (or NaCl) secretory mechanism of the small intestine at a step beyond generation of cAMP.