Cholera Toxin-induced Fluid Accumulation Research Articles

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Shank2 mutant mice display a hypersecretory response to cholera toxin

Shank2 is a PDZ (PSD-95/discs large/ZO-1)-based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2 mutant (Shank2(-/-)) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2(+/+) and Shank2(-/-) mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short-circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR-Shank2-phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin-induced fluid accumulation in the mouse intestine (∼90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens.

Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing

Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing

A novel extract SB-300 from the stem bark latex of Croton lechleri inhibits CFTR-mediated chloride secretion in human colonic epithelial cells

An oligomeric proanthocyanidin (SP-303) extracted from the bark latex of the tree Croton lechleri (family Euphorbiaceae) is a potent inhibitor of cholera toxin-induced fluid accumulation and chloride secretion. The manufacturing process for SP-303 was optimized and simplified to produce an increased yield of the herbal extract. The novel extract (named SB-300) contained on average 70.6±7.2% SP-303 by weight (mean±S.D.; n=56 lots). Here, we describe the effectiveness of SB-300 on cAMP-regulated chloride secretion, which is mediated by the cystic fibrosis transmembrane conductance regulator Cl − channel (CFTR) in human colonic T84 cells. Exposure of the apical surface to SB-300 blocked forskolin-stimulated Cl − secretion by 92.2±3.0% with a half-maximal inhibition constant ( K B) of 4.8±0.8 μM. For SP-303, stimulated Cl − currents were decreased by 98.0±7.2% and K B averaged 4.1±1.3 μM. There was no significant difference between the blocking kinetics of SP-303 and SB-300. Forskolin-stimulated whole cell Cl − currents were effectively blocked by extracellular addition of SB-300 (63±8.5%; n=3) and to a similar extent by SP-303 (83 ± 0.6%; n=2; at 50 μM each). Both extracts inhibited a time- and voltage-independent Cl − conductance, which indicated the involvement of CFTR Cl − channels. We conclude that both SP-303 (used in Provir ®) and SB-300 (used in NSF Normal Stool Formula™) are novel natural products that target the CFTR Cl − channel. SB-300 is a low cost herbal extract and may present a complementary and alternative medicine approach for the treatment of fluid loss in watery diarrhea.

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.

An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice

Background & Aims : Cholera toxin (CT) is the most recognizable enterotoxin causing secretory diarrhea, a major cause of infant morbidity and mortality throughout the world. In this study, we investigated the role of the endogenous cannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) in CT-induced fluid accumulation in the mouse small intestine. Methods : Fluid accumulation was evaluated by enteropooling; endocannabinoid levels were measured by isotope-dilution gas chromatography mass spectrometry; CB 1 receptors were localized by immunohistochemistry and their messenger RNA (mRNA) levels were quantified by reverse-transcription polymerase chain reaction (PCR). Results : Oral administration of CT to mice resulted in an increase in fluid accumulation in the small intestine and in increased levels of the endogenous cannabinoid, anandamide, and increased expression of the cannabinoid CB 1 receptor mRNA. The cannabinoid receptor agonist CP55,940 and the selective cannabinoid CB 1 receptor agonist arachidonoyl-chloro-ethanolamide inhibited CT-induced fluid accumulation, and this effect was counteracted by the CB 1 receptor antagonist SR141716A, but not by the CB 2 receptor antagonist SR144528. SR141716A, per se, but not the vanilloid VR1 receptor antagonist capsazepine, enhanced fluid accumulation induced by CT, whereas the selective inhibitor of anandamide cellular uptake, VDM11, prevented CT-induced fluid accumulation. Conclusions : These results indicate that CT, along with enhanced intestinal secretion, causes overstimulation of endocannabinoid signaling with an antisecretory role in the small intestine.

Neuronal involvement in the effect of an antisecretory factor-derived peptide on induced secretion in the porcine small intestine.

The antisecretory factor is a protein inhibiting enterotoxin-induced intestinal inflammation and hypersecretion. We studied the signaling pathway of three antisecretory factor-derived peptides (A1, A3 and A4) in the proximal and distal porcine small intestine. In vivo (ligated loops), only A3 significantly reduced the cholera toxin-induced fluid accumulation and only in proximal loops. A3 and A4 reduced Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the proximal segment, whereas A1 and A3 reduced the Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the distal segment. The secretory response to intraluminally added 5-hydroxytryptamine was not significantly inhibited by the peptides. The amount of intraluminal 5-hydroxytryptamine accumulated in cholera toxin-stimulated loops from the proximal segment was significantly reduced by A3. In vitro,the effect of A3 on secretagogue-induced increases in short-circuit current was recorded from proximal small intestine by the Ussing chamber technique. A3 decreased the tetrodotoxin sensitive effect of substance P. The in vivo results suggest that the antisecretory effect may involve inhibition of the local release of 5-hydroxytryptamine induced by cholera toxin, but not inhibition of secretory reflexes induced by 5-hydroxytryptamine. The in vitro results suggest that the effect of A3 lies beyond the surface epithelium, and involves mucosal neuronal structures.

Identification in traditional herbal medications and confirmation by synthesis of factors that inhibit cholera toxin-induced fluid accumulation.

Kampo formulations are traditional herbal medications used in China and Japan for many centuries to treat diarrheal diseases such as cholera. Our studies were undertaken to identify and verify by chemical synthesis the active components that inhibited cholera toxin (CT), the virulence factor secreted by Vibrio cholerae, the causative agent of cholera. The Kampo formulation, Daio-kanzo-to, inhibited CT activities (i.e., ADP-ribosylation, Chinese hamster ovary cell elongation); in Daio-kanzo-to, Daio (Rhei rhizoma) was responsible for this effect. Among several components purified from Daio extract, rhubarb galloyl-tannin, a compound characterized by a polygallate structure, was the most effective. To define the active component, gallate analogues similar to rhubarb galloyl-tannin were synthesized. These gallate compounds inhibited all CT activities including ADP-ribosylation, elongation of Chinese hamster ovary cells, and importantly, fluid accumulation in ileal loops. Thus, Kampo formulations or their gallate components might be effective adjunctive therapy with oral rehydration solution for the severe diarrhea of cholera.

Effects of indomethacin on Salmonella typhimurium- and cholera toxin-induced fluid accumulation in the porcine small intestine.

The effect of the cyclooxygenase and prostaglandin E2 (PGE2) synthesis inhibitor, indomethacin, on the secretory responses induced by Salmonella serotype Typhimurium (ST) and cholera toxin (CT), in the porcine small intestine was investigated. ST (10(10) colony-forming units) and CT (56 micrograms) were instilled in tied-off intestinal loops in young anaesthetized pigs receiving intravenous indomethacin in a total dose of 7.5 mg/kg, or saline. The accumulated fluid in the loops and the luminal content of endogenous secretagogues PGE2 and 5-hydroxytryptamine (5-HT) were measured. ST induced fluid accumulation in the jejunum, whereas CT induced fluid accumulation in the jejunum and ileum. Indomethacin had no effect on the secretory responses. Indomethacin had a significant effect on the luminal content of PGE2 in jejunal ST and CT loops, whereas no effect of indomethacin was observed on the luminal content of 5-HT in ST and CT loops. In ST and CT loops, an increased content of PGE2 and 5-HT compared with test loops infused with Ringer's solution was observed. These results indicate that the porcine jejunal secretory response to ST and CT does not involve prostaglandins although indomethacin has an influence on the luminal release of PGE2 but not of 5-HT.

Regional differences in the effect of cholera toxin and enterotoxigenic Escherichia coli infection on electrolyte and fluid transport in the porcine small intestine.

The regional differences in secretory and absorptive responses to cholera toxin (CT) and to infection by enterotoxigenic Escherichia coli (ETEC), producing heat-stable enterotoxins, were studied in the porcine small intestine. Proximal, mid and distal small intestine from newly weaned piglets were used. Na+ and Cl- fluxes and electrical parameters in CT-stimulated and ETEC-infected intestine were measured by the Ussing chamber technique. In addition, CT-induced fluid accumulation in ligated loops was measured. CT induced fluid accumulation, which was highest in the proximal segment and decreased in the aboral direction of the small intestine. In addition, CT induced a net Cl- secretion in the proximal and mid segments, while net Na+ absorption was reduced only in the proximal segment. The ETEC-infected intestine showed a net increase in Cl- secretion in the proximal part and abolished the net Na+ absorption in the distal segment. These results demonstrate segmental differences in the epithelial transport responses to enterotoxin from Vibrio cholerae and to ETEC infection. This needs to be taken into consideration in relation to oral rehydration studies.

The Effect of α-Trinositol on Cholera Toxin–Induced Fluid Accumulation in Pig ( Sus scrofa domesticus) Jejunum in vivo

The effect of α-trinositol (D-myo-inositol-1,2,6-trisphosphate) on cholera toxin–induced fluid accumulation (i.e., net fluid secretion) was studied in the pig jejunum in vivo. Cholera toxin caused a dose-dependent fluid accumulation in control experiments. Intravenous injection of α-trinositol produced a reduction of the response to cholera toxin with a significant maximal inhibition of 36%. However, in high concentrations of α-trinositol this inhibition was absent.

Synthesis of protein in intestinal cells exposed to cholera toxin.

The mechanism by which cyclic adenosine monophosphate (AMP), formed by intestinal epithelial cells in response to cholera toxin, ultimately results in alterations in water and electrolyte transport is poorly understood. Several studies have indicated that inhibitors of transcription or translation block much of the transport of ions and water in the intestine and edema formation in tissue elicited by cholera toxin. Data presented in this study confirmed the inhibitory effects of cycloheximide on cholera toxin-induced fluid accumulation in the rabbit intestinal loop model. Neither cycloheximide nor actinomycin D altered the amount of cyclic AMP that accumulated in intestinal cells and Chinese hamster ovary cells exposed to cholera toxin. An increase in [3H]leucine incorporation was readily demonstrable in intestinal epithelial cells from rabbits challenged with Vibrio cholerae. Similarly, intestinal epithelial cells incubated with cholera toxin for 4 hr synthesized substantially more protein than controls as determined by relative incorporation of [35S]methionine. Most of the new protein synthesized in response to cholera toxin was membrane associated and of high molecular weight. The possible significance of the toxin-induced protein relative to cholera pathogenesis was discussed.

D-Ala 2, Met 5]-Enkephalinamide: CNS-mediated inhibition of prostaglandin-stimulated intestinal fluid and ion transport in the rat

The opioid peptide [D-Ala 2, Met 5]-enkephalinamide (DAMA), a non-selective opioid agonist, has previously been shown to inhibit cholera toxin-induced fluid accumulation in the rat and dog small intestine after its intracerebroventricular (ICV) administration. In the present study, we examined the time course of the antisecretory/proabsorptive effects of ICV DAMA on net fluid and ion transport across the rat jejunum in situ during intravenous prostaglandin E 1 (PGE) infusion. Net water and NaCl absorption were measured using a standard dilution marker technique in a 15–20 cm segment of proximal jejunum in urethaneanesthetized Sprague-Dawley rats. Infusion of PGE (5 μg/kg-min) over a 2 hr period produced a decrease in fluid and ion absorption that plateaued to a steady-state within 60 min. DAMA (1 and 3 μg/rat) administered by ICV bolus 60 min after the start of PGE infusion inhibited significantly PGE-induced decreases in water and chloride absorption relative to saline-treated controls. These dose-related peptide effects were expressed 15 min after DAMA treatment and were approximately 30 min in duration; they were antagonized by naloxone (1 mg/kg, IV) given at the time of DAMA injection. These results indicate that low concentrations of DAMA administered into the central nervous system rapidly and effectively inhibit changes in intestinal transport induced by a blood-borne secretagogue through an interaction with opiate receptors.