Toxin-induced Secretion Research Articles

Aromatic substances in wheat malt inducing antisecretory factor and resistance to diarrhoea

Abstract Antisecretory factor (AF) is a protein which regulates fluid transport in the intestine and other organs. Extensive malting of wheat releases certain chemicals which induce AF and inhibit enterotoxic diarrhoea. The aim of this study was to identify AF-inducing substances in wheat malt. The active fractions of the malt leachate contained guaiacol, ferulic acid, and vanillic acid. These three 2-methyl-catechols gave >50% inhibition of cholera toxin-induced secretion in the gut. In contrast, fully methylated catechol had the opposite action; that is, they were secretory rather than antisecretory. Guaiacol and ferulic acid were further shown to induce AF in blood. Since the 2-methoxyphenol structure is present in substances binding to the vanilloid receptor, TRPV1, the specific blocker of this receptor—capsazepine—was tested. This substance exerted >50% inhibition of cholera secretion. Thus 2-methyl-catechols in wheat malt induce AF, and probably exert their effect via the TRPV1 receptor.

Calcimimetic acts on CaSR on enterocytes and enteric neurons to reverse cholera toxin-induced intestinal electrolyte secretion

Calcimimetic acts on CaSR on enterocytes and enteric neurons to reverse cholera toxin-induced intestinal electrolyte secretion

Enteroendocrine and Neuronal Mechanisms in Pathophysiology of Acute Infectious Diarrhea

While enterocyte secretion is the predominant mechanism considered responsible for secretory diarrhea in response to acute enteric infections, there are several lines of evidence that support alternative mechanisms controlling fluid and electrolyte secretion in diarrhea. To review enteroendocrine and neuronal mechanisms that participate in the development of acute infectious diarrhea. Acute infectious diarrheas due to bacterial toxins (e.g., cholera, E. coli heat-stable enterotoxin, C. difficile) and rotavirus are all associated with secretion of transmitters from enteroendocrine cells (e.g., 5-HT) and activation of afferent neurons that stimulate submucosal secretomotor neurons. The latter secrete acetylcholine (which binds to muscarinic receptors on epithelial cells) and VIP. Involvement of nerves was demonstrated by inhibition of bacterial toxin-induced secretion by hexamethonium (nicotinic), tetrodotoxin (Na(+) channel blocker), and lidocaine (visceral/mucosal afferents). Nicotinic receptors are present on secretomotoneurons and these are activated by release of acetylcholine from enteric interneurons or extrinsic efferent fibers. Specific organisms also modify other mechanisms that may contribute to development of acute diarrhea. Thus, mucin secretion, activation of motor mechanisms, increased mucosal permeability and inhibition of bile acid absorption have been reported in specific types of acute infectious diarrhea. New therapies targeting neural and transmitter mediation including 5-HT, VIP, NPY, as well as toxin receptors and channels activated during acute infectious diarrhea could usher in a novel approach to enhancing glucose-electrolyte solutions used in the treatment of acute diarrhea.

Effect of ganglionic blocking compounds on in-vivo fluid secretion in the rat small intestine.

It is well-known that enteric, secreto-motor nerves mediate cholera toxin-induced fluid secretion in the rat small intestine. This notion is, in part, derived from experiments on anaesthetized animals in which the response to cholera toxin was antagonized by the ganglionic nicotinic receptor antagonist, hexamethonium. In the current study, such anti-secretory action of ganglionic blocking compounds was analysed in an experiment designed to minimize any possible negative effect of general anaesthesia on intestinal secretion. Rats were anaesthetized with ether for 5-10 min, during which time a jejunal loop (10-12 cm) was constructed. The loop was challenged with one of the secretagogues, cholera toxin, prostaglandin E1 (PGE1) or okadaic acid. Saline (control) or either of the ganglionic blockers, hexamethonium and chlorisondamine, was administered intravenously. The rats were killed 5 h (cholera toxin) or 1.5 h (PGE1 and okadaic acid) after challenge, and the amount of fluid accumulated in the loops was determined. Cholera toxin-induced secretion was unchanged by hexamethonium but reduced by approximately 80% by chlorisondamine. The difference in effect between the two blockers might relate to the duration of ganglionic blockade. Chlorisondamine blocked secretion induced by either PGE1 or okadaic acid by approximately 60%. It is suggested that the anti-secretory effect of ganglionic blocking compounds might be a result of blockade of secreto-motor nerves but other mechanisms, for example interference with haemodynamic factors, cannot be ruled out.

Getting a Handle on Cholera and the Circuits Controlling Intestinal Motility

Getting a Handle on Cholera and the Circuits Controlling Intestinal Motility

Essential role of the glucosyltransferase activity in Clostridium difficile toxin-induced secretion of TNF-α by macrophages

Clostridium difficile causes serious and potentially fatal inflammatory diseases of the colon. Two large protein toxins, TcdA and TcdB, have been clearly implicated in pathogenesis. The goal of this study was to determine whether the glucosyltransferase activity of the toxins is critical for the induction of tumor necrosis factor-α (TNF-α), an important cytokine mediating both local and systematic inflammatory response. A dose-dependent TNF-α secretion was demonstrated in murine macrophage cell line RAW 264.7 after exposure to TcdA or TcdB. TNF-α production was blocked by anti-toxin antibodies, indicating that the cytokine-driven response is mediated by the toxins. Both toxins disrupted the cytoskeleton of host cells, while cytoskeleton disruptions using Cytochalasin-D and latrunculin B did not affect TNF-α production. The TNF-α synthesis was inhibited by reagents that target clathrin-dependent endocytosis or prevent endosomal acidification, suggesting that the endocytosis pathway is necessary for the induction of TNF-α. Furthermore, knockout of the enzymatic activity by mutating two key amino acids in the catalytic domain of TcdA abolished its cytokine-inducing activity. Our studies demonstrated a crucial role of the glucosyltransferase activity of C. difficile toxins in the induction of TNF-α in macrophages.

Ethnobotany and Natural Products: The Search for New Molecules, New Treatments of Old Diseases or a Better Understanding of Indigenous Cultures?

Results of various projects on Mexican Indian ethnobotany and some of the subsequent pharmacological and phytochemical studies are summarised focusing both on chemical-pharmacological as well as anthropological (ethnopharmacological) aspects of our research. We have identified taste and smell properties of medicinal (vs. non-medicinal) plants as important indigenous selection criteria. There exist well-defined criteria specific for each culture, which lead to the selection of a plant as a medicine. This field research has also formed a basis for studies on bioactive natural products from selected species. The bark of Guazuma ulmifolia showed antisecretory activity (cholera toxin-induced chloride secretion in rabbit distal colon in an USSING chamber). Active constituents are procyanidins with a polymerisation degree of eight or higher. Byrsonima crassifolia yielded proanthocyanidins with (+) epicatechin units and Baccharis conferta showed a dose-dependent antispasmodic effect with the effect being particularly strong in flavonoid-rich fractions. Our ethnopharmacological research led to the identification of sesquiterpene lactones (SLs) like parthenolide as potent and relatively specific inhibitors of the transcription factor NF-kappaB, an important mediator of the inflammatory process. The inhibitory effect of SLs is very strongly enhanced by the presence of such groups as the isoprenoid ring system, a lactone ring containing a conjugated exomethylene group (alpha-methylene-gamma-lactone) and an alpha,beta-unsaturated cyclopentenone or a conjugated ester moieties. Our work also elucidated the NF-kappaB inhibiting activity of the photosensitiser phaeophorbide A from Solanum diflorum (Solanaceae) in PMA induced HeLa cells. Hyptis verticillata yielded a series of lignans as well as sideritoflavone, rosmarinic acid and (R)-5-hydroxypyrrolidin-2-one and is rich in essential oil (rich in alpha-pinene, beta-pinene and thymol). Other species investigated include Begonia heracleifolia, Crossopetalum gaumerii, Epaltes mexicana, Pluchea symphytifolia and Xanthosoma robustum.

Role of mast cells and pro-inflammatory mediators on the intestinal secretion induced by cholera toxin

Recent data suggest that diarrhea caused by Vibrio cholerae involves a pro-inflammatory mediators release, such as cytokines, prostaglandin and nitric oxide. The aim of this study was to investigate the role of mast cells and their mediators in the intestinal secretion induced by cholera toxin. We examined the dose responses, time course and role of mast cells and pro-inflammatory mediators in cholera toxin intestinal secretory response, in vivo. Cholera toxin caused a dose-dependent secretion, in ligated small intestine loops, at 18 h. Rats treated with 48/80 compound or ketotifen had a significant decrease in the intestinal secretory response. Cholera toxin secretion was significantly reduced by an unspecific histamine/serotonin receptor antagonist, histamine receptor antagonist, phospholipase A 2 and cyclooxygenase inhibitors, platelet-activating factor (PAF) receptor antagonists and TNF-α synthesis blockers. On the other hand, pretreatment with a specific serotonin receptor antagonist and lipoxygenase inhibitors failed to block this effect. Analysis of the intestinal fluid from rats injected with cholera toxin, revealed that cholera toxin induces the release of IL-1β and TNF-α into fluid. The data suggest that, at least in part, mast cells are involved in cholera toxin-induced secretion, as well as point to the importance of histamine, prostaglandins, PAF, IL-1β and TNF-α in this process.

Development of microbial-human enterocyte interaction: cholera toxin.

Diarrhea in infants and children is a major health hazard worldwide. Certain toxigenic diarrheas occur more commonly and are manifested more severely during the neonatal period. We have previously studied the regulation of cholera toxin-induced secretion in animal models during development. In those studies we have shown that cholera toxin stimulates a much greater secretion by immature compared with mature small intestine, and the mechanism appears to be an up-regulation of postreceptor signal transduction molecules (adenyl cyclase and Gsalpha) leading to an elevated cAMP level. In this study, using experimental models of human intestinal development (fetal cell lines, a micro-Ussing chamber, organ cultures, and fetal intestinal xenograft transplants), we provide preliminary evidence that cholera toxin induces an enhanced secretion mediated in part by a developmental up-regulation of the cAMP response in immature versus mature human small intestine. Additional studies are needed, however, to further define whether other developmental events (e.g. receptor expression) also regulate cholera toxin-enterocyte-enhanced interaction. Nonetheless, this approach to determining the role of development in the pathophysiology of cholera in infants may help in strategies to prevent and treat this condition and other age-related intestinal infectious diseases.

Nitric oxide produced by the enterocyte is involved in the cellular regulation of ion transport.

The role of nitric oxide (NO) in the intestinal basal ion transport and under conditions of enterotoxin-induced ion secretion is controversial. Namely it is not clear whether NO enhances or counteracts intestinal ion secretion and whether the effects on transport result from a direct interaction with the enterocyte. The cell origin of NO is also unclear. We have tested the hypothesis that NO produced by the enterocyte directly regulates ion transport processes either in basal condition or in response to cholera toxin-induced secretion. Electrical variables reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers exposed to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester, in the presence or absence of cholera toxin. cAMP concentrations were also measured. NO release was determined by nitrite-nitrate concentration. NO synthase activities were assayed by Western blot analysis. Nomega-nitro-l-arginine methyl ester had a secretory effect, as judged by increased basal short-circuit current and cAMP concentration. It also increased cholera toxin-induced electrical response and cAMP production. Either cholera toxin or the cAMP analog 8-bromo-cAMP induced a rapidly progressive and Ca2+-dependent increase in NO concentration, suggesting a homeostatic up-regulation of the constitutive form of NO synthase. Western blot analysis showed an increase in constitutive NO synthase enzyme isoform. These results indicate that the enterocyte regulates its own ion transport processes, either in basal condition or in the presence of active secretion, through the activation of a constitutive NO synthase-NO pathway, functioning as a braking force of cAMP-induced ion secretion.

I, 3. The enteric nervous system and infectious diarrhea

Publisher SummaryThis chapter discusses the background knowledge about the enteric nervous system (ENS) as well as the role of ENS in secretory states of the small intestine. The chapter describes the anatomy and physiology of the ENS. A description of the experimental evidence for the involvement of ENS in secretory states of the gut, primarily in cholera toxin-induced secretion that is the most thoroughly investigated secretory state, is presented in the chapter. The chapter focuses on the involvement of ENS in rotavirus (RV) diarrhea. The involvement of the ENS in diarrhea pathophysiology opens up new potential sites of action for drugs in the treatment of intestinal secretory states. The chapter concludes with a discussion of the sites of action for the pharmacological treatment of diarrhea.

Cholera toxin-induced secretion in rats is reduced by a soluble fiber, gum arabic.

Gum arabic (GA), a soluble fiber with emulsifying properties, enhances intestinal water and electrolyte absorption in normal and secreting rats. Our aim was to assess the effect of GA, 2.5 and 5.0 g/liter, on cholera toxin-induced water and electrolyte secretion in rat jejunum in vivo. After a 2-hr exposure to cholera toxin, jejunal segments of adult rats were perfused in vivo with at plasma electrolyte solution containing GA, 0, 2.5 or 5.0 g/liter. 24Na was used as a marker of sodium influx. Cholera toxin-induced secretion was reduced by GA, 2.5 and 5.0 g/liter. 24Na secretion into the lumen was reduced by GA. GA caused a morphological expansion of intercellular spaces in the villi but not crypts. In conclusion, GA promotes lumen to blood intestinal transport of water and sodium despite cholera toxin activation. These observations support a potential role for GA in enhancing the efficacy of ORS.

Introduction. Enkephalinase inhibition: a rational approach to antisecretory therapy for acute diarrhoea

Introduction. Enkephalinase inhibition: a rational approach to antisecretory therapy for acute diarrhoea

Effect of age on vasoactive intestinal polypeptide-induced short-circuit current in porcine jejunum

Vasoactive intestinal polypeptide is a transmitter at the neuroepithelial junction of the small intestine in cholera toxin-induced secretion. We investigated whether the secretory effect in vitro of vasoactive intestinal polypeptide in porcine jejunum was changed with age. Stripped tissue preparations from three age groups, neonatal (7–11 days), young (6–8 weeks) and adult (13–15 weeks) pigs, were mounted in Ussing chambers and short-circuited. Vasoactive intestinal polypeptide produced concentration dependent increases in short-circuit current in all three age groups with EC 50 values (in nM) of 14.5±1.9, 16.2±2.0 and 147±0 in neonatal, young and adult pigs, respectively. The peak increases in short-circuit current in adult pigs were significantly decreased compared with the other two age groups. To evaluate the secretory capacity, theophylline was added to tissue preparations in which baseline short-circuit current again was established. Theophylline caused a significantly lesser increase in short-circuit current in adult pigs (25.4±2.0 μA · cm −2) than neonatal (57.1±3.6 μA · cm −2) and young pigs (63.1±2.9 μA · cm −2). In conclusion, vasoactive intestinal polypeptide showed a marked decrease in the secretory response with age in porcine jejunum, at least partly caused by a reduced secretory capacity of the enterocytes.

Water and electrolyte absorption and secretion in the small intestine

The use of gene-knockout mice permits an increased insight into the role of specific transport proteins and membrane receptors in epithelial water and electrolyte transport. Data on the secondary coupling of water transport to Na-glucose cotransport and the mechanism of action of a number of prosecretory and proabsorptive enteric neurotransmitters are reviewed. Nitric oxide and some experimental treatments with therapeutic potential for cholera toxin-induced water and electrolyte secretion are discussed. A putative role of uroguanylin in intestinal bicarbonate secretion is explored.

Effect of Saccharomyces boulardii on cAMP- and Ca2+ -dependent Cl- secretion in T84 cells.

Several reports have confirmed that the cooperative interaction between cAMP- and Ca2+-mediated transduction pathways may contribute to the stimulatory or inhibitory regulation of Cl- secretion in intestinal epithelium. Saccharomyces boulardii has been shown to inhibit cholera toxin-induced secretion in rat jejunum. We have identified a 120-kDa protein in medium conditioned by Saccharomyces boulardii that reduces cholera toxin-induced cAMP in intestinal cells. The present study evaluated the effect of medium conditioned by Saccharomyces boulardii on cAMP- and Ca2+-mediated Cl- secretion in T84 cells. Experiments performed with cAMP agonists revealed that 1 hr of preincubation of cells with medium conditioned by Saccharomyces boulardii was necessary to elicit a 40-50% reduction in receptor (cholera toxin, prostaglandin E2, and vasoactive intestinal polypeptide) and nonreceptor (forskolin) mediated cAMP synthesis and 125I efflux. Secretion induced by carbachol was inhibited when cells were pretreated for 1 hr with medium conditioned by Saccharomyces boulardii despite the absence of inhibition of Ins (1,4,5)P3. From this study we conclude that Saccharomyces boulardii exerts an inhibitory effect in vitro on Cl- secretion mediated through both cAMP- and Ca2+-mediated signaling pathways.

Calcium channels and intestinal fluid secretion: an experimental study in vivo in rats.

Several mechanisms involved in nervous secretory reflex(es) of the enteric nervous system may be dependent on the flux of calcium across the plasma membrane, which may be controlled by voltage-gated calcium channels. In this study, we investigated the importance of plasma membrane calcium channels for intestinal fluid secretion. Two types of studies were performed, in which intestinal net fluid transport in anaesthetized rats was followed with a gravimetric method. First, the effects on intestinal fluid transport of placing A23187, a calcium ionophore, in the intestinal lumen was studied. A23187 induced in a dose-dependent manner a net fluid secretion, which was abolished by giving hexamethonium (10 mg kg(-1) body wt) i.v. or placing lidocaine (1% solution) on the intestinal serosa. Nifedipine (5.75 micromol kg(-1) body wt i.v.) also abolished the fluid secretion caused by the ionophore. In the second study, the effects of various calcium channel blockers (gadolinium chloride, nifedipine, verapamil) were tested on the cholera toxin-induced secretion. It was attenuated by luminal application of gadolinium chloride (1-10 mM) or nifedipine (10-200 microM). Intravenously administered nifedipine (2.5-5.75 micromol kg(-1) body wt) abolished cholera toxin-evoked secretion dose-dependently, whereas verapamil (0.05-1 micromol kg(-1) body wt) was without consistent effect. It is concluded that the fluid secretion evoked by placing A23187 in the intestinal lumen in vivo was induced via an activation of the enteric nervous system. Cholera secretion was attenuated or abolished by calcium channel blockers of the L- or N-type.

Effect of granisetron on cholera toxin-induced enteric secretion

range 7·7 to 3·7]) was reversed to net water absorption by pre-treatment with granisetron (1·3 [ 2·0 to 5·0]; p<0·004 Mann Whitney) (figure). Net electrolyte movement paralleled net water movement. This represents the prevention by granisetron of a median net shift in water movement of 6·7 mL cm h (about 3·3 L/h of secretion when extrapolated to the whole small intestine) and is similar to the degree of reversal of secretion seen when perfusing with a glucose-electrolyte oral rehydration solution. This study provides supporting evidence for the role of 5-HT in cholera and, since 5-HT3 receptors are neuronally located, confirms the significance of neuronal mechanisms in intestinal secretion. Eherer et al studied the effect of the 5-HT3 antagonists, tropisetron and ondansetron, and tropisetron in combination with the 5-HT2 antagonist, ketanserin, on cholera toxin-induced secretion in a similar human perfusion model but found no reduction in secretion; on the contrary, tropisetron with ketanserin and ondansetron alone appeared to enhance secretion. Differing receptor binding profiles and potencies of the various 5-HT3 receptor antagonists may account for these findings. We do not consider that changes in jejunal motility would account for our findings. Granisetron is not thought to alter jejunal motility and this is confirmed by our finding that the proximal and distal port [C]-PEG recoveries (mean of 80% [SD 15·1] and 59% [SD 23·6] respectively) were similar to those in previous studies. Furthermore, the 30 cm test segment and perfusion rate of 15 mL/min, set to mimic the pathophysiological secretory state, minimise the reliance upon peristalsis for transit. Oral rehydration solutions remain the cornerstone of treatment for cholera and other acute diarrhoeal states. Nonetheless, highly selective agents such as granisetron, targeted at the pathophysiological processes which drive secretion, are likely to emerge as potent anti-secretory treatments.

Nitric oxide counteracts 5-hydroxytryptamine- and cholera toxin-induced fluid secretion and enhances the effect of oral rehydration solution

The effects of pharmacological modulation of the nitric oxide (NO) pathway on intestinal fluid transport were studied in a model of ligated jejunal loops of anaesthetized rats in vivo. Close intraarterial infusion of 5-hydroxytryptamine (5-HT) (0.16 μg/min) induced net fluid secretion. Intravenous infusion of the NO synthase inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME) (0.55 mg/kg per min) reversed net fluid absorption in controls to net secretion and significantly enhanced 5-HT-induced fluid secretion. 5-HT-induced net fluid secretion was inhibited by intravenous infusion of l-arginine (8.88 mg/kg per min), sodium nitroprusside (22.2 μg/kg per min), or 3-morpholino sydnonimine (SIN-1) (22.2 μg/kg per min). Intraluminal instillation of cholera toxin (0.5 μg/ml) induced net secretion, which was significantly enhanced by l-NAME and reduced by l-arginine. Another series of experiments was performed using a model of luminally perfused jejunal loops. Cholera toxin (10 μg/ml) induced profuse net fluid secretion also in this model. l-Arginine and sodium nitroprusside significantly enhanced net fluid absorption compared to controls and abolished the secretory effect of cholera toxin. Luminal perfusion with oral rehydration solution enhanced net absorption of fluid in controls and reversed cholera toxin-induced secretion to absorption. Intravenous infusion, but not intraluminal administration, of l-arginine significantly enhanced the antisecretory effect of oral rehydration solution. These results give further support to the existence of an intestinal NO-mediated proabsorptive tone, which also downregulates fluid secretion elicited by different enterotoxins or mediators of secretion. Intravenous administration of exogenous sources of NO counteracts intestinal fluid accumulation and augments the antisecretory effect of oral rehydration solution, findings which may lead to therapeutic consequences.

Evidence for a nonneural electrogenic effect of cholera toxin on human isolated ileal mucosa.

Cholera toxin-induced intestinal secretion in intact rats requires a functioning myenteric plexus. The aim of this investigation was to determine whether neural elements were essential for cholera toxin to produce a secretory effect in human isolated ileum. Mucosal preparations were mounted in Ussing chambers. Cholera toxin was applied apically and short-circuit current monitored for 3 hr, at which point forskolin was given. Cholera toxin (10 microg/ml) induced a tetrodotoxin-insensitive increase in short-circuit current in muscle-stripped preparations of human ileum. The increase was not additive with the action of forskolin (25 microM). Cholera toxin exerts a marked nonneural secretory effect in human ileal mucosa in vitro, probably by the same mechanism as forskolin, namely elevation of cyclic AMP.