cyclic-gmp and Diarrhea

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function.

Topics: Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Escherichia coli Infections; Gastrointestinal Microbiome; Humans; Inflammasomes; Inflammation; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Salmonella Infections

cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field.

Topics: Amino Acid Sequence; Animals; Cyclic GMP; Diarrhea; Epithelium; Genetic Pleiotropy; Humans; Molecular Sequence Data; Myocardium; Natriuretic Peptides; Olfactory Receptor Neurons; Photoreceptor Cells, Vertebrate; Receptors, Guanylate Cyclase-Coupled

Many medications induce diarrhea as a side effect, which can be a major obstacle to therapeutic efficacy and also a life-threatening condition. Secretory diarrhea can be caused by excessive fluid secretion in the intestine under pathological conditions. The cAMP/cGMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) is the primary chloride channel at the apical membrane of intestinal epithelial cells and plays a major role in intestinal fluid secretion and homeostasis. CFTR forms macromolecular complexes at discreet microdomains at the plasma membrane, and its chloride channel function is regulated spatiotemporally through protein-protein interactions and cAMP/cGMP-mediated signaling. Drugs that perturb CFTR-containing macromolecular complexes in the intestinal epithelium and upregulate intracellular cAMP and/or cGMP levels can hyperactivate the CFTR channel, causing excessive fluid secretion and secretory diarrhea. Inhibition of CFTR chloride-channel activity may represent a novel approach to the management of drug-induced secretory diarrhea.

Topics: Animals; Cell Membrane; Cyclic AMP; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Drug-Related Side Effects and Adverse Reactions; Humans

Cellular microbiology is a new discipline that is emerging at the interface between cell biology and microbiology. The application of molecular techniques to the study of bacterial pathogenesis has made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. The use of microbial pathogens to address questions in cell physiology is just now yielding promising applications and striking results.

Topics: Amino Acid Sequence; Animals; Bacterial Infections; Calcium; Cells; Cyclic GMP; Diarrhea; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Models, Biological; Molecular Sequence Data; Natriuretic Peptides; Peptides; Sequence Alignment; Signal Transduction

The guanylin family of bioactive peptides consists of three endogenous peptides, including guanylin, uroguanylin and lymphoguanylin, and one exogenous peptide toxin produced by enteric bacteria. These small cysteine-rich peptides activate cell-surface receptors, which have intrinsic guanylate cyclase activity, thus modulating cellular function via the intracellular second messenger, cyclic GMP. Membrane guanylate cyclase-C is an intestinal receptor for guanylin and uroguanylin that is responsible for stimulation of Cl- and HCO3- secretion into the intestinal lumen. Guanylin and uroguanylin are produced within the intestinal mucosa to serve in a paracrine mechanism for regulation of intestinal fluid and electrolyte secretion. Enteric bacteria secrete peptide toxin mimics of uroguanylin and guanylin that activate the intestinal receptors in an uncontrolled fashion to produce secretory diarrhea. Opossum kidney guanylate cyclase is a key receptor in the kidney that may be responsible for the diuretic and natriuretic actions of uroguanylin in vivo. Uroguanylin serves in an endocrine axis linking the intestine and kidney where its natriuretic and diuretic actions contribute to the maintenance of Na+ balance following oral ingestion of NaCl. Lymphoguanylin is highly expressed in the kidney and myocardium where this unique peptide may act locally to regulate cyclic GMP levels in target cells. Lymphoguanylin is also produced in cells of the lymphoid-immune system where other physiological functions may be influenced by intracellular cyclic GMP. Observations of nature are providing insights into cellular mechanisms involving guanylin peptides in intestinal diseases such as colon cancer and diarrhea and in chronic renal diseases or cardiac disorders such as congestive heart failure where guanylin and/or uroguanylin levels in the circulation and/or urine are pathologically elevated. Guanylin peptides are clearly involved in the regulation of salt and water homeostasis, but new findings indicate that these novel peptides have diverse physiological roles in addition to those previously documented for control of intestinal and renal function.

Topics: Amino Acid Sequence; Animals; Colonic Neoplasms; Cyclic GMP; Diarrhea; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Mucosa; Kidney Diseases; Molecular Sequence Data; Natriuretic Peptides; Peptides

This review discuss some recent findings in the study of the regulation of the permeability of the intestinal epithelial layer. Comparison of electrical phenomena and transport of macromolecules suggests that secretory activity and increased transepithelial transport of macromolecules are related when secretion is mediated by the Ca2+ and PKC dependent pathways. The transport of the macromolecules is via the transcellular and via the paracellular route. The barrier function of the intestinal epithelium may be diminished during nervous (acetylcholine)- and immuno-(histamine) mediated secretion. It is hypothesised that some bacterial toxins may also induce Ca2+ and PKC dependent secretion and thereby can reduce the epithelial barrier. The cAMP and cGMP mediated secretion, which can be recognised by their long-lasting transepithelial potential changes, are not coupled to increased transepithelial transport of macromolecules. Some forms of secretory diarrhea may therefore be related to the development of food-allergy or inflammation.

Topics: Biological Transport, Active; Calcium; Cyclic AMP; Cyclic GMP; Diarrhea; Electrophysiology; Epithelium; Horseradish Peroxidase; Humans; Intestinal Mucosa; Intestines; Macromolecular Substances; Permeability; Protein Kinase C; Tight Junctions

Topics: Animals; Chloramines; Colitis; Cyclic GMP; Diarrhea; Disease Models, Animal; Free Radicals; Humans; Hydrogen Peroxide; Inflammatory Bowel Diseases; Ion Transport; Nitric Oxide; Nitrogen; Rats; Reactive Oxygen Species

Heat-stable enterotoxin, produced by Escherichia coli, binds to particulate guanylate cyclase to increase cyclic GMP in intestinal cells. This in turn stimulates the cyclic-GMP- or cyclic-AMP-dependent protein kinase, activating the same chloride channel that is defective in cystic fibrosis. It is possible that the relatively high prevalence of cystic fibrosis in humans results from its protective effect against diarrhea.

Topics: Animals; Bacterial Toxins; Chloride Channels; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterotoxins; Escherichia coli Infections; Escherichia coli Proteins; Humans; Ion Transport; Membrane Proteins

Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrheal diseases, including traveler's diarrhea and epidemic diarrhea in infants and newborns. The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon. Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine. Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen. These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway. The release of archidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa. Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Calcium; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Proteins; Humans; Intestinal Secretions; Molecular Sequence Data; Phosphatidylinositols; Protein Kinases

The etiological factors involved in diarrhea are multiple. Also the mechanisms and mediators involved are multiple: intracellular mediators (Ca, cAMP, cGMP, calmodulin, phospholipids), extracellular mediators (hormones, neurotransmitters, prostaglandins, enterotoxins...), intramural blood flow and oxygen, intestinal motility (local- and peristaltic motility). Till now, antidiarrheals are not so versatile that they provide a solution to all types of diarrhea. The mechanisms of action of fluid replacement therapy, loperamide, alpha 2 agonist and some nonsteroidal anti-inflammatory substances are reviewed.

Topics: Acetylcholine; Animals; Antidiarrheals; Bacterial Toxins; Biological Transport; Calcium; Catecholamines; Chlorides; Cyclic AMP; Cyclic GMP; Diarrhea; Fluid Therapy; Glucagon; Histamine; Intestinal Absorption; Intestinal Mucosa; Intestinal Secretions; Intestines; Lipid Metabolism; Loperamide; Regional Blood Flow; Serotonin; Sodium; Somatostatin; Substance P; Vasoactive Intestinal Peptide; Water-Electrolyte Balance

The physiology of intestinal electrolyte transport is currently an area of intense research interest. Also, reports regularly appear that define possible roles of various endocrine, paracrine, and neurohumoral substances in regulating intestinal ion and water flux. A vast body of knowledge has appeared recently that focuses on the action of specific intracellular mediators or second messengers and certain biochemical events that are thought to be involved in this transport process. This area of research has drawn the attention of the clinical investigator as well as the basic scientist because of the implications of these findings to the understanding of secretory disorders of the gastrointestinal tract, in particular diarrheal disease. The purpose of this review is to focus on recent findings reported in three major areas: the physiology of intestinal electrolyte transport and its regulation; the pathophysiology of secretory diarrhea; and current thoughts and practices in the therapeutic approach to the disease.

Topics: Acid-Base Equilibrium; Adrenergic alpha-Agonists; Animals; Arachidonic Acids; Carbachol; Chlorides; Cholera Toxin; Cyclic AMP; Cyclic GMP; Diarrhea; Electric Stimulation; Electrolytes; Hydroxyeicosatetraenoic Acids; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Leukotrienes; Models, Biological; Narcotics; Phenothiazines; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphorylation; Protein Kinase C; Receptors, Cholinergic; Receptors, Muscarinic; Renin-Angiotensin System; Sodium; Sodium-Potassium-Exchanging ATPase; Tetradecanoylphorbol Acetate

Topics: Cyclic AMP; Cyclic GMP; Diarrhea; Humans; Intestinal Absorption; Intestinal Secretions; Prostaglandins; Vasoactive Intestinal Peptide

The intestinal epithelial cell, or enterocyte, performs a dual function: absorption and secretion. Absorption takes place in the villi and secretion in the crypts (and perhaps also in the villi). Both functions are controlled by complex extra- and intracellular systems. The extracellular system is mediated by neuromediators released in the subcellular space. Some mediators, like the vasoactive intestinal peptide (VIP) or the prostaglandins, stimulate secretion while others, like enkephalins, stimulate absorption. All these mediators act on intracellular metabolic processes by activating "second messengers" (e.g. amp or intracellular calcium) which in turn regulate the enterocyte transport system. Diarrheogenic processes are frequently due to disturbances in enterocyte control with abnormal stimulation of the secretory processes. Some pancreatic cancers and sympathicoblastomas release VIP into the blood stream in sufficient amounts to stimulate cAMP production by the enterocytes and, consequently, secretion. The cholera toxin and the E-coli thermolabile toxin can also induce stimulation of intracellular cAMP production. Conversely, the most promising antidiarrhoeal drugs are antisecretory agents, like opiates, which stimulate the enterocyte absorption processes.

Topics: Animals; Calcium; Cyclic AMP; Cyclic GMP; Diarrhea; Enkephalins; Epithelial Cells; Epithelium; Humans; Intestinal Absorption; Intestinal Mucosa; Intestines; Neurotransmitter Agents; Peptides; Somatostatin; Vasoactive Intestinal Peptide

Topics: Adenylyl Cyclases; Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Guanylate Cyclase; Humans; Mice; Vibrio cholerae

A detailed review of the pathophysiology of diarrhoeal diseases is presented. Recent developments in the understanding of the mechanisms by which water absorption occurs via electrolyte transport are detailed. The secretion of Cl across the apical membrane in vivo is accompanied by a flow of Na as a counter ion, probably via the paracellular pathway. Normal regulation of active intestinal ion transport depends on cyclic AMP and cyclic GMP as well as intracellular calcium. In diarrhoeal diseases, some portion of the gut is usually found to be in a secretory rather than absorptive condition. This may be due to changes in active ion transport (decrease in Na absorption or increase in Cl secretion), alterations in intestinal motility and/or luminal osmolarity or to an increase in tissue hydrostatic pressure.

Topics: Calcium; Cyclic AMP; Cyclic GMP; Diarrhea; Electrolytes; Gastrointestinal Motility; Humans; Hydrostatic Pressure; Intestinal Absorption; Intestinal Mucosa; Osmolar Concentration; Water

Enteric infections cause more than a billion episodes of diarrhoeal disease in humans each year killing many millions of people, especially young children, in developing countries. Recent progress, reviewed in this article, has enabled that a specific pathogen now can be isolated in the majority of patients with acute diarrhoea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents. Based on this understanding it now seems possible to devise new techniques for the treatment and prevention of diarrhoeal disease to complement those based on fluid replacement therapy and sanitation; prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed.. Enteric infections cause more than 1 billion episodes of diarrheal disease in humans each year killing many millions of people, especially young children, in developing countries. Recent progress, reviewed here, has enabled that a specific pathogen can now be isolated in the majority of patients with acute diarrhea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents. Based on this understanding, it now seems possible to devise new techniques for the treatment and prevention of diarrheal disease to complement those based on fluid replacement therapy and sanitation. Prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed.

Topics: Antidiarrheals; Bacterial Vaccines; Calcium; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Dysentery, Amebic; Escherichia coli Infections; G(M1) Ganglioside; Humans; Reoviridae Infections; Rotavirus; Viral Vaccines

Previous experimental studies suggest that bile salt-induced colonic fluid secretion is mediated by adenosine 3':5'-phosphate (cyclic AMP). Two biopsy specimens of colonic mucosa were obtained endoscopically before and after different periods of therapy (five, 10, or 15 days), from each of 21 patients receiving chenodeoxycholic acid. A rise of cyclic AMP intracellular levels was found, but only after five and 10 days of treatment was the increase statistically significant when compared with basal levels. Similar changes were observed for guanosine 3':5'-phosphate (cyclic GMP), but percentage increases were higher than for cyclic AMP. Initial diarrhoea disappeared spontaneously, and at 15 days the levels of both cyclic nucleotides were not significantly different from basal levels. Our findings suggest that colonic adaptation to increase in luminal bile salt levels is related to changes in intracellular levels of cyclic nucleotides and support the hypothesis that not only cyclic AMP, but also cyclic GMP may play an important role in producing bile salt-induced diarrhoea in man.

Topics: Adult; Chenodeoxycholic Acid; Cholelithiasis; Colon; Cyclic AMP; Cyclic GMP; Diarrhea; Female; Humans; Intestinal Mucosa; Male; Middle Aged; Time Factors

Endogenous peptides and structurally similar bacterial heat-stable enterotoxins (ST) bind guanylate cyclase-C (GC-C), resulting in fluid homeostasis or diarrhea, respectively. In this issue of Cell Host & Microbe, Carey et al., show how bats have evolutionarily maintained homeostatic signaling while avoiding pathogenic effects of ST.

Topics: Animals; Bacterial Toxins; Chiroptera; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterocytes; Enterotoxigenic Escherichia coli; Enterotoxins; Guanylate Cyclase; Protein Binding; Signal Transduction; Sodium-Hydrogen Exchangers

Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder.. We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin.. Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation.. We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator-dependent chloride secretion, the driver of persistent diarrhea.

Topics: Abnormalities, Multiple; Cyclic GMP; Diarrhea; Gain of Function Mutation; Heterocyclic Compounds, 4 or More Rings; Humans; Metabolism, Inborn Errors; Receptors, Enterotoxin

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxins that cause dehydration by disrupting intestinal water absorption. We investigated patterns of genetic variation in mammalian guanylate cyclase-C (GC-C), an intestinal receptor targeted by bacterially encoded heat-stable enterotoxins (STa), to determine how host species adapt in response to diarrheal infections. Our phylogenetic and functional analysis of GC-C supports long-standing evolutionary conflict with diarrheal bacteria in primates and bats, with highly variable susceptibility to STa across species. In bats, we further show that GC-C diversification has sparked compensatory mutations in the endogenous uroguanylin ligand, suggesting an unusual scenario of pathogen-driven evolution of an entire signaling axis. Together, these findings suggest that conflicts with diarrheal pathogens have had far-reaching impacts on the evolution of mammalian gut physiology.

Topics: Animals; Bacterial Toxins; Chiroptera; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterocytes; Enterotoxigenic Escherichia coli; Enterotoxins; Guanylate Cyclase; Natriuretic Peptides; Protein Binding; Receptors, Enterotoxin; Signal Transduction; Sodium-Hydrogen Exchangers; Vibrio cholerae

Topics: Bacterial Toxins; Caco-2 Cells; Cell Line, Tumor; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Diarrhea; Enterotoxigenic Escherichia coli; Enterotoxins; Epithelial Cells; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae Proteins; Humans; Jejunum; Membrane Glycoproteins; Peptide Hydrolases; Propionates; Quinolines; Virulence Factors

Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease.

Topics: Analysis of Variance; Animals; Bacterial Toxins; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Disease Models, Animal; Enterotoxigenic Escherichia coli; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Escherichia coli Proteins; Humans; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Transgenic; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

Ulcerative colitis (UC) belongs to inflammatory bowel disorders, a group of gastrointestinal disorders that can produce serious recurring diarrhea in affected patients. The mechanism for UC- and inflammatory bowel disorder-associated diarrhea is not well understood. The cystic fibrosis transmembrane-conductance regulator (CFTR) chloride channel plays an important role in fluid and water transport across the intestinal mucosa. CFTR channel function is regulated in a compartmentalized manner through the formation of CFTR-containing macromolecular complexes at the plasma membrane. In this study, we demonstrate the involvement of a novel macromolecular signaling pathway that causes diarrhea in UC. We found that a nitric oxide-producing enzyme, inducible nitric oxide synthase (iNOS), is overexpressed under the plasma membrane and generates compartmentalized cGMP in gut epithelia in UC. The scaffolding protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) bridges iNOS with CFTR, forming CFTR-NHERF2-iNOS macromolecular complexes that potentiate CFTR channel function via the nitric oxide-cGMP pathway under inflammatory conditions both in vitro and in vivo. Potential disruption of these complexes in Nherf2(-/-) mice may render them more resistant to CFTR-mediated secretory diarrhea than Nherf2(+/+) mice in murine colitis models. Our study provides insight into the mechanism of pathophysiologic occurrence of diarrhea in UC and suggests that targeting CFTR and CFTR-containing macromolecular complexes will ameliorate diarrheal symptoms and improve conditions associated with inflammatory bowel disorders.

Topics: Animals; Cell Membrane; Cells, Cultured; Colitis, Ulcerative; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Disease Models, Animal; Humans; Male; Mice, Inbred C57BL; Mice, Knockout; Phosphoproteins; Signal Transduction; Sodium-Hydrogen Exchangers

Based on ethnopharmacological indications that Mentha species may be used in the treatment of gastrointestinal diseases, this study aimed to characterize the gastroprotective mechanisms of menthol (ME), the major compound of the essential oil from species of the genus Mentha. The gastroprotective action of ME was analyzed in gastric ulcers that were induced by ethanol or indomethacin in Wistar male rats. The mechanisms responsible for the gastroprotective effect were assessed by analyzing the amount of mucus secreted, involvement of non-protein sulfhydryl (NP-SH) compounds, involvement of calcium ion channels and NO/cGMP/K(+)ATP pathway, gastric antisecretory activity and the prostaglandin E2 (PGE2) production. The anti-diarrheal activity and acute toxicity of ME were also evaluated. Oral treatment with ME (50mg/kg) offered 88.62% and 72.62% of gastroprotection against ethanol and indomethacin, respectively. There was an increased amount of mucus and PGE2 production. The gastroprotective activity of ME involved NP-SH compounds and the stimulation of K(+)ATP channels, but not the activation of calcium ion channels or the production of NO. The oral administration of ME induced an antisecretory effect as it decreased the H(+) concentration in gastric juice. ME displayed anti-diarrheal and antiperistaltic activity. There were no signs of toxicity in the biochemical analyses performed in the rats' serum. These results demonstrated that ME provides gastroprotective and anti-diarrheal activities with no toxicity in rats.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcium Channels; Castor Oil; Cyclic GMP; Diarrhea; Dinoprostone; Ethanol; Indomethacin; Male; Menthol; Nitric Oxide; Potassium Channels; Rats; Rats, Wistar; Signal Transduction; Stomach Ulcer; Sulfhydryl Compounds

Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis.. We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells.. We identified a heterozygous missense mutation (c.2519G→T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members.. Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest [Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.).

Topics: Chronic Disease; Cyclic GMP; Diarrhea; Female; Genetic Linkage; Heterozygote; Humans; Male; Mutation, Missense; Pedigree; Polymorphism, Single Nucleotide; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Down-Regulation; Enterotoxins; Escherichia coli Proteins; Female; Gastrointestinal Hormones; Genes, Modifier; HEK293 Cells; Heterozygote; Humans; Intestinal Mucosa; Intestinal Obstruction; Male; Meconium; Mice; Molecular Sequence Data; Mutation; Natriuretic Peptides; Pedigree; Phenotype; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.. In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.. Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.

Topics: Animals; Bacterial Toxins; Cell Line; Cloning, Molecular; Cyclic AMP; Cyclic GMP; Diarrhea; Enterotoxigenic Escherichia coli; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Proteins; Plasmids; Prevalence; Swine; Time Factors; Virulence

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

Topics: Animals; Caco-2 Cells; Cyclic AMP; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterotoxins; Exocytosis; HEK293 Cells; Humans; Jejunum; Mice; Mice, Inbred BALB C; Qa-SNARE Proteins; Rats; Rats, Sprague-Dawley

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.

Topics: Adenylyl Cyclase Inhibitors; Animals; Antidiarrheals; Bacterial Toxins; Cell Line; Cricetinae; Cyclic AMP; Cyclic GMP; Diarrhea; Enterotoxins; Enzyme Inhibitors; Escherichia coli Proteins; Guanylate Cyclase; Heterocyclic Compounds, 4 or More Rings; Humans; Rats; Small Molecule Libraries

The facultative pathogen Vibrio cholerae can exist in both the human small bowel and in aquatic environments. While investigation of the infection process has revealed many factors important for pathogenesis, little is known regarding transmission of this or other water-borne pathogens. Using a temporally controlled reporter of transcription, we focus on bacterial gene expression during the late stage of infection and identify a unique class of V. cholerae genes specific to this stage. Mutational analysis revealed limited roles for these genes in infection. However, using a host-to-environment transition assay, we detected roles for six of ten genes examined for the ability of V. cholerae to persist within cholera stool and/or aquatic environments. Furthermore, passage through the intestinal tract was necessary to observe this phenotype. Thus, V. cholerae genes expressed prior to exiting the host intestinal tract are advantageous for subsequent life in aquatic environments.

Topics: Animals; Animals, Suckling; Cholera; Cyclic GMP; Diarrhea; Gene Expression Regulation, Bacterial; Genes, Bacterial; Humans; Mice; Osmolar Concentration; Reverse Transcriptase Polymerase Chain Reaction; Vibrio cholerae; Water Microbiology

Although the phenomenon of opioid tolerance and dependence has been widely investigated, neither opioid nor non-opioid mechanisms are completely understood. In view of the modulation of 5-HT transport into presynaptic terminals in the brain by nitric oxide (NO) via cGMP, and the existence of a tonic 5-HTergic inhibition of dopamine release, the present study investigated the effect of fluoxetine, a selective serotonin reuptake inhibitor, and NO modulators L-N(G)-nitroarginine methyl ester (L-NAME; NO synthase inhibitor) and L-Arginine (substrate for nitric oxide synthase) alone or in combination against morphine tolerance and dependence. Animals developed tolerance to the antinociceptive effect of morphine (10 mg/kg s.c. twice daily) on day 3 and the degree of tolerance was further enhanced on days 9 and 10. The development of tolerance to the antinociceptive effect of morphine was delayed by prior administration of fluoxetine (10 mg/kg i.p, twice daily for 9 days) and L-NAME (10 mg/kg i.p. twice daily for 9 days) alone or in combination. It was accentuated by L-Arginine (50 mg/kg i.p. twice daily for 9 days) alone or in combination with fluoxetine (10 mg/kg i.p. twice daily for 9 days). Similarly, fluoxetine (10 mg/kg i.p.) or L-NAME (10 mg/kg i.p.), when administered acutely on day 10, reversed morphine-induced tolerance. L-Arginine (50 mg/kg i.p.) however, when administered acutely on day 10, accentuated morphine tolerance. Fluoxetine (10 mg/kg i.p. twice daily for 9 days) suppressed the development of morphine dependence as assessed by naloxone (2 mg/kg i.p.)-precipitated withdrawal jumps. This suppression of dependence was potentiated by L-NAME (10 mg/kg i.p. twice daily for 9 days) and reversed by L-Arginine (50 mg/kg i.p. twice daily for 9 days), respectively. Acute administration of the respective drugs on day 10 modulated morphine dependence in a similar fashion. L-Arginine also reversed fluoxetine-induced weight loss in morphine-dependent animals. The present study demonstrated that fluoxetine suppressed the dependence and development of tolerance to the antinociceptive effect of morphine. Fluoxetine-induced suppression was potentiated by L-NAME and accentuated by L-Arginine. The results therefore suggest that a complex phenomenon such as morphine tolerance and dependence might involve close interplay of the NO-c GMP/5-HT/DA receptor system. To the best of the authors' knowledge, this is the first report to suggest targeting this cascade for ame

Topics: Animals; Arginine; Body Weight; Cyclic GMP; Depression, Chemical; Diarrhea; Dopamine; Drug Interactions; Drug Tolerance; Female; Fluoxetine; Male; Mice; Morphine Dependence; Naloxone; Narcotic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Selective Serotonin Reuptake Inhibitors; Serotonin; Substance Withdrawal Syndrome

Various invasive pathogens cause diarrhea, but the mechanism(s) are poorly understood. We hypothesized that nitric oxide and prostaglandins might modulate chloride secretory and barrier properties of the infected intestinal epithelium and that diarrhea is caused, in part, by altered expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2).. Studies were conducted in human intestinal epithelial cell lines (HT29/cl.19A, Caco-2, and T84). Cells were infected with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]). Infected cells and controls were tested for transepithelial resistance, chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate, and protein expression.. Cells infected with EIEC or SD, but not uninfected controls or ST/LA-exposed monolayers, showed a progressive reduction in transepithelial resistance starting at 6-12 hours. Infected HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an increase in total nitrite. Expression of iNOS, and consequently COX-2, was also increased, followed by increased production of prostaglandins and cyclic nucleotides. Furthermore, basal and stimulated chloride secretory responses to various agonists were enhanced in HT29/cl.19A and Caco-2 cells after infection with enteroinvasive bacteria, and this effect was reversed for some agonists by iNOS or COX-2 inhibitors. Increased expression of cystic fibrosis transmembrane conductance regulator and NKCC1 was also observed in EIEC or SD-infected cells vs. controls, secondary to NO synthase activity.. Up-regulation of iNOS and COX-2 by enteroinvasive bacteria can modulate chloride secretion and barrier function in intestinal epithelial cells. Thus, these enzymes represent possible therapeutic targets in infectious diarrhea.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Biological Transport; Caco-2 Cells; Chlorides; Cyclic AMP; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Dinoprostone; Electric Impedance; Enzyme Inhibitors; Epithelial Cells; Escherichia coli Infections; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; HT29 Cells; Humans; Intestinal Mucosa; Isoenzymes; Membrane Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrobenzenes; Peroxynitrous Acid; Prostaglandin-Endoperoxide Synthases; Salmonella Infections; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Sulfonamides; Up-Regulation; Water-Electrolyte Balance

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.

Topics: Bacterial Toxins; Cyclic GMP; Diarrhea; Enterotoxins; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Humans; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [125I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a Kd of 0.5 x 10(-10) M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [125I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.

Topics: Bacterial Toxins; Binding Sites; Carrier Proteins; Cell Membrane; Colon; Colonic Neoplasms; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Guanylate Cyclase; Humans; Kinetics; Neoplasm Proteins; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Tumor Cells, Cultured

Cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP. Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates. These results indicate that cGKII plays a central role in diverse physiological processes.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bacterial Toxins; Body Water; Bone Development; Crosses, Genetic; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diarrhea; Dwarfism; Enterotoxins; Escherichia coli Proteins; Female; Gene Deletion; Growth Plate; Intestinal Mucosa; Male; Mice; Mice, Inbred C57BL; Osteogenesis; Signal Transduction

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Base Sequence; Child; Cloning, Molecular; Conjugation, Genetic; Cyclic GMP; Diarrhea; DNA, Bacterial; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Genes, Bacterial; Humans; Ileum; In Vitro Techniques; Intestinal Mucosa; Male; Molecular Sequence Data; Multigene Family; Oligodeoxyribonucleotides; Open Reading Frames; Rabbits; Restriction Mapping; Sequence Homology, Amino Acid

To clarify the kinetics of cell membrane and intracellular mediators in the process of auranofin (AF)-induced diarrhea, we perfused electrolyte solution containing the oral gold preparation AF, which is a treatment for rheumatoid arthritis, through the rat jejunum, and studied net water and electrolyte transport, Na+, K(+)-ATPase activity, and c-AMP and c-GMP concentrations in the jejunal mucosa. In addition, change in Ca+ concentration in isolated intestinal cells was evaluated using fura-2-acetoxyl-methyl ester. AF significantly suppressed water and electrolyte transport. Mucosal secretion was increased due to elevation of the intracellular Ca+ concentration early in the perfusion period, then due to reduction in the Na+, K(+)-ATPase activity, and increase in the c-AMP concentration late in the perfusion period. Therefore, these cell membranes and intracellular mediators are considered to be involved in the mechanism of AF-induced diarrhea.

Topics: Animals; Auranofin; Biological Transport; Calcium; Cyclic AMP; Cyclic GMP; Diarrhea; In Vitro Techniques; Intestinal Mucosa; Jejunum; Male; Rats; Rats, Inbred Strains; Sodium-Potassium-Exchanging ATPase

Escherichia coli heat-stable enterotoxins (ST) are classified into STa and STb according to their physicochemical and biologic characteristics. STa induces diarrhea, activating the guanylate cyclase-cGMP system. ST-like enterotoxins can be produced by bacteria other than E. coli, including Klebsiella pneumoniae. A Klebsiella ST has previously been shown to share some chemical and immunologic characteristics with E. coli ST. Aiming to define better the nature of Klebsiella ST, we have screened 237 children with diarrhea and 179 controls for ST-producing Klebsiella, using the SMA. We detected 26 Klebsiella strains from patients, two of which were positive in the SMA, and 36 from controls, all negative for ST. A partial purification was performed using an acetone precipitation followed by ultrafiltration and gel filtration techniques. Klebsiella toxin was heat-stable, methanol-soluble, sensitive to mercaptoethanol, active at acid pH values, but not at pH greater than 8. The time course of Klebsiella toxin in the SMA resembled that of E. coli STa. Klebsiella ST caused reduced Na absorption and net Cl secretion in rabbit ileal mucosa mounted in Ussing chambers. It was found to increase the cGMP but not the cAMP concentration. Finally, Klebsiella ST did not react with anti-E. coli STa MAb in a competitive ELISA. We conclude that K. pneumoniae may induce diarrhea through the production of an STa similar but not identical to E. coli STa.

Topics: Bacterial Toxins; Child; Child, Preschool; Cyclic AMP; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli Proteins; Humans; Infant; Infant, Newborn; Intestinal Mucosa; Ion Channels; Klebsiella pneumoniae

The effects of intestinal resection on several intestinal parameters have been studied in the large intestine of rats one month after the surgical operation. The results show that both 50% and 80% distal small bowel resection increased net fluid absorption and mucosal permeability and caused expansion of the intercellular spaces of the large intestine. The increase in net fluid absorption was dependent upon the extent of the intestine removed. The cAMP and cGMP content of cecal and colonic mucosa were significantly reduced after jejunoilectomy. Changes in nucleotide levels were dependent on the length of the intestine resected. On the other hand, mucosa Na,K-ATPase specific activity was only increased in the cecum after 80% intestinal resection. The results are discussed in terms of adaptation to prevent diarrhea.

Topics: Adenosine Triphosphatases; Animals; Cecum; Cyclic AMP; Cyclic GMP; Diarrhea; Ileum; Intestinal Mucosa; Intestine, Small; Jejunum; Male; Rats; Rats, Inbred Strains; Water

Azodisalicylate, used to treat ulcerative colitis, causes diarrhea in up to 12.5% of patients. We compared the in vitro effects of azodisalicylate, sulfasalazine, and 5-aminosalicylic acid on rabbit intestinal electrolyte transport. Distal ileal mucosae mounted in Ussing chambers were exposed to varying concentrations of the drugs. Mucosal addition of azodisalicylate (greater than 5 mM) caused the greatest anion-dependent increase in short-circuit current of 83 microA/cm2 (ED50 = 0.3 mM). Isotope flux measurements suggest that azodisalicylate may stimulate predominantly electrogenic HCO3 secretion and induces net NaCl secretion. In contrast, serosal addition of azodisalicylate and sulfasalazine (greater than 5 mM) decreased short-circuit current, and 5-aminosalicylic acid had no effect. Azodisalicylate had no effect on ion transport in distal colon. The effects of azodisalicylate in ileum were not inhibited with piroxicam (an inhibitor of cyclooxygenase). Mucosal cyclic adenosine monophosphate and cyclic guanosine monophosphate levels were unchanged after ileal exposure to azodisalicylate. Azodisalicylate appears to be a mechanistically unusual secretagogue, possibly explaining the increased incidence of diarrhea seen in patients taking the drug.

Topics: Aminosalicylic Acids; Animals; Colon; Cyclic AMP; Cyclic GMP; Diarrhea; Dose-Response Relationship, Drug; Electrolytes; Ileum; Male; Rabbits

Topics: Adenylyl Cyclases; Adult; Aged; Colitis, Ulcerative; Cyclic AMP; Cyclic GMP; Diarrhea; Female; Humans; Intestinal Mucosa; Male; Middle Aged; Rectum

The study was made on 59 chinchilla rabbits. S. typhimurium 1847 live culture was introduced into the lumen of an isolated loop of the thin intestine. The activity of adenylate cyclase (AC), guanylate cyclase (GC), the levels of cyclic adenosine 3,5-monophosphate (cAMP), cyclic guanosine 3,5-monophosphate (cGMP), the activity of cAMP- and cGMP-phosphodiesterases were determined in the mucous membrane of the ligated part of the intestine. Considerable fluid accumulation in the loop, activation of AC and cGMP-phosphodiesterase, a rise in the level of cAMP and a drop in the level of cGMP in the mucosa of the ligated part of the intestine were registered. In one group of the animals phosphadene and in the other group unitiol were introduced into the infected intestinal loop; as a result, a decrease in the accumulation of fluid in the loop, on the average, by 40% and a tendency to an increase in the level of cAMP and a drop in the level of cGMP in the mucous membrane of the ligated part of the intestine were observed. Changes in the level of cGMP play, seemingly, a more important role in the development of diarrhea in salmonellosis.

Topics: Adenosine Monophosphate; Adenylyl Cyclases; Animals; Cyclic AMP; Cyclic GMP; Diarrhea; Dimercaprol; Drug Evaluation, Preclinical; Guanylate Cyclase; In Vitro Techniques; Intestinal Mucosa; Intestine, Small; Male; Rabbits; Salmonella Infections, Animal; Salmonella typhimurium; Unithiol

Cardinal signs of red mold toxicosis in man and farm animals are vomiting, nausea, diarrhea, and food refusal. The red mold toxicosis has been suggested to be induced by trichothecenes, which are produced by Fusarium fungi. Fusarenon-X (F-X) is one of the trichothecene mycotoxins. The ip injection of F-X to rats causes an expansion of the small intestine and watery diarrhea. In this study, we measured the concentrations of protein, sodium, potassium, and calcium in the serum of rat treated with F-X for the sake of demonstrating the loss of serum protein and the decreases of serum sodium and calcium by F-X. Since it is well known that some diarrheal diseases are due to the increase of cyclic nucleotide level in the intestinal mucosa, we also measured cyclic AMP and cyclic GMP levels in the intestinal mucosa. It was demonstrated that F-X did not increase the cyclic AMP and cyclic GMP levels in the jejunal and the ileal mucosa at 8 and 24 hr after F-X treatment. The results obtained in this work suggest that F-X-induced diarrhea is not mediated by the cyclic nucleotide system.

Topics: Animals; Blood Proteins; Cyclic AMP; Cyclic GMP; Diarrhea; Electrolytes; Fusarium; Ileum; Intestinal Mucosa; Jejunum; Male; Mycotoxins; Rats; Rats, Inbred Strains; Sesquiterpenes; Trichothecenes

We studied possible mechanisms of magnesium sulfate (MgSO4)-induced diarrhea. In vivo perfusion of hamster small intestine with an isotonic electrolyte solution containing 50 mM MgSO4 produced nearly three times as much fluid secretion as did a solution containing an equiosmotic amount of mannitol. We found that magnesium was absorbed at a faster rate than mannitol under these conditions, suggesting that differences in solute permeability do not explain the differences in secretory rates. Magnesium ion rather than sulfate appeared largely responsible for the effect as replacement of sulfate with chloride did not diminish the response. MgSO4 perfusion of a proximal intestinal segment did not affect water transport in an isolated distal segment suggesting that release of cholecystokinin or alterations in serum levels of other hormones were not responsible. Intestinal permeability, morphology, and cyclic nucleotide levels were normal after MgSO4 perfusion. Thus, MgSO4-induced diarrhea cannot be explained by the usual mechanisms, and additional processes responsible for intestinal secretion must exist.

Topics: Animals; Cricetinae; Cyclic AMP; Cyclic GMP; Diarrhea; Intestinal Mucosa; Intestine, Small; Inulin; Magnesium Sulfate; Male; Mannitol; Mesocricetus; Osmosis; Permeability

Subcutaneously administered chlorpromazine reduced intestinal fluid accumulation induced by Escherichia coli heat-stable enterotoxin in infant mice. The antisecretory effect of chlorpromazine, although dose related, was, even with high doses, less than that observed with respect to cholera toxin. Whereas 100 micrograms chlorpromazine abolished cholera toxin-induced intestinal secretion almost completely, 500 microgram chlorpromazine (equivalent to 200 microgram/g body wt) lowered secretion induced by heat-stable enterotoxin by only 41%. The effect of chlorpromazine on intestinal secretion was quantitatively similar regardless of whether heat-stable enterotoxin or the cyclic GMP analogue, 8-Br-cyclic GMP, was the secretagogue. This finding, which suggested that the inhibitory effect of chlorpromazine on heat-stable enterotoxin was independent of guanylate cyclase, was confirmed by assaying this enzyme in intestinal homogenates from mice that had been inoculated with chlorpromazine, and also in experiments in which chlorpromazine was added to guanylate cyclase assay mixtures in vitro. Caution is advised before chlorpromazine is routinely adopted for the treatment of all syndromes of watery diarrhea.

Topics: Animals; Chlorpromazine; Cholera Toxin; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Guanylate Cyclase; Hot Temperature; Intestinal Mucosa; Intestines; Mice; Rabbits

Topics: Adenylyl Cyclases; Animals; Cholera; Cholera Toxin; Cyclic AMP; Cyclic GMP; Diarrhea; Drug Stability; Electrolytes; Enterotoxins; Enzyme Activation; Escherichia coli; Ileum; Intestinal Mucosa; Rabbits

Topics: Animals; Bacterial Toxins; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Guanosine Monophosphate; Intestinal Mucosa; Mice; Rabbits

Topics: Adolescent; Adult; Cholera; Colitis, Ulcerative; Cyclic AMP; Cyclic GMP; Diarrhea; Female; Humans; Male; Middle Aged

A case of chronic secretory diarrhea with elevated plasma vasoactive intestinal peptide (VIP) and serum gastrin levels is described. Plasma secretin, glucagon, insulin, and cyclic adenosine and guanine monophosphate (cAMP and (CGMP) concentrations were normal. Administration of a prostaglandin synthetase inhibitor failed to decrease the volume of diarrhea. There was no evidence of laxative abuse, antral cell hyperplasia, gastric hypersecretion, or pancreatic hypersecretion. The pancreatic histology was interpreted as islet cell hyperplasia. Jejunal tissue cAMP and cGMP concentrations were in the same range as those obtained from three control subjects. This report suggests that cyclic nucleotides may not mediate intestinal secretion in hormone-induced diarrhea.

Topics: Aged; Cyclic AMP; Cyclic GMP; Diarrhea; Female; Humans; Jejunum; Vasoactive Intestinal Peptide

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adult; Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Diarrhea, Infantile; Enterotoxins; Epithelial Cells; Epithelium; Escherichia coli Infections; Fluorides; Guanosine Triphosphate; Guanylate Cyclase; Humans; Infant; Intestinal Mucosa; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes; Prostaglandins; Temperature; Tritium