Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding of Escherichia coli strain 431 heat-stable enterotoxin (STa) and activation of intestinal particulate guanylate cyclase by E. coli STa were studied with rat intestinal epithelial cells and brush border membranes (BBMs). The rates of guanylate cyclase stimulation by 431 STa in cells and BBMs were rapid, with maximal levels of cyclic GMP observed within 5 min. Specific binding of 125I-labeled STa from E. coli 431 (431 125I-STa) and activation of guanylate cyclase by unlabeled 431 STa were observed with intestinal BBMs; however, neither was detected with membranes from nonintestinal tissues. The STa receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a nondenaturing dipolar ionic detergent, in yields of approximately 50%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the detergent-solubilized receptor-431 125I-STa complex, followed by autoradiography, showed that 431 125I-STa bound to a single BBM component with a molecular weight of about 100,000. Binding of 431 STa to its solubilized receptor was saturable, specific, and essentially irreversible. Pretreatment of the soluble receptor with trypsin and pronase but not chymotrypsin decreased binding of 431 125I-STa. The 431 STa-receptor complex was dissociated by boiling in the presence of 1% sodium dodecyl sulfate, incubation with 0.5 M acetic acid, or reduction with dithiothreitol. In contrast to the residual particulate guanylate cyclase activity of detergent-treated membranes, solubilized guanylate cyclase was not stimulated by STa. Membrane structure appears to play an important role in the coordination of STa binding and stimulation of guanylate cyclase activity.
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PMID:Solubilization and partial characterization of the intestinal receptor for Escherichia coli heat-stable enterotoxin. 615 10

Diarrheagenic strains of Escherichia coli have been shown to produce a heat-stable enterotoxin (ST) that simulates guanylate cyclase, increases short-circuit current (Isc), and inhibits active Cl absorption in the intestine. In rabbit ileum, the ion transport effects are smaller than those produced by cAMP-related agonists. Because ST may be a selective cGMP agonist, we further explored its mode of action in rabbit ileum. ST inhibits net Na and net Cl absorption. ST also inhibits the same fraction of Cl influx across the brush border that theophylline inhibits. At maximal doses, ST and 8-bromo-cGMP (8-Br-cGMP) had nearly equal, nonadditive effects of Isc that were about 66% of that produced by 8-Br-cAMP. ST increased mucosal cGMP concentration 16-fold, whereas epinephrine, an inhibitor of secretion, increased cGMP concentration by only 30%. This is insufficient to alter ion transport because doses of ST that increased cGMP concentration by 100% failed to alter Cl fluxes. Furthermore, epinephrine did not increase cGMP concentration in isolated enterocytes. We conclude that 1) cGMP mediates ST effects on ion transport, and 2) although ST and cAMP-related agonists have the same antiabsorptive effects, ST is less effective in stimulating electrogenic Cl secretion.
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PMID:cGMP modulation of ileal ion transport: in vitro effects of Escherichia coli heat-stable enterotoxin. 628 16

The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of brush border membranes reduced binding of 431 125I-STa by about 30%, suggesting that the STa receptor was a protein or a glycoprotein. The putative STa receptor was radiolabeled with 431 125I-STa and solubilized with sodium deoxycholate. One major radioactive band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. These data suggested that STas bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.
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PMID:Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes. 653 47

Escherichia coli heat stable enterotoxin (STa) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared STa binding and affinity crosslinking of STa receptors in human small intestine to those in the Caco-2 human colon carcinoma cell line. STa had similar kinetics of binding in human intestinal and Caco-2 brush border membranes. In both human intestine and Caco-2 brush border membranes, multiple specifically radiolabeled bands, including a 140-165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned STa receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco-2 cells could be explained by the existence of several members of a family of STa receptors and/or the existence of smaller STa binding proteins generated by the protease cleavage of a larger complete STa receptor.
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PMID:Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco-2). 810 Feb 32

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors. 810 72

Guanylin, a bioactive peptide, has recently been isolated from the intestine; this peptide activates intestinal guanylate cyclase (i.e., guanylate cyclase C) and thus is potentially involved in the regulation of water/electrolyte transport in the gastrointestinal mucosa. As yet, the cells involved in synthesis, storage, or secretion of guanylin have not been identified by immunocytochemistry. We raised antisera against guanylin and investigated the entire gastrointestinal tract of guinea pigs by light and electron microscopical immunocytochemistry. Extracts of various intestinal segments and plasma analyzed on a Western blot revealed a peptide band corresponding to the molecular mass of guanylin. Localization studies in the entire digestive tract showed that guanylin is exclusively confined to enterochromaffin (EC) cells. Remarkably, most EC cells contacted the gut lumen by cell processes that were highly immunoreactive for guanylin. In addition to the well known secretion in an endocrine fashion, EC cells by circumstantial evidence may release guanylin into the gut lumen to activate guanylate cyclase C that is immediately located on the brush border of adjacent enterocytes. The unique localization of guanylin in EC cells may indicate that these cells are involved in the regulation of fluid secretion in the gastrointestinal mucous membrane.
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PMID:Enterochromaffin cells of the digestive system: cellular source of guanylin, a guanylate cyclase-activating peptide. 815 83

Regulation of intestinal epithelial differentiation is critical to normal function, malignant transformation, and healing. However, the intracellular regulation of intestinal epithelial differentiation is incompletely understood. We studied the effects of intracellular cyclic AMP and cyclic GMP on brush border enzyme activity in the human Caco-2 intestinal epithelial cell using pharmacologic agonists and antagonists of cAMP and cGMP mediated pathways as probes. The stable cyclic nucleotide analogs dibutyryl cAMP and dibutyryl cGMP selectively decreased Caco-2 dipeptidyl dipeptidase specific activity while increasing alkaline phosphatase. The inhibitors of adenylate and guanylate cyclase KT5720 and KT5823 each exerted the opposite effects. Combinations of dibutyryl cAMP and dibutyryl cGMP demonstrated synergistic effects on each brush border enzyme but KT5720 and KT5823 were less than additive. Thus, cAMP and CGMP may regulate human intestinal epithelial differentiation by interacting pathways.
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PMID:Regulation of human Caco-2 intestinal epithelial brush border enzyme activity by cyclic nucleotides. 861 19

The heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli are classified into two groups, methanol-soluble (STI) and methanol-insoluble (STII) enterotoxins. These are distinct toxins with unique properties. Their features in common include heat-stability, low molecular weight, secretion from the bacteria, and ability to induce fluid secretion from the intestine. STI is an 18- or 19-amino acid extracellular peptide with three intramolecular disulfide bonds, which is produced by proteolytic cleavage of 72 amino acid precursor. The STI in the lumen of the intestine binds to specific protein receptors (guanylate cyclase C) located in the brush border membrane and leads to elevation of intracellular cyclic GMP level. Several factors involved in the activation of guanylate cyclase by STI have been identified. Elevation of cyclic GMP level induces intestinal fluid secretion by stimulation of chloride secretion. Cystic fibrosis transmembrane conductance regulator, which is a chloride channel, might be involved in chloride secretion. In contrast, STII is a 48-amino acid peptide with two intramolecular disulfide bonds, which results from 71 amino acid precursor. Compared with STI, the steps that lead to intestinal fluid secretion by STII are not well established. It has been proposed that sulfatide in the brush border is a receptor for STII and that the STII bound to the receptor opens GTP-binding regulatory protein-linked calcium channels. These actions of STII induce not only stimulation of the production of secretagogues such as prostaglandin E2 and serotonin, but also activation of the calcium-calmodulin-dependent protein kinase II in the cells.
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PMID:Properties and actions of heat-stable enterotoxin of Escherichia coli. 1099 26

Inhibition of proximal tubular phosphate (Pi) reabsorption involves, as far as we know, brush border membrane retrieval of the type IIa Na/Pi-cotransporter. The aim of the present study was to analyze whether intracellular cGMP-mediated regulation of Pi reabsorption also involves retrieval of the type IIa Na/Pi-cotransporter, as previously shown for cAMP. Atrial natriuretic peptide (ANP) and nitric oxide (NO) were used to stimulate guanylate cyclase. In vivo perfusion of mice kidneys with either ANP or NO donors resulted in a downregulation of type IIa Na/Pi-cotransporters on the brush border membranes of proximal tubules. These effects were mimicked by activation of protein kinase G with 8Br-cGMP. In in-vitro-perfused mice proximal tubules, ANP was effective when added either to the apical or basolateral perfusate, suggesting the presence of receptors on both membrane sites. The effects of ANP and NO were blocked by the protein kinase G inhibitor LY 83553. Parallel experiments in OK cells, a renal proximal tubule model, provided similar information. Our findings document that cGMP-mediated regulation (ANP and NO) of type IIa Na/Pi-cotransporters also takes place via internalization of the transporter protein.
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PMID:Regulation of the renal type IIa Na/Pi cotransporter by cGMP. 1171 58

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.
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PMID:Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea. 1855 51


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