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Leonard R Forte - One of the best experts on this subject based on the ideXlab platform.
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REVIEW 4 uanylin: a peptide regulator of epithelial transport1
2013Co-Authors: Leonard R Forte, Mark Cur. C. RieAbstract:ABSTRACT Regulation of intestinal salt and water transport is critical to the maintenance of fluid volume. Control of this life-sustaining activity is mediated by the concerted actions of hormones, neurotransmitters, and locally acting factors, The intestinal peptide Guanylin is ideally suited to play a pivotal role in this regulation. Guanylin is produced by the epithelium and appears to be secreted mucosally to act locally on an apical receptor. The Guanylin receptor is a member of the guanylate cyclase (GC-C) family of proteins. Elevation of intracellular cyclic GMP by Guanylin mediates the stimulation of C1 secretion, which results in the increased intestinal fluid secretion. ProGuanylin is found in the circulation and GC-C occurs in other epithelia, suggesting that Guanylin plays an endocrine role by regulating the function of tissues such as the kidney and liver. UroGuanylin is a structurally related peptide that is abundant in urine, has biological activity similar to Guanylin, and appears to be made by the intestine. This peptide may link the intestine and kidney in an endocrine pathway for control of renal salt excretion. Overproduction of Guanylin/uroGuanylin would be expected to elicit secretory diarrhea similar to that caused by the bacteria that produce peptide analogs of these endogenous peptide hormones. This unique molecular mimicry has provided clues leading to the discovery of Guanylin and insight into the mechanism of action of these intestinal peptides. The discoveries of Guanylin and uroGuanylin have provided exciting opportunities for further enhancing our understanding of epithelial transport and function.- Forte, L. R., Currie, M. C. Guanylin: a peptide regulator of epithelial transport
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interaction of atrial natriuretic peptide urodilatin Guanylin and uroGuanylin in the isolated perfused rat kidney
Regulatory Peptides, 2006Co-Authors: Messias S Santosneto, Helena Serra Azul Monteiro, Andre F Carvalho, Leonard R Forte, Manassés C. FontelesAbstract:Abstract Escherichia coli heat-stable enterotoxin (STa), Guanylin and uroGuanylin are novel natriuretic and kaliuretic peptides that bind to and activate membrane guanylate cyclase (GC) receptors such as GC-C and OK-GC that are expressed in the kidney and intestine. Atrial natriuretic peptide (ANP) and its renal form (urodilatin, UROD) elicit natriuretic effects by activation of a different membrane guanylate cyclase, GC-A. Experiments were done in perfused rat kidneys to search for possible synergistic interactions between ANP, UROD, Guanylin and uroGuanylin on renal function. Pretreatment with ANP (0.03 nM) enhanced Guanylin (0.19 μM) natriuretic activity (%ENa+; from 18.5 ± 4.25 to 31.5 ± 1.69, P
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UroGuanylin and Guanylin peptides: pharmacology and experimental therapeutics.
Pharmacology & therapeutics, 2004Co-Authors: Leonard R ForteAbstract:Guanylin, uroGuanylin, and the bacterial heat-stable enterotoxin (ST) peptides comprise a new family of cyclic guanosine 3'-5' monophosphate (cGMP)-regulating agonists. The discovery of Guanylin and uroGuanylin peptides stems from studies of cellular mechanisms underlying a form of secretory diarrhea caused by enteric bacteria. Guanylin, uroGuanylin, and microbial ST peptides activate a common apical membrane receptor-guanylate cyclase (R-GC) that elicits large increases in the intestinal secretion of chloride and bicarbonate via the intracellular second messenger, cGMP. Guanylin and uroGuanylin were isolated from rat jejunum and opossum urine, respectively. These peptides are endogenous peptide hormones that physiologically regulate R-GC signaling proteins in target cells. Physiological roles for these peptides include the regulation of epithelial cell balance in the intestinal epithelium and modulation of sodium balance through actions in the kidney. The Guanylin-uroGuanylin-ST peptides are candidate therapeutic agents targeting receptors in the intestine, kidney, and other epithelia. For example, uroGuanylin has anti-tumor actions in an animal model for human colon cancer. The ST peptides can be used as diagnostic agents to detect secondary colon cancers by single photon-emitting computed tomography (SPECT) imaging, thus localizing metastatic forms of colon cancer. Other examples of potential therapeutic applications for the Guanylin family of cGMP-regulating agonists are: (1) the irritable bowel syndrome (IBS) with constipation, (2) salt-dependent forms of high blood pressure, (3) liver regeneration and repair, and (4) respiratory diseases such as asthma. Competitive pharmacological antagonists of bacterial ST peptides offer a means for treating the diarrhea caused by ST-secreting strains of enteric bacteria.
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Guanylin and its lysine-containing analogue in the isolated perfused rat kidney: interaction with chymotrypsin inhibitor.
Pharmacology & toxicology, 2003Co-Authors: Messias S. Santos-neto, Helena Serra Azul Monteiro, Andre F Carvalho, Leonard R Forte, Stephen L Carrithers, Richard Greenberg, Manassés C. FontelesAbstract:Guanylin and uroGuanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GMP. Their natriuretic and kaliuretic activities are well documented. Since Guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of Guanylin. Using the isolated perfused rat kidney, Guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-Guanylin (0.1-2.5 microg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 microg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 microg/ml), Guanylin (0.3 microg/ml) was placed into solution. A maximal decrease in fractional Na+ reabsorption (%TNa+) was achieved at 1.0 microg/ml of lys-Guanylin (from 73.25+/-2.29 to 54.97+/-0.10, P
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Guanylin and its lysine containing analogue in the isolated perfused rat kidney interaction with chymotrypsin inhibitor
Pharmacology & Toxicology, 2003Co-Authors: Messias S Santosneto, Helena Serra Azul Monteiro, Andre F Carvalho, Leonard R Forte, Richard N. Greenberg, Stephen L Carrithers, Manassés C. FontelesAbstract:Guanylin and uroGuanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GMP Their natriuretic and kaliuretic activities are well documented. Since Guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of Guanylin. Using the isolated perfused rat kidney, Guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-Guanylin (0.1-2.5 μg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 μg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 μg/ml), Guanylin (0.3 μg/ml) was placed into solution. A maximal decrease in fractional Na + reabsorption (%TNA + ) was achieved at 1.0 μg/ml of lys-Guanylin (from 73.25±2.29 to 54.97±0.10, P<0.05). Lys-Guanylin (1.0 μg/ml) also decreased fractional K reabsorption (%TK + ) from 59.26±1.91 to 30.75±0.78 (P<0.05). Chymostatin had no detectable effects in electrolyte reabsorption in this assay. When introduced after chymostatin, Guanylin lowered %TNa + (from 81.2±1.86 to 72.6±2.45, P<0.05) and %TK + (from 69.4±4.12 to 65.8±2.81, P<0.05). At this subthreshold concentration, Guanylin alone lacks effects in %TNa + or %TK + . Furthermore, the ability of both peptides to promote increases in intestinal fluid secretion was evaluated in the in vivo suckling mouse model. When administered per os, Guanylin failed to stimulate intestinal secretion. When chymostatin was present in the test solution, Guanylin induced intestinal secretion in this assay. In marked contrast, lys-Guanylin alone induced diarrhoea in the suckling mouse. The present paper concludes that Guanylin undergoes metabolism in target tissues such as the intestine and kidney and its lysine-containing analogue retains full biological activity.
Yalcin Cetin - One of the best experts on this subject based on the ideXlab platform.
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Guanylin and functional coupling proteins in the hepatobiliary system of rat and guinea pig
Histochemistry and Cell Biology, 2012Co-Authors: Karen Schwabe, Yalcin CetinAbstract:Guanylin, a bioactive intestinal peptide, is involved in the cystic fibrosis transmembrane conductance (CFTR)-regulated electrolyte/water secretion in various epithelia. In the present work we report on the expression and cellular localization of Guanylin and its affiliated signaling and effector proteins, including guanylate cyclase C (Gucy2c), Proteinkinase GII (Pkrg2), CFTR and the solute carrier family 4, anion exchanger, member 2 (Slc4a2) in the hepatobiliary system of rat and guinea pig. Localization studies in the liver and the gallbladder revealed that Guanylin is located in the secretory epithelial cells of bile ducts of the liver and of the gallbladder, while Gucy2c, Pkrg2, CFTR, and Slc4a2 are confined exclusively to the apical membrane of the same epithelial cells. Based on these findings, we assume that Guanylin is synthesized as an intrinsic peptide in epithelial cells of the hepatobiliary system and released luminally into the hepatic and cystic bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.
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Guanylin regulates chloride secretion in the human gallbladder via the bile fluid.
Gastroenterology, 2004Co-Authors: Hasan Kulaksiz, Yalcin Cetin, Thorsten Schlenker, Daniel Rost, Adolf Stiehl, Martin Volkmann, Thomas Lehnert, Wolfgang StremmelAbstract:Abstract Background & Aims: The biliary epithelium of bile ducts and gallbladder modifies the composition of primary hepatic bile by absorption and secretion of an electrolyte-rich fluid. The underlying transport mechanisms, however, are still incompletely understood. We investigated the expression, the cellular localization, and the functional role of Guanylin, a bioactive intestinal peptide involved in the cystic fibrosis transmembrane conductance regulator (CFTR)-regulated electrolyte/water secretion, in the human gallbladder. Methods: Peptide-specific antibodies were raised to localize Guanylin and its affiliated signaling proteins, i.e., the Guanylin receptor, guanylate cyclase C (GC-C), cGMP-dependent protein kinase type II (cGKII), and CFTR in the human gallbladder and cholangiocarcinoma cells (Mz-Cha-1) by RT-PCR, Western blot, and immunocytochemistry. A sensitive ELISA was used to assess the range of Guanylin concentration in human bile fluid. The functional role of Guanylin was investigated in subconfluent Mz-Cha-1 cell monolayers by isotope efflux experiments. Results: Guanylin and its affiliated signaling proteins are highly expressed in the human gallbladder. Guanylin is localized to secretory epithelial cells of the gallbladder and is present in the bile, whereas GC-C, cGKII, and CFTR are confined exclusively to the apical membrane of the same epithelial cells. Functional studies in Mz-Cha-1 cells identify Guanylin as a specific regulator of biliary Cl − secretion that very likely is mediated by an intracellular increase of cGMP-concentration. Conclusions: Based on the present findings and on the functional role of Guanylin in other epithelia, it is likely that gallbladder epithelial cells synthesize and release Guanylin into the bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.
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Guanylin and functional coupling proteins in the human salivary glands and gland tumors : expression, cellular localization, and target membrane domains.
The American journal of pathology, 2002Co-Authors: Hasan Kulaksiz, Elisabeth Rehberg, Wolfgang Stremmel, Yalcin CetinAbstract:Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated secretion of an electrolyte-rich fluid is a major but incompletely understood function of the salivary glands. We provide molecular evidence that Guanylin, a bioactive intestinal peptide involved in the CFTR-regulated secretion of electrolyte/water in the gut epithelium, is highly expressed in the human parotid and submandibular glands and in respective clinically most relevant tumors. Moreover, in the same organs we identified expression of the major components of the Guanylin signaling pathway, ie, Guanylin-receptor guanylate cyclase-C, cGKII, and CFTR, as well as of the epithelial Cl − /HCO 3 − anion exchanger type 2 (AE2). At the cellular level, Guanylin is localized to epithelial cells of the ductal system that, based on its presence in the saliva, is obviously released into the salivary gland ducts. The Guanylin-receptor guanylate cyclase-C, cGKII, CFTR, and AE2 are all confined exclusively to the apical membrane of the same duct cells. These findings implicate Guanylin as intrinsic regulator of electrolyte secretion in the salivary glands. We assume that duct epithelial cells synthesize and release Guanylin into the saliva to regulate electrolyte secretion in the ductal system by an intraductal luminocrine signaling pathway. Moreover, the high expression of Guanylin in pleomorphic adenoma and Warthin tumors (cystadenolymphoma), the most common neoplasms of salivary glands, predicts Guanylin as a significant marker in tumor pathology.
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Guanylin in the human pancreas a novel luminocrine regulatory pathway of electrolyte secretion via cgmp and cftr in the ductal system
Histochemistry and Cell Biology, 2001Co-Authors: Hasan Kulaksiz, Andreas Schmid, Matthias Honscheid, R Eissele, Jurgen Klempnauer, Yalcin CetinAbstract:Cystic fibrosis transmembrane conductance regulator (CFTR) is a channel and regulator protein that is crucially involved in transepithelial ion transport. In the exocrine pancreas, the CFTR-mediated secretion of an electrolyte-rich fluid is a major but as yet incompletely understood function. We show here that the peptide Guanylin is a specific activator of CFTR function in the human pancreas implicating regulation of pancreatic electrolyte secretion. Guanylin and its affiliated signaling and effector proteins including guanylate cyclase C, cGMP-dependent protein kinase II, CFTR, and the epithelial $$Cl^-/HCO_3^-$$ exchanger, anion exchanger 2, are highly expressed in the human pancreas. Guanylin is localized specifically to the typical centroacinar cells and proximal duct cells which, based on its additional presence in the pancreatic juice, is obviously released luminally into the pancreatic ducts. The Guanylin receptor and the respective functional downstream proteins are all confined to the apical membrane of the duct cells implicating an as yet unknown route of luminal regulatory pathway of electrolyte secretion in the ductal system. Functional studies in two different human pancreatic duct cell lines expressing the CFTR Cl– channel that is functionally intact in CAPAN-1 cells but defective (ΔF508) in CFPAC-1 cells clearly identify Guanylin as a specific regulator of pancreatic CFTR channel function. Whole-cell patch-clamp recordings in CAPAN-1 cells revealed that forskolin induces an increase of Cl– conductance mediated by cAMP. In contrast, Guanylin increased Cl– conductance in the same cells via cGMP but not cAMP; the respective membrane current was largely blockable by the sulfonylurea glibenclamide. In CFPAC-1 cells, however, neither Guanylin nor forskolin produced a current activation. Based on the present findings we conclude that Guanylin is an intrinsic pancreatic regulator of Cl– current activation in pancreatic duct cells via cGMP and CFTR. Remarkably, in the pancreas Guanylin may exert its function through an intriguing luminocrine mode via the pancreatic juice.
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Guanylin and uroGuanylin in the parotid and submandibular glands: potential intrinsic regulators of electrolyte secretion in salivary glands.
Histochemistry and cell biology, 2001Co-Authors: Hasan Kulaksiz, Tindaro G Renda, Ulrich Rausch, Rosa Vaccaro, Yalcin CetinAbstract:The intestinal peptides Guanylin and uroGuanylin regulate the electrolyte/water transport in the gastrointestinal epithelium via activation of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis gene product. Because a major but incompletely understood function of the salivary glands is the CFTR-mediated secretion of an electrolyte-rich fluid, we investigated the rat and guinea pig parotid and submandibular glands for expression, cellular distribution, and subcellular localization of Guanylin and uroGuanylin. RT-PCR analyses with Guanylin and uroGuanylin-specific primers revealed that both peptides are highly expressed in the parotid and submandibular glands. At the translational level, western blotting analyses with peptide-specific Guanylin and uroGuanylin antibodies identified the expected 12.5-kDa immunoreactive peptides in these organs. At the cellular level, Guanylin and uroGuanylin were exclusively confined to epithelial cells of the intralobular and interlobular ducts. At the subcellular level, the immunoreactivities were localized by preembedding immunoelectron microscopy to small vesicles which were concentrated at the apical part of the secretory epithelial cells. The expression and cell-specific localization of Guanylin and uroGuanylin in the salivary glands indicate that these peptides may be specifically involved in the regulation of CFTR-mediated electrolyte/water secretion in the salivary gland ductal system.
Mitchell B. Cohen - One of the best experts on this subject based on the ideXlab platform.
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TNFα suppresses Guanylin expression.
2013Co-Authors: Eleana Harmel-laws, Mitchell B. Cohen, Elizabeth A Mann, Kris A. SteinbrecherAbstract:Goblet cell-like HT29-18-N2 cells were treated with 10 ng/ml TNFα or 100 U/ml IFNγ for 24 hours and Guanylin mRNA and protein were measured. (A) Realtime RT-PCR analysis indicated that, unlike other goblet cell genes such as Muc2 and TFF3, Guanylin expression was substantially depressed by TNFα. n = 4 per group; *p
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Effect of secretagogues and pH on intestinal transport in Guanylin-deficient mice.
Biochimica et biophysica acta, 2004Co-Authors: Alan N Charney, Kris A. Steinbrecher, Richard W Egnor, Mitchell B. CohenAbstract:The small and large intestine secrete Guanylin, a peptide homologous to heat stable enterotoxin (STa) elaborated by enterotoxigenic Escherichia coli. Guanylin's role in intestinal electrolyte transport was investigated in Guanylin-deficient knockout mice and heterozygous littermate controls. Segments of mid-jejunum, distal ileum, and proximal and distal colon were studied in Ussing chambers in HCO3- Ringer under short circuit conditions. We found that (1) under basal conditions, all segments in control and knockout mice absorb Na+, and the knockout mouse proximal colon secretes Cl-; (2) all segments except the jejunum of knockout mice respond by increasing absorption in response to reductions in pH from 7.6 to 7.1; (3) all segments exhibit decreased absorption in response to 1 mM cAMP; (4) the jejunum and ileum of knockout and control mice, and the proximal colon of control mice (but not knockout mice) respond to the mucosal addition of 50 nM STa with decreases in absorption; and (5) mucosal Guanylin caused similar decreases in proximal colon absorption in control and Guanylin-deficient mice. These findings suggest that Guanylin deficiency causes basal Cl- secretion and reduced responsiveness to STa in mouse proximal colon. The effectiveness of Guanylin in this segment suggests a difference in the intestinal secretory actions of STa and Guanylin.
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Coordinate upregulation of Guanylin and uroGuanylin expression by hypertonicity in HT29-18-N2 cells
American journal of physiology. Cell physiology, 2002Co-Authors: Kris A. Steinbrecher, Jeffrey A. Rudolph, Guangju Luo, Mitchell B. CohenAbstract:Guanylin and uroGuanylin are particulate guanylate cyclase-activating peptides that are secreted from the epithelia of the intestine, kidney, pancreas, and salivary gland. These peptides elicit chloride and bicarbonate secretion via the cystic fibrosis transmembrane conductance regulator. To test the hypothesis that hypertonicity mediates an increase in Guanylin and uroGuanylin mRNA, we subjected HT29-18-N2 to osmotic stress. Guanylin and uroGuanylin RNA were increased substantially in the presence of hypertonicity but only with solutes that were relatively impermeable to the cell membrane. This hypertonicity-mediated increase was transcriptional and did not require protein synthesis. Herbimycin A and mitogen-activated protein kinase inhibitors SB-203580 and PD-98059 had no effect on basal or induced levels of Guanylin or uroGuanylin. Both staurosporine and prolonged exposure to phorbol ester reduced basal levels and completely blocked hypertonicity-related increases in Guanylin or uroGuanylin RNA. These data suggest that serine/theonine protein kinases, possibly protein kinase C (PKC), mediate the hypertonicity-associated increase in Guanylin and uroGuanylin RNA. We conclude that Guanylin and uroGuanylin are released in response to hypertonic stress and that regulation of these genes may be mediated by PKC isoforms.
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Targeted Inactivation of the Mouse Guanylin Gene Results in Altered Dynamics of Colonic Epithelial Proliferation
The American journal of pathology, 2002Co-Authors: Kris A. Steinbrecher, David P. Witte, Steve A. Wowk, Jeffrey A. Rudolph, Mitchell B. CohenAbstract:Heat-stable enterotoxin (STa), elaborated by enterotoxigenic Echerichia coli, is a worldwide cause of secretory diarrhea in infants and travelers. Both STa and Guanylin, a peptide structurally similar to STa, increase intracellular cGMP levels after binding to the same intestinal receptor, guanylate cyclase C (GC-C). Distinct from its role as an intestinal secretagogue, Guanylin may also have a role in intestinal proliferation, as Guanylin expression is lost in intestinal adenomas. To determine the function of Guanylin in intestinal epithelia, Guanylin null mice were generated using a Cre/loxP-based targeting vector. Guanylin null mice grew normally, were fertile and showed no signs of malabsorption. However, the levels of cGMP in colonic mucosa of Guanylin null mice were significantly reduced. The colonic epithelial cell migration rate was increased and increased numbers of colonocytes expressing proliferating cell nuclear antigen (PCNA) were present in crypts of Guanylin null mice as well. The apoptotic index was similar in Guanylin null mice and littermate controls. We conclude from these studies that loss of Guanylin results in increased proliferation of colonic epithelia. We speculate that the increase in colonocyte number is related to decreased levels of cGMP and that this increase in proliferation plays a role in susceptibility to intestinal adenoma formation and/or progression.
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increases in Guanylin and uroGuanylin in a mouse model of osmotic diarrhea are guanylate cyclase c independent
Gastroenterology, 2001Co-Authors: Kris A. Steinbrecher, Elizabeth A Mann, Ralph A Giannella, Mitchell B. CohenAbstract:Abstract Background & Aims: Guanylin and uroGuanylin are peptide hormones that are homologous to the diarrhea-causing Escherichia coli enterotoxins. These secretagogues are released from the intestinal epithelia into the intestinal lumen and systemic circulation and bind to the receptor guanylate cyclase C (GC-C). We hypothesized that a hypertonic diet would result in osmotic diarrhea and cause a compensatory down-regulation of Guanylin/uroGuanylin. Methods: Gut-to-carcass weights were used to measure fluid accumulation in the intestine. Northern and/or Western analysis was used to determine the levels of Guanylin, uroGuanylin, and GC-C in mice with osmotic diarrhea. Results: Wild-type mice fed a polyethylene glycol or lactose-based diet developed weight loss, diarrhea, and an increased gut-to-carcass ratio. Unexpectedly, 2 days on either diet resulted in increased Guanylin/uroGuanylin RNA and prohormone throughout the intestine, elevated uroGuanylin RNA, and prohormone levels in the kidney and increased levels of circulating prouroGuanylin. GC-C–deficient mice given the lactose diet reacted with higher gut-to-carcass ratios. Although they did not develop diarrhea, GC-C–sufficient and—deficient mice on the lactose diet responded with elevated levels of Guanylin and uroGuanylin RNA and protein. A polyethylene glycol drinking water solution resulted in diarrhea, higher gut-to-carcass ratios, and induction of Guanylin and uroGuanylin in both GC-C heterozygous and null animals. Conclusions: We conclude that this model of osmotic diarrhea results in a GC-C–independent increase in intestinal fluid accumulation, in levels of these peptide ligands in the epithelia of the intestine, and in prouroGuanylin in the kidney and blood. GASTROENTEROLOGY 2001;121:1191-1202
Mark G. Currie - One of the best experts on this subject based on the ideXlab platform.
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Structure and activity of uroGuanylin and Guanylin from the intestine and urine of rats
The American journal of physiology, 1997Co-Authors: Xiaohui Fan, Roslyn M. London, William J. Krause, Ronald H. Freeman, Sammy L. Eber, Mark G. Currie, F. Kent Hamra, Christine E. Smith, Leonard R ForteAbstract:UroGuanylin and Guanylin are related peptides that activate common guanylate cyclase signaling molecules in the intestine and kidney. UroGuanylin was isolated from urine and duodenum but was not detected in extracts from the colon of rats. Guanylin was identified in extracts from small and large intestine but was not detected in urine. UroGuanylin and Guanylin have distinct biochemical and chromatographic properties that facilitated the separation, purification, and identification of these peptides. Northern assays revealed that mRNA transcripts for uroGuanylin were more abundant in small intestine compared with large intestine, whereas Guanylin mRNA levels were greater in large intestine relative to small intestine. Synthetic rat uroGuanylin and Guanylin had similar potencies in the activation of receptors in T84 intestinal cells. Production of uroGuanylin and Guanylin in the mucosa of duodenum is consistent with the postulate that both peptides influence the activity of an intracellular guanosine 3',5'-cyclic monophosphate signaling pathway that regulates the transepithelial secretion of chloride and bicarbonate in the intestinal epithelium.
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Regulation of intestinal uroGuanylin/Guanylin receptor-mediated responses by mucosal acidity
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: F. Kent Hamra, Mark G. Currie, Sammy L. Eber, David T. Chin, Leonard R ForteAbstract:Guanylin and uroGuanylin are intestinal peptides that stimulate chloride secretion by activating a common set of receptor–guanylate cyclase signaling molecules located on the mucosal surface of enterocytes. High mucosal acidity, similar to the pH occurring within the fluid microclimate domain at the mucosal surface of the intestine, markedly enhances the cGMP accumulation responses of T84 human intestinal cells to uroGuanylin. In contrast, a mucosal acidity of pH 5.0 renders Guanylin essentially inactive. T84 cells were used as a model epithelium to further explore the concept that mucosal acidity imposes agonist selectivity for activation of the intestinal receptors for uroGuanylin and Guanylin, thus providing a rationale for the evolution of these related peptides. At an acidic mucosal pH of 5.0, uroGuanylin is 100-fold more potent than Guanylin, but at an alkaline pH of 8.0 Guanylin is more potent than uroGuanylin in stimulating intracellular cGMP accumulation and transepithelial chloride secretion. The relative affinities of uroGuanylin and Guanylin for binding to receptors on the mucosal surface of T84 cells is influenced dramatically by mucosal acidity, which explains the strong pH dependency of the cGMP and chloride secretion responses to these peptides. The Guanylin-binding affinities for peptide–receptor interaction were reduced by 100-fold at pH 5 versus pH 8, whereas the affinities of uroGuanylin for these receptors were increased 10-fold by acidic pH conditions. Deletion of the N-terminal acidic amino acids in uroGuanylin demonstrated that these residues are responsible for the increase in binding affinities that are observed for uroGuanylin at acidic pH. We conclude that Guanylin and uroGuanylin evolved distinctly different structures, which enables both peptides to regulate, in a pH-dependent fashion, the activity of receptors that control intestinal salt and water transport via cGMP.
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regulation of intestinal uroGuanylin Guanylin receptor mediated responses by mucosal acidity
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Kent F Hamra, Mark G. Currie, Sammy L. Eber, David T. Chin, Leonard R ForteAbstract:Guanylin and uroGuanylin are intestinal peptides that stimulate chloride secretion by activating a common set of receptor–guanylate cyclase signaling molecules located on the mucosal surface of enterocytes. High mucosal acidity, similar to the pH occurring within the fluid microclimate domain at the mucosal surface of the intestine, markedly enhances the cGMP accumulation responses of T84 human intestinal cells to uroGuanylin. In contrast, a mucosal acidity of pH 5.0 renders Guanylin essentially inactive. T84 cells were used as a model epithelium to further explore the concept that mucosal acidity imposes agonist selectivity for activation of the intestinal receptors for uroGuanylin and Guanylin, thus providing a rationale for the evolution of these related peptides. At an acidic mucosal pH of 5.0, uroGuanylin is 100-fold more potent than Guanylin, but at an alkaline pH of 8.0 Guanylin is more potent than uroGuanylin in stimulating intracellular cGMP accumulation and transepithelial chloride secretion. The relative affinities of uroGuanylin and Guanylin for binding to receptors on the mucosal surface of T84 cells is influenced dramatically by mucosal acidity, which explains the strong pH dependency of the cGMP and chloride secretion responses to these peptides. The Guanylin-binding affinities for peptide–receptor interaction were reduced by 100-fold at pH 5 versus pH 8, whereas the affinities of uroGuanylin for these receptors were increased 10-fold by acidic pH conditions. Deletion of the N-terminal acidic amino acids in uroGuanylin demonstrated that these residues are responsible for the increase in binding affinities that are observed for uroGuanylin at acidic pH. We conclude that Guanylin and uroGuanylin evolved distinctly different structures, which enables both peptides to regulate, in a pH-dependent fashion, the activity of receptors that control intestinal salt and water transport via cGMP.
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Guanylyl cyclase receptors and Guanylin-like peptides in reptilian intestine
General and comparative endocrinology, 1997Co-Authors: William J. Krause, Ronald H. Freeman, Sammy L. Eber, Mark G. Currie, F. Kent Hamra, Leonard R ForteAbstract:Abstract Receptors for Guanylin and uroGuanylin were identified on the mucosal surface of enterocytes lining the intestine of the bobtail skink ( Tiliqua rugosa ), king's skink ( Egernia kingii ), and knight anole ( Anolis equestris ) by receptor autoradiography using 125 I-ST ( Escherichia coli heat-stable enterotoxin) as the radioligand. Specific, high-affinity binding of 125 I-ST to receptors was found on the microvillus border of enterocytes and little or no specific binding of 125 I-ST was observed in other strata comprising the gut wall. The American alligator ( Alligator mississippensis ) also exhibited receptor binding, but unlike the other three species had relatively high levels of apparent nonspecific binding. A comparison of intestinal cGMP accumulation responses between the American alligator and the knight anole demonstrated a greater magnitude of cGMP responses to ST and Guanylin in vitro in the knight anole relative to the tissue cGMP accumulation responses of alligators. Treatment with ST resulted in markedly greater tissue cGMP accumulation responses in both species compared to treatment with Guanylin. To complete a paracrine signaling pathway in reptilian intestine, Guanylin-like peptides that stimulated cGMP accumulation in human T 84 intestinal cells were isolated from the intestinal mucosa of alligators. We conclude that functional receptor-guanylyl cyclases and one or more endogenous Guanylin/uroGuanylin-like peptides occur in the intestinal tract of reptiles as well as in the intestines of mammals and birds. Thus, higher vertebrates have a conserved signaling pathway that regulates intestinal function through the first-messenger peptides, Guanylin and/or uroGuanylin, and the intracellular second messenger, cGMP.
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Opossum colonic mucosa contains uroGuanylin and Guanylin peptides
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1996Co-Authors: F K Hamra, William J. Krause, Ronald H. Freeman, Sammy L. Eber, Mark G. Currie, Christine E. Smith, Leonard R ForteAbstract:UroGuanylin and Guanylin are structurally related peptides that activate an intestinal form of membrane guanylate cyclase (GC-C). Guanylin was isolated from the intestine, but uroGuanylin was isolated from urine, thus a tissue source for uroGuanylin was sought. In these experiments, uroGuanylin and Guanylin were separated and purified independently from colonic mucosa and urine of opossums. Colonic, urinary, and synthetic forms of uroGuanylin had an isoelectric point of approximately 3.0, eluted from C18 reverse-phase high-performance liquid chromatography (RP-HPLC) columns at 8-9% acetonitrile, elicited greater guanosine 3', 5'-cyclic monophosphate (cGMP) responses in T84 cells at pH 5.5 than pH 8, and were not cleaved and inactivated by pretreatment with chymotrypsin. In contrast, colonic, urinary, and synthetic Guanylin had an isoelectric point of approximately 6.0, eluted at 15-16% acetonitrile on C18 RP-HPLC columns, stimulated greater cGMP responses in T84 cells at pH 8 than pH 5.5, and were inactivated by chymotrypsin, which hydrolyzed the Phe-Ala or Try-Ala bonds within Guanylin. UroGuanylin joins Guanylin as an intestinal peptide that may participate in an intrinsic pathway for cGMP-mediated regulation of intestinal salt and water transport. Moreover, uroGuanylin and Guanylin in urine may be derived from the intestinal mucosa, thus implicating these peptides in an endocrine mechanism linking the intestine with the kidney.
Hasan Kulaksiz - One of the best experts on this subject based on the ideXlab platform.
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Guanylin regulates chloride secretion in the human gallbladder via the bile fluid.
Gastroenterology, 2004Co-Authors: Hasan Kulaksiz, Yalcin Cetin, Thorsten Schlenker, Daniel Rost, Adolf Stiehl, Martin Volkmann, Thomas Lehnert, Wolfgang StremmelAbstract:Abstract Background & Aims: The biliary epithelium of bile ducts and gallbladder modifies the composition of primary hepatic bile by absorption and secretion of an electrolyte-rich fluid. The underlying transport mechanisms, however, are still incompletely understood. We investigated the expression, the cellular localization, and the functional role of Guanylin, a bioactive intestinal peptide involved in the cystic fibrosis transmembrane conductance regulator (CFTR)-regulated electrolyte/water secretion, in the human gallbladder. Methods: Peptide-specific antibodies were raised to localize Guanylin and its affiliated signaling proteins, i.e., the Guanylin receptor, guanylate cyclase C (GC-C), cGMP-dependent protein kinase type II (cGKII), and CFTR in the human gallbladder and cholangiocarcinoma cells (Mz-Cha-1) by RT-PCR, Western blot, and immunocytochemistry. A sensitive ELISA was used to assess the range of Guanylin concentration in human bile fluid. The functional role of Guanylin was investigated in subconfluent Mz-Cha-1 cell monolayers by isotope efflux experiments. Results: Guanylin and its affiliated signaling proteins are highly expressed in the human gallbladder. Guanylin is localized to secretory epithelial cells of the gallbladder and is present in the bile, whereas GC-C, cGKII, and CFTR are confined exclusively to the apical membrane of the same epithelial cells. Functional studies in Mz-Cha-1 cells identify Guanylin as a specific regulator of biliary Cl − secretion that very likely is mediated by an intracellular increase of cGMP-concentration. Conclusions: Based on the present findings and on the functional role of Guanylin in other epithelia, it is likely that gallbladder epithelial cells synthesize and release Guanylin into the bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.
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clara cell impact in air side activation of cftr in small pulmonary airways
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Hasan Kulaksiz, Andreas Schmid, Matthias Honscheid, Annette Ramaswamy, Y. CetinAbstract:The Clara cells are nonciliated, nonmucous, secretory cells containing characteristic peptidergic granules; they constitute up to 80% of the epithelial cell population of the distal airways. Despite this exposed histotopology and abundance within the terminal airways where fluid secretion is of pivotal importance, the functional role of the Clara cells remained poorly understood. At the transcriptional, translational, and cellular levels, we provide evidence that the Clara cells are well equipped with the bioactive peptide Guanylin and proteins of the cGMP-signaling system including guanylate cyclase C, cGMP-dependent protein kinase II, and cystic fibrosis transmembrane conductance regulator (CFTR) together with the two CFTR scaffolding proteins EBP50/NHERF and E3KARP/NHERF-2 that are essential for proper function of CFTR. Guanylin was localized to secretory granules underneath the apical membrane of Clara cells and was, in addition, detected in high concentrations in bronchoalveolar lavage fluid, predicting release of the peptide luminally into the bronchiolar airways. On the other hand, the Guanylin-receptor guanylate cyclase C, CFTR, and proteins linked to CFTR activation and function were all confined to the adluminal membrane of Clara cells, implicating an intriguing air-side route of action of Guanylin. Whole-cell patch-clamp recordings in the Clara cell line H441 revealed that Guanylin activates CFTR Cl− conductance via the cGMP but not the cAMP-signaling pathway. Hence, in the critical location of distal airways in situ, the Clara cells may play the outstanding role of CFTR-dependent regulation of epithelial electrolyte/water secretion through a sophisticated paracrine/luminocrine mode of Guanylin-induced CFTR activation.
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Guanylin and functional coupling proteins in the human salivary glands and gland tumors : expression, cellular localization, and target membrane domains.
The American journal of pathology, 2002Co-Authors: Hasan Kulaksiz, Elisabeth Rehberg, Wolfgang Stremmel, Yalcin CetinAbstract:Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated secretion of an electrolyte-rich fluid is a major but incompletely understood function of the salivary glands. We provide molecular evidence that Guanylin, a bioactive intestinal peptide involved in the CFTR-regulated secretion of electrolyte/water in the gut epithelium, is highly expressed in the human parotid and submandibular glands and in respective clinically most relevant tumors. Moreover, in the same organs we identified expression of the major components of the Guanylin signaling pathway, ie, Guanylin-receptor guanylate cyclase-C, cGKII, and CFTR, as well as of the epithelial Cl − /HCO 3 − anion exchanger type 2 (AE2). At the cellular level, Guanylin is localized to epithelial cells of the ductal system that, based on its presence in the saliva, is obviously released into the salivary gland ducts. The Guanylin-receptor guanylate cyclase-C, cGKII, CFTR, and AE2 are all confined exclusively to the apical membrane of the same duct cells. These findings implicate Guanylin as intrinsic regulator of electrolyte secretion in the salivary glands. We assume that duct epithelial cells synthesize and release Guanylin into the saliva to regulate electrolyte secretion in the ductal system by an intraductal luminocrine signaling pathway. Moreover, the high expression of Guanylin in pleomorphic adenoma and Warthin tumors (cystadenolymphoma), the most common neoplasms of salivary glands, predicts Guanylin as a significant marker in tumor pathology.
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Guanylin in the human pancreas a novel luminocrine regulatory pathway of electrolyte secretion via cgmp and cftr in the ductal system
Histochemistry and Cell Biology, 2001Co-Authors: Hasan Kulaksiz, Andreas Schmid, Matthias Honscheid, R Eissele, Jurgen Klempnauer, Yalcin CetinAbstract:Cystic fibrosis transmembrane conductance regulator (CFTR) is a channel and regulator protein that is crucially involved in transepithelial ion transport. In the exocrine pancreas, the CFTR-mediated secretion of an electrolyte-rich fluid is a major but as yet incompletely understood function. We show here that the peptide Guanylin is a specific activator of CFTR function in the human pancreas implicating regulation of pancreatic electrolyte secretion. Guanylin and its affiliated signaling and effector proteins including guanylate cyclase C, cGMP-dependent protein kinase II, CFTR, and the epithelial $$Cl^-/HCO_3^-$$ exchanger, anion exchanger 2, are highly expressed in the human pancreas. Guanylin is localized specifically to the typical centroacinar cells and proximal duct cells which, based on its additional presence in the pancreatic juice, is obviously released luminally into the pancreatic ducts. The Guanylin receptor and the respective functional downstream proteins are all confined to the apical membrane of the duct cells implicating an as yet unknown route of luminal regulatory pathway of electrolyte secretion in the ductal system. Functional studies in two different human pancreatic duct cell lines expressing the CFTR Cl– channel that is functionally intact in CAPAN-1 cells but defective (ΔF508) in CFPAC-1 cells clearly identify Guanylin as a specific regulator of pancreatic CFTR channel function. Whole-cell patch-clamp recordings in CAPAN-1 cells revealed that forskolin induces an increase of Cl– conductance mediated by cAMP. In contrast, Guanylin increased Cl– conductance in the same cells via cGMP but not cAMP; the respective membrane current was largely blockable by the sulfonylurea glibenclamide. In CFPAC-1 cells, however, neither Guanylin nor forskolin produced a current activation. Based on the present findings we conclude that Guanylin is an intrinsic pancreatic regulator of Cl– current activation in pancreatic duct cells via cGMP and CFTR. Remarkably, in the pancreas Guanylin may exert its function through an intriguing luminocrine mode via the pancreatic juice.
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Guanylin and uroGuanylin in the parotid and submandibular glands: potential intrinsic regulators of electrolyte secretion in salivary glands.
Histochemistry and cell biology, 2001Co-Authors: Hasan Kulaksiz, Tindaro G Renda, Ulrich Rausch, Rosa Vaccaro, Yalcin CetinAbstract:The intestinal peptides Guanylin and uroGuanylin regulate the electrolyte/water transport in the gastrointestinal epithelium via activation of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis gene product. Because a major but incompletely understood function of the salivary glands is the CFTR-mediated secretion of an electrolyte-rich fluid, we investigated the rat and guinea pig parotid and submandibular glands for expression, cellular distribution, and subcellular localization of Guanylin and uroGuanylin. RT-PCR analyses with Guanylin and uroGuanylin-specific primers revealed that both peptides are highly expressed in the parotid and submandibular glands. At the translational level, western blotting analyses with peptide-specific Guanylin and uroGuanylin antibodies identified the expected 12.5-kDa immunoreactive peptides in these organs. At the cellular level, Guanylin and uroGuanylin were exclusively confined to epithelial cells of the intralobular and interlobular ducts. At the subcellular level, the immunoreactivities were localized by preembedding immunoelectron microscopy to small vesicles which were concentrated at the apical part of the secretory epithelial cells. The expression and cell-specific localization of Guanylin and uroGuanylin in the salivary glands indicate that these peptides may be specifically involved in the regulation of CFTR-mediated electrolyte/water secretion in the salivary gland ductal system.