uroguanylin and guanylin

Guanylate cyclase C (GC-C) receptor is a transmembrane receptor, predominantly expressed in intestinal epithelial cells, which is considered to play a main role in homeostasis and function of the digestive tract. The endogenous ligands for this receptor are the paracrine hormones uroguanylin and guanylin. Upon ligand binding, GC-C receptors increase cyclic guanosine monophosphate (cGMP) levels, regulating a variety of key cell-type specific processes such as chloride and bicarbonate secretion, epithelial cell growth, regulation of intestinal barrier integrity and visceral sensitivity. It has been suggested that GC-C acts as an intestinal tumor suppressor with the potential to prevent the initiation and progression of colorectal cancer. In fact, loss of ligand expression is a universal step in sporadic colorectal carcinogenesis. Interestingly, the role of GC-C is not limited to the digestive tract but it has been extended to several other systems such as the cardiovascular system, kidney, and the central nervous system, where it has been involved in a gut-hypothalamus endocrine axis regulating appetite. Objetive: In this review we summarize the physiology of the GC-C receptor and its ligands, focusing on newly developed drugs like linaclotide, and their suggested role to reverse/prevent the diseases in which the receptor is involved.. Available data points toward a relationship between uroguanylin and guanylin and their receptor and pathological processes like gastrointestinal and renal disorders, colorectal cancer, obesity, metabolic syndrome and mental disorders among others. Recent pharmacological developments in the regulation of GC-receptor may involve further improvements in the treatment of relevant diseases.

Topics: Animals; Colorectal Neoplasms; Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Kidney Diseases; Natriuretic Peptides; Obesity; Protein Binding; Protein Transport; Receptors, Peptide; Signal Transduction

Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

Topics: Animals; Carcinogenesis; Cell Cycle; Colonic Neoplasms; Cyclic GMP; Enterotoxins; Gastrointestinal Hormones; Genomics; Homeostasis; Hormones; Humans; Inflammation; Ligands; Mutation; Natriuretic Peptides; Paracrine Communication; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Treatment Outcome

Asthma is a prevalent respiratory disorder triggered by a variety of inhaled environmental factors, such as allergens, viruses, and pollutants. Asthma is characterized by an elevated activation of the smooth muscle surrounding the airways, as well as a propensity of the airways to narrow excessively in response to a spasmogen (i.e. contractile agonist), a feature called airway hyperresponsiveness. The level of airway smooth muscle (ASM) activation is putatively controlled by mediators released in its vicinity. In asthma, many mediators that affect ASM contractility originate from inflammatory cells that are mobilized into the airways, such as eosinophils. However, mounting evidence indicates that mediators released by remote organs can also influence the level of activation of ASM, as well as its level of responsiveness to spasmogens and relaxant agonists. These remote mediators are transported through circulating blood to act either directly on ASM or indirectly via the nervous system by tuning the level of cholinergic activation of ASM. Indeed, mediators generated from diverse organs, including the adrenals, pancreas, adipose tissue, gonads, heart, intestines, and stomach, affect the contractility of ASM. Together, these results suggest that, apart from a paracrine mode of regulation, ASM is subjected to an endocrine mode of regulation. The results also imply that defects in organs other than the lungs can contribute to asthma symptoms and severity. In this review, I suggest that the endocrine mode of regulation of ASM contractility is overlooked.

Topics: Adaptation, Physiological; Adiponectin; Androgens; Animals; Asthma; Bronchodilator Agents; Dehydroepiandrosterone; Epinephrine; Estrogens; Female; Fibrin; Gastrin-Releasing Peptide; Gastrointestinal Hormones; Glucocorticoids; Humans; Insulin; Leptin; Lung; Male; Muscle Contraction; Muscle, Smooth; Natriuretic Peptides; Progesterone; Respiratory Physiological Phenomena; Respiratory System; Theophylline; Thyroid Hormones; Urokinase-Type Plasminogen Activator

Guanylin (GN) and uroguanylin (UGN) are low-molecular-weight peptide hormones produced mainly in the intestinal mucosa in response to oral salt load. GN and UGN (guanylin peptides) induce secretion of electrolytes and water in both intestine and kidney. Thought to act as "intestinal natriuretic factors", GN and UGN modulate renal salt secretion by both endocrine mechanisms (linking the digestive system and kidney) and paracrine/autocrine (intrarenal) mechanisms. The cellular function of GN and UGN in intestine and proximal tubule is mediated by guanylyl cyclase C (GC-C)-, cGMP-, and G protein-dependent pathways, whereas, in principal cells of the cortical collecting duct (CCD), these peptide hormones act via GC-C-independent signaling through phospholipase A2 (PLA2). The Cl(-)/HCO(-)3 exchanger pendrin (SLC26A4), encoded by the PDS gene, is expressed in non-α intercalated cells of the CCD. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. Our recent studies have provided evidence that pendrin-mediated anion exchange in the CCD is regulated at the transcriptional level by UGN. UGN exerts an inhibitory effect on the pendrin gene promoter likely via heat shock factor 1 (HSF1) action at a defined heat shock element (HSE) site. Recent studies have unraveled novel roles for guanylin peptides in several organ systems including involvement in appetite regulation, olfactory function, cell proliferation and differentiation, inflammation, and reproductive function. Both the guanylin system and pendrin have also been implicated in airway function. Future molecular research into the receptors and signal transduction pathways involved in the action of guanylin peptides and the pendrin anion exchanger in the kidney and other organs, and into the links between them, may facilitate discovery of new therapies for hypertension, heart failure, hepatic failure and other fluid retention syndromes, as well as for diverse diseases such as obesity, asthma, and cancer.

Topics: Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Mucosa; Kidney Tubules, Collecting; Membrane Transport Proteins; Natriuretic Peptides; Signal Transduction; Sulfate Transporters; Transcription, Genetic

This review provides an overview of the molecular mechanisms of K transport in the mammalian connecting tubule (CNT) and cortical collecting duct (CCD), both nephron segments responsible for the regulation of renal K secretion. Aldosterone and dietary K intake are two of the most important factors regulating K secretion in the CNT and CCD. Recently, angiotensin II (AngII) has also been shown to play a role in the regulation of K secretion. In addition, genetic and molecular biological approaches have further identified new mechanisms by which aldosterone and dietary K intake regulate K transport. Thus, the interaction between serum-glucocorticoid-induced kinase 1 (SGK1) and with-no-lysine kinase 4 (WNK4) plays a significant role in mediating the effect of aldosterone on ROMK (Kir1.1), an important apical K channel modulating K secretion. Recent evidence suggests that WNK1, mitogen-activated protein kinases such as P38, ERK, and Src family protein tyrosine kinase are involved in mediating the effect of low K intake on apical K secretory channels.

Topics: Aldosterone; Angiotensin II; Animals; Extracellular Signal-Regulated MAP Kinases; Gastrointestinal Hormones; Immediate-Early Proteins; Kidney Tubules, Collecting; Natriuretic Peptides; p38 Mitogen-Activated Protein Kinases; Potassium; Potassium Channels, Inwardly Rectifying; Potassium, Dietary; Protein Serine-Threonine Kinases; src-Family Kinases

Guanylyl cyclase C (GCC) is the receptor specifically expressed by intestinal cells for the paracrine hormones guanylin and uroguanylin and diarrheagenic bacterial heat-stable enterotoxins. This tissue-specific receptor coordinates lineage-dependent regulation of epithelial homeostasis, and its disruption contributes to intestinal tumorigenesis. It coordinates regenerative and metabolic circuits by restricting the cell cycle and proliferation and programming metabolic transitions central to organizing the dynamic crypt-surface axis. Further, mice deficient in GCC signaling are more susceptible to colon cancer induced by Apc mutations or the carcinogen azoxymethane. Moreover, guanylin and uroguanylin are gene products most commonly lost, early, in colon cancer in animals and humans. The role of GCC as a tumor suppressing receptor regulating proliferation and metabolism, together with the universal loss of guanylin and uroguanylin in tumorigenesis, suggests a model in which colorectal cancer is a paracrine hormone deficiency syndrome. In that context, activation of GCC reverses the tumorigenic phenotype by limiting growth of colorectal cancer cells by restricting progression through the G1/S transition and reprogramming metabolic circuits from glycolysis to oxidative phosphorylation, limiting bioenergetic support for rapid proliferation. These observations suggest a pathophysiological hypothesis in which GCC is a lineage-dependent tumor suppressing receptor coordinating proliferative homeostasis whose dysregulation through hormone loss contributes to neoplasia. The correlative therapeutic hypothesis suggests that colorectal cancer is a disease of hormone insufficiency that can be prevented or treated by oral supplementation with GCC ligands.

Topics: Administration, Oral; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Epithelial Cells; Gastrointestinal Hormones; Guanylate Cyclase; Hormone Replacement Therapy; Humans; Interphase; Intestine, Large; Mice; Natriuretic Peptides; Organ Specificity; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

Agonists of guanylyl-C receptor, such as guanylin/uroguanylin, are correlated not only with the intestinal cell epithelial physiology but also with the colorectal cancer tumorigenesis. Activation of the second intracellular messenger cyclic guanosine monophosphate by guanylyl cyclase-C receptor results in a complex intracellular signalling cascade involving the phosphodiesterase, the ion channels and the protein kinase. After an analytical review of relevant new knowledge, new diagnostic and therapeutic approaches for colorectal cancer are discussed.

Topics: Animals; Colorectal Neoplasms; Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Ion Channels; Natriuretic Peptides; Phosphoric Diester Hydrolases; Protein Kinases; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

Guanylin peptides are secreted from the intestine and influence electrolyte and water transport in intestine and kidney, suggesting that these peptides act as intestinal natriuretic peptides. This review presents recent research on renal guanylin and uroguanylin effects.. After salty meals guanylin peptides are produced in the intestine activating anion secretion and inhibiting sodium absorption. In the kidney guanylin peptides induce saluresis and diuresis. The signaling of guanylin peptides in the intestine is well known, involving guanylate cyclase C and increases in cellular cGMP concentrations. As in the intestine in proximal tubule cells a cGMP and guanylate cyclase C-dependent signaling pathway exists. In guanylate cyclase C-deficient mice, renal effects are unaltered, which could be by explained by recently described new cGMP-independent signaling pathways. In proximal tubules, Uroguanylin activates a pertussis toxin-sensitive receptor. Another cGMP-independent signaling pathway of guanylin peptides involving phospholipase A2 and arachidonic acid is shown for principal cells of human and mouse cortical collecting ducts.. Mechanisms and sites of renal actions of guanylin peptides are still not completely understood. Renal receptors for guanylin peptides are probably G-protein-coupled. The influences of guanylin peptides on natriuresis, kaliuresis, and diuresis are complex and only further detailed studies will allow a complete understanding of the function of these peptides.

Topics: Amino Acid Sequence; Animals; Biological Transport; Electrolytes; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Kidney; Mice; Models, Biological; Molecular Sequence Data; Natriuretic Peptides; Sequence Alignment; Signal Transduction

Ingestion of a salty meal induces secretion of guanylin (GN) and uroguanylin (UGN) into the intestinal lumen, where they inhibit Na+ absorption and induce Cl-, HCO3-, and water secretion. Simultaneously, these hormones stimulate renal electrolyte excretion by inducing natriuresis, kaliuresis, and diuresis. GN and UGN therefore participate in the prevention of hypernatremia and hypervolemia after salty meals. The signaling pathway of GN and UGN in the intestine is well known. They activate enterocytes via guanylate cyclase C (GC-C), which leads to cGMP-dependent inhibition of Na+/H+ exchange and activation of the cystic fibrosis transmembrane regulator. In GC-C-deficient mice, GN and UGN still produce renal natriuresis, kaliuresis, and diuresis, suggesting different signaling pathways in the kidney compared with the intestine. Signaling pathways for GN and UGN in the kidney differ along the various nephron segments. In proximal tubule cells, a cGMP- and GC-C-dependent signaling was demonstrated for both peptides. In addition, UGN activates a pertussis toxin-sensitive G-protein-coupled receptor. A similar dual signaling pathway is also known for atrial natriuretic peptide. Recently, a cGMP-independent signaling pathway for GN and UGN was also shown in principal cells of the human and mouse cortical collecting duct. Because GN and UGN activate different signaling pathways in specific organs and even within the kidney, this review focuses on more recent findings on cellular effects and signaling mechanisms of these peptides and their pathophysiologic implications in the intestine and the kidney.

Topics: Animals; Biological Transport; Gastrointestinal Hormones; Humans; Hypernatremia; Intestinal Absorption; Kidney Tubules, Collecting; Kidney Tubules, Proximal; Mice; Natriuretic Peptides; Receptors, Peptide; Sensitivity and Specificity; Signal Transduction; Sodium, Dietary; Water Intoxication; Water-Electrolyte Imbalance

Topics: Animals; Gastrointestinal Hormones; Humans; Natriuretic Peptides; Salts

Guanylins and uroguanylins are natriuretic peptides with different effects in many of tissues. In context with guanylins, the intestine-renal axis is presented. The overproduction of guanylin or uroguanylin leads to secondary diarrhea with stimulation of Cl(-) secretion. A diet high in salt lead especially to increased guanylin and uroguanylin secretion. Interesting applications with guanylins measurement could to be in hypertension diagnosis, monitoring of heart dysfunction treatment, intensive care etc.

Topics: Gastrointestinal Hormones; Humans; Natriuretic Peptides

In contrast to their prohormones the mature peptide hormones guanylin and uroguanylin are not able to fold to their native disulfide connectivities upon oxidative folding. Structural properties of both peptide hormones and their precursor proteins as well as the role of their prosequences in proper disulfide coupled folding are reviewed. In addition, the structural behavior of a proguanylin mutant that closely resembles prouroguanylin has been investigated to gain further insight into structural properties of this homologous precursor protein.

Topics: Amino Acid Sequence; Disulfides; Gastrointestinal Hormones; Humans; Isomerism; Models, Molecular; Molecular Sequence Data; Natriuretic Peptides; Oxidation-Reduction; Peptides; Protein Folding; Protein Precursors; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment

After a salty meal, stimulation of salt excretion via the kidney is a possible mechanism to prevent hypernatremia and hypervolemia. Besides the well known hormonal regulators of salt and water excretion in the distal nephron, arginine vasopressin and aldosterone, guanylin (GN) peptides produced in the intestine were proposed to be intestinal natriuretic peptides. These peptides inhibit Na+ absorption in the intestine and induce natriuresis, kaliuresis and diuresis in the kidney. The signaling pathway of GN peptides in the intestine is well known. They activate enterocytes via guanylate cyclase C (GC-C) and increase the cellular concentration of cGMP which leads to secretion of Cl-, HCO3- and water into the intestinal lumen and to inhibition of Na+ absorption. Guanylin peptides are filtered in the glomerulus, and additionally synthesized and excreted by tubular cells. They activate receptors located in the luminal membrane of the tubular cells along the nephron. In GC-C deficient mice renal effects of GN peptides are retained. In human, rat, and opossum proximal tubule cells, a cGMP-dependent signaling was demonstrated, but in addition GN peptides apparently also activate a PT-sensitive G-protein coupled receptor. A similar dual signaling pathway is also known for other natriuretic peptides like atrial natriuretic peptide. A cGMP-independent signaling pathway of GN peptides is also shown for principal cells of the human cortical collecting duct where the final hormonal regulation of electrolyte homeostasis takes place. This review will focus on the current knowledge on renal actions of GN peptides and specifically address novel GC-C- and cGMP-independent signaling mechanisms.

Topics: Amino Acid Sequence; Animals; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Kidney; Kidney Tubules, Collecting; Kidney Tubules, Proximal; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sequence Homology, Amino Acid; Signal Transduction; Sodium, Dietary

Topics: Animals; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Water-Electrolyte Balance

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.

Topics: Amino Acid Sequence; Animals; Gastrointestinal Diseases; Gastrointestinal Hormones; Humans; Molecular Sequence Data; Natriuretic Peptides; Peptides; Receptors, Peptide

Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Polyps; Mice; Mice, Knockout; Models, Biological; Molecular Sequence Data; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

The 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. Guanylin and uroguanylin are novel peptides that were first isolated from rat jejunum and opossum urine, respectively. They bind to and activate guanylyl cyclase-C (GC-C) receptors to regulate intestinal and renal fluid and electrolyte transport through the second messenger, cyclic guanosine 3',5'-monophosphate (GMP). Heat-stable enterotoxins produced by pathogenic bacteria have close structural similarities to guanylin and uroguanylin, and they use this mimicry to act on GC-C, causing life-threatening secretory diarrhea. Guanylin primarily is restricted to the intestine, whereas uroguanylin is present in the stomach, kidney, lung, and pancreas, in addition to intestine. Guanylin and uroguanylin are secreted into the intestinal lumen and blood in response to sodium chloride administration. These peptides function in salt and water transport in the intestine and kidney by luminocrine and endocrine actions. The guanylin family is involved in the pathophysiology of some gastrointestinal, renal, and heart diseases.

Topics: Animals; Digestive System; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Natriuretic Peptides; Opossums; Peptides; Rats; RNA, Messenger; Water-Electrolyte Balance

More than 20% of adults in industrialized countries display arterial pressure outside the normal physiological range. For most individuals, the molecular basis of hypertension remains unknown. In some hypertensive persons, a postprandial natriuretic response, normally elicited by a salty meal, is diminished and contributes to body sodium accumulation and plasma volume expansion. An important physiological mechanism ensuring the increased salt excretion following ingestion of salt is based on a luminocrine and endocrine secretion of novel small intestinal peptides--guanylins. Membrane guanylate cyclase receptors mediate effects of these peptides that provide a novel link between the intestine and kidney by means of circulating molecular guanylin forms. It can be expected that the emergence of the novel guanylin signaling pathways will energize search for molecular defects causing hypertension.

Topics: Animals; Gastrointestinal Hormones; Humans; Hypertension; Intestines; Kidney; Natriuresis; Natriuretic Peptides; Peptides; Postprandial Period

The guanylin family of cGMP-regulating peptides has three subclasses of peptides containing either three intramolecular disulfides found in bacterial heat-stable enterotoxins (ST), or two disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These small, heat-stable peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity. Two receptor GC signaling molecules have been identified that are highly expressed in the intestine (GC-C) and/or the kidney (OK-GC) and are selectively activated by the guanylin peptides. Stimulation of cGMP production in renal target cells by guanylin peptides in vivo or ex vivo elicits a long-lived diuresis, natriuresis, and kaliuresis. Activation of GC-C receptors in target cells of intestinal mucosa markedly stimulates the transepithelial secretion of Cl(-) and HCO(-)/(3), causing enhanced secretion of fluid and electrolytes into the intestinal lumen. Bacterial ST peptides act as mimics of guanylin and uroguanylin in the intestine, which provide a cellular mechanism underlying the diarrhea caused by ST-secreting strains of Escherichia coli. Uroguanylin and guanylin may participate in a novel endocrine axis linking the digestive system and kidney as a physiological mechanism that influences Na(+) homeostasis. Guanylin, uroguanylin, and/or lymphoguanylin may also serve within intrarenal signaling pathways controlling cGMP production in renal target cells. Thus we propose that guanylin regulatory peptides participate in a complex multifactorial biological process that evolved to regulate the urinary excretion of NaCl when dietary salt levels exceed the body's physiological requirements. This highly integrated and redundant mechanism allows the organism to maintain sodium balance by eliminating excess NaCl in the urine. Uroguanylin, in particular, may be a prototypical "intestinal natriuretic hormone."

Topics: Animals; Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Mucosa; Kidney; Natriuretic Peptides; Peptides; Receptors, Peptide; 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

Topics: Animals; Gastrointestinal Hormones; Humans; Natriuretic Peptides; Peptides; Sodium Chloride; Water; Water-Electrolyte Balance

Guanylin and uroguanylin are novel peptides that are first isolated from rat jejunum and opossum urine, respectively. They bind to and activate guanylyl cyclase-C (GC-C) to regulate intestinal and renal fluid and electrolyte transport through the second messenger, cyclic GMP. Heat-stable enterotoxins produced by pathogenic bacteria have close structural similarities to guanylin and uroguanylin, and they use this mimicry to act on GC-C, causing life-threatening secretory diarrhea. Guanylin primarily is restricted to the intestine, whereas uroguanylin is present in the stomach kidney, lung and pancreas in addition to intestine. Guanylin and uroguanylin in the intestine are secreted into the lumen and blood in response to sodium chloride administration. These peptides will function in salt and water transport in the intestine and kidney by luminocrine and/or endocrine actions. Guanylin peptide family links the intestine with the kidney and could play the physiological roles in the control of water and electrolyte balance.

Topics: Amino Acid Sequence; Animals; Cyclic GMP; Digestive System; Enterotoxins; Gastrointestinal Hormones; Guanylate Cyclase; Homeostasis; Kidney; Molecular Sequence Data; Natriuretic Peptides; Peptides; Rats; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Second Messenger Systems; Sequence Homology, Amino Acid; Water-Electrolyte Balance

Topics: Animals; Enterochromaffin Cells; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Intestine, Small; Natriuretic Peptides; Peptides; RNA, Messenger; Tissue Distribution

Guanylin and uroguanylin are newly discovered, related peptides that activate common guanylyl cyclase signaling molecules and via 3', 5'-guanosine cyclic monophosphate regulate the activity of a variety of tissues and organs. Additionally, the message for both peptides is expressed in a variety of tissues and organs, including the intestinal tract and kidney, and thus may serve as part of a functional endocrine axis linking these two major organ systems in fluid/volume homeostasis. This manuscript reviews the discovery and nature of the guanylin and uroguanylin peptides, their actions on the intestinal mucosa and kidney, the distribution and molecular biology of the guanylyl cyclase C receptor, and explores the future directions of this rapidly developing, expanding field of inquiry.

Topics: Amino Acid Sequence; Animals; Cyclic GMP; Female; Gastrointestinal Hormones; Guanylate Cyclase; Humans; In Vitro Techniques; Intestinal Mucosa; Kidney Cortex; Male; Marsupialia; Molecular Sequence Data; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

Guanylin and uroguanylin are small, heat-stable peptides that were initially isolated from rat jejunum and opossum urine, respectively. Both peptides bind to and activate a common set of apical membrane receptors that contain a guanylate cyclase catalytic domain within the receptor molecule. The guanylin/uroguanylin receptors are found on the luminal surface of epithelial cells lining the intestinal tract and renal proximal tubules as well as in other organs. Activation of receptor-guanylate cyclase signaling molecules by uroguanylin or guanylin elicits large increases in guanosine cyclic 3'-5' monophosphate (cGMP) production. Intracellular accumulation of this second messenger in target cells leads to the stimulation of intestinal chloride secretion, culminating in the enhancement of salt and water secretion into the intestinal lumen as well as increases in urinary sodium, potassium, and water excretion by actions of cGMP in the renal tubules. Uroguanylin and guanylin are produced throughout the intestinal mucosa and, surprisingly, uroguanylin messenger RNA (mRNA) is also expressed in both atria and ventricles of the heart. Both proguanylin and prouroguanylin are inactive polypeptides, and activation is accomplished by cleavage and release of the COOH-terminal peptides, guanylin and uroguanylin. Uroguanylin is postulated to function as an intestinal natriuretic hormone because: (1) prouroguanylin and uroguanylin both circulate in the plasma of normal animals; (2) uroguanylin is the predominant peptide agonist appearing in the filtrate and, thus, in urine; (3) the receptors for uroguanylin are localized to the apical membranes of renal tubular cells; (4) uroguanylin is substantially more potent than guanylin in eliciting a natriuresis; and (5) uroguanylin is expressed in the duodenum and myocardium, which are appropriate sites in the body for the production and release of a hormone that acts as a natriuretic agonist in vivo. The hypothesis that uroguanylin links the intestine with the kidney in an endocrine axis also predicts that the secretion of uroguanylin from the intestinal mucosa will be influenced by dietary levels of salt. Accordingly, plasma levels of uroguanylin or prouroguanylin should be influenced by oral salt loads. Future investigations will focus on the basic endocrinology of uroguanylin to provide answers to this intriguing question. In conclusion, uroguanylin is a candidate for a physiological role as an intestinal natriuretic hormone. K

Topics: Animals; Base Sequence; Gastrointestinal Hormones; Humans; Intestines; Kidney; Molecular Sequence Data; Natriuresis; Natriuretic Peptides; Peptides; Receptors, Peptide; Water-Electrolyte Balance

To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB.. Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects on food intake and glucose regulation. The effect of pro-UGN or UGN overexpression, using adeno-associated virus (AAV) vectors, was assessed in diet-induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas.. GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV-mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice.. GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes.

Topics: Adult; Animals; Body Weight Maintenance; Diabetes Mellitus; Enteroendocrine Cells; Female; Gastric Bypass; Gastrointestinal Hormones; Gene Expression Regulation; Guanylate Cyclase-Activating Proteins; Humans; Male; Mice; Middle Aged; Natriuretic Peptides; Obesity

Guanylate cyclase C (GUCY2C) is a tumor-suppressing receptor silenced by loss of expression of its luminocrine hormones guanylin and uroguanylin early in colorectal carcinogenesis. This observation suggests oral replacement with a GUCY2C agonist may be an effective targeted chemoprevention agent. Linaclotide is an FDA-approved oral GUCY2C agonist formulated for gastric release, inducing fluid secretion into the small bowel to treat chronic idiopathic constipation. The ability of oral linaclotide to induce a pharmacodynamic response in epithelial cells of the colorectum in humans remains undefined. Here, we demonstrate that administration of 0.87 mg of oral linaclotide daily for 7 days to healthy volunteers, after oral colon preparation with polyethylene glycol solution (MoviPrep), activates GUCY2C, resulting in accumulation of its product cyclic (c)GMP in epithelial cells of the cecum, transverse colon, and distal rectum. GUCY2C activation by oral linaclotide was associated with homeostatic signaling, including phosphorylation of vasodilator-stimulated phosphoprotein and inhibition of proliferation quantified by reduced Ki67-positive epithelial cells. In the absence of the complete oral colonoscopy preparation, linaclotide did not alter cGMP production in epithelial cells of the colorectum, demonstrating that there was an effect related to the laxative preparation. These data show that the current FDA-approved formulation of oral linaclotide developed for small-bowel delivery to treat chronic idiopathic constipation is inadequate for reliably regulating GUCY2C in the colorectum to prevent tumorigenesis. The study results highlight the importance of developing a novel GUCY2C agonist formulated for release and activity targeted to the large intestine for colorectal cancer prevention.

Topics: Administration, Oral; Animals; Cell Adhesion Molecules; Colon; Colonoscopy; Colorectal Neoplasms; Cyclic GMP; Epithelial Cells; Gastrointestinal Hormones; Guanylyl Cyclase C Agonists; Healthy Volunteers; Humans; Ki-67 Antigen; Microfilament Proteins; Natriuretic Peptides; Peptides; Phosphoproteins; Phosphorylation; Polyethylene Glycols; Receptors, Enterotoxin; Rectum

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.

Topics: Acinetobacter; Animals; Antibodies, Bacterial; Antibodies, Neutralizing; Antigens, Bacterial; Bacterial Toxins; Bacteriophages; Cross Reactions; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Escherichia coli Vaccines; Female; Gastrointestinal Hormones; Immunization; Mice; Mice, Inbred BALB C; Natriuretic Peptides; Toxoids; Vaccines, Subunit; Vaccines, Virus-Like Particle

Diarrhea is one of the most important causes of mortality in the developing world, being responsible for 2.5 million deaths each year. Many of these deaths are caused by enterotoxigenic strains of bacteria, like

Topics: Bacterial Toxins; Cell Line; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterotoxins; Enzyme Inhibitors; Escherichia coli Proteins; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Natriuretic Peptides; Pyrimidinones; Receptors, Enterotoxin

Infection with enterotoxigenic

Topics: Animals; Antibodies, Bacterial; Antibodies, Neutralizing; Bacterial Toxins; Cross Reactions; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Escherichia coli Proteins; Escherichia coli Vaccines; Gastrointestinal Hormones; Humans; Immunization; Mice; Mice, Inbred BALB C; Natriuretic Peptides; Swine

Heat-stable toxin (STa)-producing enterotoxigenic

Topics: Animals; Antibodies, Bacterial; Antibodies, Neutralizing; Antigens, Bacterial; Bacterial Toxins; Child; Cross Reactions; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Female; Gastrointestinal Hormones; Hot Temperature; Humans; Immunization; Mice; Mutation; Natriuretic Peptides; Toxoids

High doses of ionizing radiation induce acute damage to epithelial cells of the gastrointestinal (GI) tract, mediating toxicities restricting the therapeutic efficacy of radiation in cancer and morbidity and mortality in nuclear disasters. No approved prophylaxis or therapy exists for these toxicities, in part reflecting an incomplete understanding of mechanisms contributing to the acute radiation-induced GI syndrome (RIGS). Guanylate cyclase C (GUCY2C) and its hormones guanylin and uroguanylin have recently emerged as one paracrine axis defending intestinal mucosal integrity against mutational, chemical, and inflammatory injury. Here, we reveal a role for the GUCY2C paracrine axis in compensatory mechanisms opposing RIGS. Eliminating GUCY2C signaling exacerbated RIGS, amplifying radiation-induced mortality, weight loss, mucosal bleeding, debilitation, and intestinal dysfunction. Durable expression of GUCY2C, guanylin, and uroguanylin mRNA and protein by intestinal epithelial cells was preserved following lethal irradiation inducing RIGS. Oral delivery of the heat-stable enterotoxin (ST), an exogenous GUCY2C ligand, opposed RIGS, a process requiring p53 activation mediated by dissociation from MDM2. In turn, p53 activation prevented cell death by selectively limiting mitotic catastrophe, but not apoptosis. These studies reveal a role for the GUCY2C paracrine hormone axis as a novel compensatory mechanism opposing RIGS, and they highlight the potential of oral GUCY2C agonists (Linzess; Trulance) to prevent and treat RIGS in cancer therapy and nuclear disasters.

Topics: Animals; Apoptosis; Cell Proliferation; Colonic Neoplasms; Female; Gamma Rays; Gastrointestinal Hormones; Gastrointestinal Tract; Humans; Irritable Bowel Syndrome; Lymphoma; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Natriuretic Peptides; Paracrine Communication; Radiation Injuries, Experimental; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Tumor Cells, Cultured

The gastrointestinal tract, the key interface between ingested nutrients and the body, plays a critical role in regulating energy homeostasis. Gut-derived signals convey information regarding incoming nutrients to the brain, initiating changes in eating behavior and energy expenditure, to maintain energy balance. Here we review hormonal, neural, and nutrient signals emanating from the gastrointestinal tract and evidence for their role in controlling feeding behavior. Mechanistic studies that have utilized pharmacologic and/or transgenic approaches targeting an individual hormone/mediator have yielded somewhat disappointing body weight changes, often leading to the hormone/mediator in question being dismissed as a potential obesity therapy. However, the recent finding of sustained weight reduction in response to systemic administration of a long-acting analog of the gut-hormone glucagon-like peptide-1 highlights the therapeutic potential of gut-derived signals acting via nonphysiologic mechanisms. Thus, we also review therapeutics strategies being utilized or developed to leverage gastrointestinal signals in order to treat obesity.

Topics: Animals; Apolipoproteins A; Calcium-Binding Proteins; Cholecystokinin; DNA-Binding Proteins; Eating; Energy Metabolism; Enteroendocrine Cells; Gastrointestinal Hormones; Gastrointestinal Tract; Ghrelin; Glucagon-Like Peptide 1; Homeostasis; Humans; Leptin; Natriuretic Peptides; Nerve Tissue Proteins; Neurons, Afferent; Neurotensin; Nucleobindins; Obesity; Oxyntomodulin; Peptide YY; Receptors, G-Protein-Coupled

Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.

Topics: Animals; Cell Count; Colon; Duodenum; Female; Gastrointestinal Hormones; Humans; In Situ Hybridization; Intestinal Mucosa; Natriuretic Peptides; Rats; Rats, Sprague-Dawley

Uroguanylin and guanylin are secreted by intestinal epithelial cells as prohormones postprandially and act on the hypothalamus to induce satiety. The impact of obesity and obesity-associated type 2 diabetes (T2D) on proguanylin and prouroguanylin expression/secretion as well as the potential role of guanylin and uroguanylin in the control of lipolysis in humans was evaluated.. Circulating and gastrointestinal expression of proguanylin (GUCA2A) and prouroguanylin (GUCA2B) were measured in 134 subjects. In addition, plasma proguanylin and prouroguanylin were measured before and after weight loss achieved either by Roux-en-Y gastric bypass (RYGB) (n=24) or after a conventional diet (n=15). The effect of guanylin and uroguanylin (1-100 nmol l(-1)) on lipolysis was determined in vitro in omental adipocytes.. Circulating concentrations of prouroguanylin, but not proguanylin, were decreased in obesity in relation to adiposity. Weight loss achieved by RYGB increased plasma proguanylin and prouroguanylin. Obese T2D individuals showed higher expression of intestinal GUCA2A as well as of the receptors of the guanylin system, GUCY2C and GUCY2D, in omental adipocytes. The incubation with guanylin and uroguanylin significantly stimulated lipolysis in differentiated omental adipocytes, as evidenced by hormone-sensitive lipase phosphorylation at Ser563, an increase in fatty acids and glycerol release together with an upregulation of several lipolysis-related genes, including AQP3, AQP7, FATP1 or CD36.. Both guanylin and uroguanylin trigger lipolysis in human visceral adipocytes. Given the lipolytic action of the guanylin system on visceral adipocytes, the herein reported decrease of circulating prouroguanylin concentrations in obese patients may have a role in excessive fat accumulation in obesity.

Topics: Adipocytes; Adult; Carrier Proteins; Diabetes Mellitus, Type 2; Diet, Reducing; Female; Gastric Bypass; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Intra-Abdominal Fat; Lipolysis; Natriuretic Peptides; Obesity; Satiation; Signal Transduction; Sterol Esterase; Weight Loss

The transmembrane receptor guanylate cyclase-C (GC-C) signaling pathway has been implicated in several gastrointestinal disorders. Activation of GC-C via guanylin (Gn) and uroguanylin (Ugn) regulates intestinal fluid and electrolyte homeostasis. However, how it regulates the pathogenesis of inflammatory bowel disease (IBD) is still unclear. Here, we investigated the activation of GC-C signaling in ulcerative colitis (UC) of different clinical severities. A total of 60 UC patients and 20 normal controls were recruited. Evaluation of the UC disease activity index (DAI) was performed using a modified Mayo scoring system. The expression of GC-C, Gn and Ugn in the colonic mucosa was measured by quantitative real-time PCR and Western blot. We found that the UC patients had significantly lower expression of GC-C, Gn and Ugn than the controls. Furthermore, there were significant differences for GC-C, Gn and Ugn expression for the UC groups of Grade 1, 2 and 3, and their expression levels were reduced with increases in their DAI. Taken together, our results demonstrate that GC-C, Gn and Ugn are downregulated in UC, and this downregulation is more significant with aggravation of the clinical condition. Therefore, the GC-C signaling pathway may be implicated in the progression of UC.

Topics: Adult; Colitis, Ulcerative; Down-Regulation; Female; Gastrointestinal Hormones; Humans; Male; Middle Aged; Natriuretic Peptides; Receptors, Atrial Natriuretic Factor; Signal Transduction

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt's base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus-surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt-villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Topics: Animals; Cell Lineage; Epithelial Cells; Gastrointestinal Hormones; Intestinal Mucosa; Intestines; Mice; Mice, Inbred Strains; Natriuretic Peptides; Signal Transduction

Functional gastrointestinal disorders (FGID) and inflammatory bowel diseases (IBD) are the most frequent pathologic conditions affecting the gastrointestinal (GI) tract and both significantly reduce patients' quality of life. Recent studies suggest that guanylyl cyclase C (GC-C) expressed in the GI tract constitutes a novel pharmacological target in the treatment of FGID and IBD. Endogenous GC-C agonists - guanylin peptides: guanylin and uroguanylin, by the regulation of water and electrolyte transport, are involved in the maintenance of homeostasis in the intestines and integrity of the intestinal mucosa. Linaclotide, a synthetic agonist of GC-C was approved by Food and Drug Administration and European Medicines Agency as a therapeutic in constipation-predominant irritable bowel syndrome (IBS-C) and chronic idiopathic constipation (CIC). Lately, several preclinical and clinical trials focused on assessment of therapeutic properties of synthetic agonists of uroguanylin, plecanatide, and SP-333. Plecanatide is currently tested as a potential therapeutic in diseases related to constipation and SP-333 is a promising drug in ulcerative colitis treatment.. Here, we discuss the most recent findings and future trends on the development of GC-C agonists and their use in clinical trials.

Topics: Clinical Trials as Topic; Constipation; Female; Gastroenterology; Gastrointestinal Diseases; Gastrointestinal Hormones; Humans; Inflammatory Bowel Diseases; Male; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Cloning, Molecular; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Gene Expression Regulation, Bacterial; Humans; Models, Molecular; Natriuretic Peptides; Protein Binding; Protein Conformation

The physiological effects of guanylin (GN) and uroguanylin (UGN) on fluid and electrolyte transport in the teleost fish intestine have yet to be thoroughly investigated. In the present study, the effects of GN, UGN, and renoguanylin (RGN; a GN and UGN homolog) on short-circuit current (Isc) and the transport of Cl-, Na+, bicarbonate (HCO3-), and fluid in the Gulf toadfish (Opsanus beta) intestine were determined using Ussing chambers, pH-stat titration, and intestinal sac experiments. GN, UGN, and RGN reversed the Isc of the posterior intestine (absorptive-to-secretory), but not of the anterior intestine. RGN decreased baseline HCO3- secretion, but increased Cl- and fluid secretion in the posterior intestine. The secretory response of the posterior intestine coincides with the presence of basolateral NKCC1 and apical cystic fibrosis transmembrane conductance regulator (CFTR), the latter of which is lacking in the anterior intestine and is not permeable to HCO3- in the posterior intestine. However, the response to RGN by the posterior intestine is counterintuitive given the known role of the marine teleost intestine as a salt- and water-absorbing organ. These data demonstrate that marine teleosts possess a tissue-specific secretory response, apparently associated with seawater adaptation, the exact role of which remains to be determined.

Topics: Animals; Batrachoidiformes; Bicarbonates; Chlorides; Cloning, Molecular; DNA, Complementary; Eels; Gastrointestinal Hormones; Intestines; Membrane Proteins; Natriuretic Peptides; Saccharomyces cerevisiae Proteins; Sodium; Water; Water-Electrolyte Balance

Guanylin (GN), uroguanylin (UGN) and the GC-C receptor have been associated with two endocrine axes: the salt and water homeostasis regulating enterorenal axis and the recently described appetite-regulating UGN/GC-C extraintestinal axis. The present work assessed the mRNA expression levels of GN peptides system (GPS) in a model of diet-induced obesity. Male C57BL/6J mice were submitted to either a high-fat high-simple carbohydrate diet (obese) or a normal diet (control). The renal and intestinal GN, UGN and GC-C receptor mRNA expression were evaluated by reverse transcriptase quantitative polymerase chain reaction in both groups, during normo-saline (NS) and high-saline (HS) diet. The diet-induced obesity was accompanied by glucose intolerance and insulin resistance as well as by a significant increase in blood pressure. During NS diet, obese mice presented reduced mRNA expression of GN in ileum and colon, UGN in duodenum, ileum and colon and GC-C in duodenum, jejunum, ileum and colon. This was accompanied by increased UGN mRNA expression in renal cortex. During HS diet, obese mice presented reduced mRNA expression of GN in jejunum as well as reduced mRNA expression of UGN and GC-C in duodenum, jejunum and colon. The data obtained suggest that, in a mouse model of diet-induced obesity, a down-regulation of intestinal mRNA expression of GN, UGN and its GC-C receptor is accompanied by a compensatory increase of renal UGN mRNA expression. We hypothesize that the decrease in gene expression levels of intestinal GPS may contribute to the development of hypertension and obesity during hypercaloric diet intake.

Topics: Animals; Diet; Gastrointestinal Hormones; Gene Expression Profiling; Hypertension; Intestines; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Natriuretic Peptides; Real-Time Polymerase Chain Reaction

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Topics: Cell Line; Cell Membrane; Endoplasmic Reticulum; Gastrointestinal Hormones; Glycosylation; Humans; Ligands; Mannose-Binding Lectins; Membrane Transport Proteins; Natriuretic Peptides; Protein Binding; Protein Folding; Protein Structure, Tertiary; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

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

Guanylate cyclase C (GUCY2C or GC-C) and its ligands, guanylin (GUCA2A or Gn) and uroguanylin (GUCA2B or Ugn), are expressed in intestinal epithelial cells and regulate ion secretion, intestinal barrier function, and epithelial monolayer homeostasis via cGMP-dependent signaling pathways. The aim of this study was to determine whether GC-C and its ligands direct the course of intestinal inflammation. In this article, we show that dextran sodium sulfate (DSS)-induced clinical disease and histological damage to the colonic mucosa were significantly less severe in GC-C(-/-) mice and moderately reduced in Gn(-/-) animals. Relative to wild-type controls, GC-C(-/-) and Gn(-/-) mice had reduced apoptosis and increased proliferation of intestinal epithelial cells during DSS colitis. Basal and DSS-induced production of resistin-like molecule β (RELMβ) was substantially diminished in GC-C(-/-) mice. RELMβ is thought to stimulate cytokine production in macrophages in this disease model and, consistent with this, TNF-α and IFN-γ production was minimal in GC-C(-/-) animals. RELMβ and cytokine levels were similar to wild-type in Gn(-/-) mice, however. Colonic instillation of recombinant RELMβ by enema into GC-C(-/-) mice restores sensitivity to DSS-mediated mucosal injury. These findings demonstrate a novel role for GC-C signaling in facilitating mucosal wounding and inflammation, and further suggest that this may be mediated, in part, through control of RELMβ production.

Topics: Animals; Colonic Diseases; Gastrointestinal Hormones; Guanylate Cyclase; Hormones, Ectopic; Inflammation; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Intestinal Mucosa; Mice; Mice, Knockout; Natriuretic Peptides; Tumor Necrosis Factor-alpha

Midbrain dopamine neurons regulate many important behavioral processes, and their dysfunctions are associated with several human neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and schizophrenia. Here, we report that these neurons in mice selectively express guanylyl cyclase-C (GC-C), a membrane receptor previously thought to be expressed mainly in the intestine. GC-C activation potentiates the excitatory responses mediated by glutamate and acetylcholine receptors via the activity of guanosine 3',5'-monophosphate-dependent protein kinase (PKG). Mice in which GC-C has been knocked out exhibit hyperactivity and attention deficits. Moreover, their behavioral phenotypes are reversed by ADHD therapeutics and a PKG activator. These results indicate important behavioral and physiological functions for the GC-C/PKG signaling pathway within the brain and suggest new therapeutic targets for neuropsychiatric disorders related to the malfunctions of midbrain dopamine neurons.

Topics: Amphetamine; Animals; Attention; Attention Deficit Disorder with Hyperactivity; Behavior, Animal; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Dopamine; Enzyme Activation; Gastrointestinal Hormones; Glycine; Impulsive Behavior; Ligands; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Natriuretic Peptides; Neurons; Patch-Clamp Techniques; Receptors, Enterotoxin; Receptors, Glutamate; Receptors, Guanylate Cyclase-Coupled; Receptors, Muscarinic; Receptors, Peptide; Resorcinols; Signal Transduction; Substantia Nigra; Ventral Tegmental Area

Guanylate Cyclase C (GC-C) expression in the intestine plays a role in the regulation of fluid and ion transport, as well as epithelial cell apoptosis and proliferation. In the adult rat liver, GC-C expression is increased in response to injury. We hypothesized that GC-C is required for repair/recovery from liver injury.. We subjected wild type (WT) and GC-C deficient mice to acute liver injury with a single injection of the hepatotoxin carbon tetrachloride. Changes in the level of expression of GC-C and its ligands uroguanylin and guanylin were quantified by real-time PCR. Liver morphology, and hepatocyte necrosis, apoptosis and proliferation, were examined at 1-3 days post-injury in mice on a mixed genetic background. Survival was followed for 14 days after carbon tetrachloride injection in wild type and GC-C deficient mice on both a mixed genetic background and on an inbred C57BL6/J background.. GC-C deficient mice on the mixed genetic background nearly all died (median survival of 5 days) following carbon tetrachloride injection while WT littermates experienced only 35% mortality. Elevated levels of TUNEL-positive hepatocyte death on post-injury day 1, increased apoptosis on day 2, and increased areas of centrilobular necrosis on days 2 and 3, were evident in livers from GC-C null mice compared to WT. Collectively these data suggest increased hepatocyte death in the GC-C null mice in the early time period after injury. This corresponds temporally with increased expression of GC-C and its ligands guanylin and uroguanylin in post-injury WT mouse liver. The hepatocyte proliferative response to injury was the same in both genotypes. In contrast, there was no difference in survival between GC-C null and WT mice on the inbred C57BL/6 J background in response to acute liver injury.. Signalling via GC-C promotes hepatocyte survival in vivo and is required for effective recovery from acute toxic injury to the liver in a strain-specific manner.

Topics: Animals; Apoptosis; Carbon Tetrachloride; Cell Proliferation; Chemical and Drug Induced Liver Injury; Female; Gastrointestinal Hormones; Guanylate Cyclase; Hepatocytes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Natriuretic Peptides; Survival Rate

Three guanylin-like peptides, guanylin, uroguanylin and renoguanylin and two guanylate cyclase type C (GC-C) receptor isoforms were cloned and sequenced from the European eel (Anguilla anguilla). All peptides and both receptors (GC-C1 and GC-C2) were predominantly expressed within the intestine and kidney of both sexually immature yellow, and sexually maturing, migratory silver eels. The derived amino acid sequences for the pre-prohormones and guanylate cyclase isoforms had structural features in common with sequences previously reported for guanylin-like peptides and guanylate cyclases from teleost fish and other species in general. The highest sequence homologies for the prohormones were found within the active, 15-16 amino acid C-terminal peptide domain, whereas the guanylate cyclase receptors exhibited highest homology throughout the transmembrane domain and intracellular region of the protein comprising the kinase homology, oligomerisation/coiled-coil and catalytic domains. In both yellow and silver eels, seawater (SW) acclimation induced sustained increases in the expression of uroguanylin and GC-C1 mRNAs within the intestine but no significant changes were found in the abundance of mRNAs for guanylin, renoguanylin or GC-C2. Likewise there were no significant changes in expression of any of the prohormone or receptor mRNAs within the renal kidney following transfer to SW. The results suggest that uroguanylin and GC-C1 are key components of a cGMP signalling system that may play an important role within intestinal enterocytes for the regulation of salt and water absorption in the SW-acclimated eel.

Topics: Acclimatization; Amino Acid Sequence; Anguilla; Animals; Gastrointestinal Hormones; Guanylate Cyclase; Intestinal Mucosa; Kidney; Molecular Sequence Data; Natriuretic Agents; Natriuretic Peptides; Protein Isoforms; RNA, Messenger; Seawater; Sequence Alignment; Water-Electrolyte Balance

The mammalian nose employs several olfactory subsystems to recognize and transduce diverse chemosensory stimuli. These subsystems differ in their anatomical position within the nasal cavity, their targets in the olfactory forebrain, and the transduction mechanisms they employ. Here we report that they can also differ in the strategies they use for stimulus coding. Necklace glomeruli are the sole main olfactory bulb (MOB) targets of an olfactory sensory neuron (OSN) subpopulation distinguished by its expression of the receptor guanylyl cyclase GC-D and the phosphodiesterase PDE2, and by its chemosensitivity to the natriuretic peptides uroguanylin and guanylin and the gas CO(2). In stark contrast to the homogeneous sensory innervation of canonical MOB glomeruli from OSNs expressing the same odorant receptor (OR), we find that each necklace glomerulus of the mouse receives heterogeneous innervation from at least two distinct sensory neuron populations: one expressing GC-D and PDE2, the other expressing olfactory marker protein. In the main olfactory system it is thought that odor identity is encoded by a combinatorial strategy and represented in the MOB by a pattern of glomerular activation. This combinatorial coding scheme requires functionally homogeneous sensory inputs to individual glomeruli by OSNs expressing the same OR and displaying uniform stimulus selectivity; thus, activity in each glomerulus reflects the stimulation of a single OSN type. The heterogeneous sensory innervation of individual necklace glomeruli by multiple, functionally distinct, OSN subtypes precludes a similar combinatorial coding strategy in this olfactory subsystem.

Topics: Animals; Exonucleases; Gastrointestinal Hormones; Guanylate Cyclase; Mice; Natriuretic Peptides; Neurons; Olfactory Bulb; Receptors, Odorant

The heat-stable enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. (125)I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant (125)I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that (125)I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bacterial Toxins; Bicarbonates; Cystic Fibrosis Transmembrane Conductance Regulator; Duodenum; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Glyburide; Guanylate Cyclase; In Vitro Techniques; Mice; Mice, Knockout; Microvilli; Natriuretic Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sodium-Hydrogen Exchangers

The peptide hormones guanylin and uroguanylin and their receptor, guanylate cyclase C (GC-C), are expressed in pancreatic duct cells. In colon cancer, guanylin peptides are shown to exert strong anti-tumor activity through the GC-C pathway. The objective of this study was to analyze the role of guanylin and uroguanylin in human pancreatic cancer.. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to show the expression of guanylin, uroguanylin and GC-C in specimens of human pancreatic cancer, chronic pancreatitis donor and in pancreatic tumor cell lines. The presence of guanylins and GC-C in tumor cell lines and in pancreatic cancer tissues was shown by immunofluorescence and immunohistochemistry. The effect of guanylin and uroguanylin on cell cycle and cell death of pancreatic cancer cells was investigated by fluorescence activated cell sorter (FACS) analysis using annexin and propidium iodide. In addition, the growth inhibitory effect of guanylins on pancreatic cancer cells was assessed using the MTT assay.. Guanylin, uroguanylin and GC-C were expressed at mRNA and protein levels in pancreatic cancer and cancer cell lines. As shown by QRT-PCR, GC-C expression was significantly up-regulated in pancreatic cancer compared with that in healthy pancreatic tissues (p<0.00001) and chronic pancreatitis (p<0.05). Guanylin and uroguanylin were not up-regulated in pancreatic cancer. The MTT assay revealed significant inhibition of pancreatic cancer cell proliferation by uroguanylin in a dose-dependent fashion, whereas Panc1 and Capan1 cell lines were significantly inhibited already at the lowest uroguanylin concentration (2 nM, p<0.05).. Our data suggest therapeutic properties of uroguanylin in pancreatic cancer via GC-C-dependent mechanisms. In addition, determination of GC-C expression might be a useful marker for differentiation between pancreatic cancer and chronic pancreatitis.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Division; Cell Line, Tumor; Chronic Disease; Female; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Male; Middle Aged; Natriuretic Peptides; Pancreas; Pancreatic Neoplasms; Pancreatitis; Polymerase Chain Reaction

Uroguanylin and guanylin are endogenous ligands for guanylate cyclase C, an upstream regulator of the cystic fibrosis transmembrane resistance (CFTR) anion channel, and both peptides increase intestinal anion export in vitro. We have compared the effects of close intra-arterial and luminal administration of uroguanylin and guanylin on duodenal bicarbonate secretion in vivo and studied the interactions with melatonin and cholinergic stimulation.. Lewis x Dark Agouti rats were anaesthetized and a segment of the proximal duodenum with intact blood supply was cannulated in situ. Mucosal bicarbonate secretion (pH stat) was continuously recorded and peptides were infused intra-arterially or added to the luminal perfusate.. Intra-arterial (50-1000 pmol kg(-1) h(-1)) as well as luminal administration (50-500 nmol L(-1)) of guanylin or uroguanylin caused dose-dependent increases in the duodenal secretion. Luminal administration induced more rapidly appearing rises in secretion and the two peptides induced secretory responses of similar shape and magnitude. The melatonin MT(2)-selective antagonist luzindole (600 nmol kg(-1)) significantly depressed the response to intra-arterial guanylins but did not affect secretion induced by luminal guanylins. Similarly, the muscarinic antagonist atropine (0.75 micromol kg(-1) followed by 0.15 micromol kg(-1) h(-1)) abolished the response to intra-arterial uroguanylin but caused only slight suppression of the response to luminal uroguanylin.. Intra-arterial as well as luminal uroguanylin and guanylin are potent stimuli of duodenal mucosal bicarbonate secretion in vivo. The response to luminal guanylins reflects an action at apical receptors. Stimulation by parenteral guanylins, in contrast, is under cholinergic influence and interacts with melatonin produced by mucosal enteroendocrine cells.

Topics: Animals; Arteries; Bicarbonates; Cytoprotection; Duodenum; Gastrointestinal Hormones; Intestinal Mucosa; Male; Natriuretic Peptides; Rats; Rats, Inbred Lew

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 microM) natriuretic activity (%ENa(+); from 18.5+/-4.25 to 31.5+/-1.69, P<0.05, 120 min) and its kaliuretic activity (%EK(+); from 24.5+/-4.43 to 50.6+/-3.84, P<0.05, 120 min). Furthermore, ANP increased the natriuretic (29.05+/-3.00 to 37.8+/-2.95, P<0.05, 120 min) and kaliuretic (from 33.2+/-3.52 to 42.83+/-2.45, P<0.05, 120 min) responses of perfused kidneys treated with low-dose (0.06 microM) uroguanylin. In contrast, ANP clearly inhibited the uroguanylin-induced (0.31 microM) increase in %ENa(+) (from 35.9+/-2.37 to 14.8+/-1.93, P<0.05, 120 min), and in %EK(+) (from 51.0+/-4.43 to 38.8+/-3.61, P<0.05, 120 min). UROD (0.03 nM) also enhanced the guanylin-induced natriuresis (to %ENa(+)=31.0+/-1.93, P<0.05, 120 min) and kaliuresis (to %EK(+)=54.2+/-3.61, P<0.05, 120 min), and inhibited the %ENa(+) of uroguanylin (0.31 microM) to 17.9+/-1.67 as well as its %EK(+) to 24.3+/-3.13 (both at 120 min, P<0.05). The synergism between ANP and UROD with either guanylin or uroguanylin at sub-threshold doses and the unexpected antagonism between ANP and UROD with uroguanylin at a pharmacological dose point to possible interactions between natriuretic peptide receptor (NPR) and uroguanylin/guanylin receptor signaling pathways. The interactions herein described may play a contributory role in the regulation of kidney function in many pathophysiological states, such as in the saliuresis following ingestion of salty meals.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Gastrointestinal Hormones; Glomerular Filtration Rate; Guanylate Cyclase; Kidney; Male; Natriuretic Peptides; Opossums; Peptide Fragments; Peptides; Perfusion; Rats; Rats, Wistar; Time Factors

Guanylin and uroguanylin are peptides synthesized in the intestine and kidney that are postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of guanylin and uroguanylin in the regulation of sodium excretion, we used gene-targeted mice in which the uroguanylin, guanylin or the peptide receptor guanylate cyclase C gene expression had been ablated.. Metabolic balance studies demonstrated that there was impaired excretion of a sodium load in uroguanylin (but not in guanylin or guanylate cyclase C) knockout mice. Uroguanylin-dependent natriuresis occurred without an increase in circulating prouroguanylin. A distinct morphological phenotype was present in the proximal convoluted tubules of uroguanylin knockout animals after an enteral salt loading. Marked vacuolization of the proximal convoluted tubule epithelial cells was observed by using light and electron microscopy. There was also a change in the distribution of the sodium hydrogen exchanger 3 (NHE3) after an enteral salt loading. In wild-type animals, there was a partial redistribution of NHE3 from the villus fraction to the less accessible submicrovillus membrane compartment, but this effect was less apparent in uroguanylin knockout animals, presumably resulting in greater Na/H exchange.. Together, these findings further establish a role for uroguanylin in fluid homeostasis and support a role for uroguanylin as an integral component of a signaling mechanism that mediates changes in Na excretion in response to an enteral salt loading. Proximal tubular NHE3 activity is a possible target for uroguanylin-mediated changes in Na excretion.

Topics: Analysis of Variance; Animals; Biomarkers; Blotting, Western; Cyclic GMP; Enteral Nutrition; Fluorescent Antibody Technique, Indirect; Gastrointestinal Hormones; Guanylate Cyclase; Kidney Tubules, Proximal; Mice; Mice, Knockout; Microscopy, Electron; Models, Animal; Natriuresis; Natriuretic Peptides; Potassium Channels; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sodium Chloride, Dietary; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Time Factors; Water-Electrolyte Balance

We have shown that the kinetics of conversion of intestinal crypt cell populations to a partially or wholly mutant phenotype are consistent with a model in which each crypt contains an infrequently dividing 'deep' stem cell that is the progenitor of several more frequently dividing 'proximate' stem cells. An assumption of our model is that each deep stem cell exists in a growth inhibitory niche. We have used information from the literature to develop a model for a quiescent intestinal stem cell niche. This niche is postulated to be primarily defined by an enteroendocrine cell type that maintains stem cell quiescence by secretion of growth inhibitory peptides such as somatostatin and guanylin/uroguanylin. Consistent with this model, there is evidence that the proteins postulated as defining a growth-inhibitory stem cell niche can act as intestinal tumour suppressors. Confirmation that a growth-inhibitory niche does exist would have important implications for our understanding of intestinal homeostasis and tumorigenesis.

Topics: Animals; Cell Differentiation; Cell Lineage; Enterocytes; Enteroendocrine Cells; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Models, Biological; Natriuretic Peptides; Somatostatin; Stem Cells

The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists.

Topics: Amino Acid Sequence; Gastrointestinal Hormones; Indicators and Reagents; Isomerism; Molecular Sequence Data; Natriuretic Peptides; Oligopeptides; Peptides; Sequence Alignment; Sequence Homology, Amino Acid

Guanylin and uroguanylin link intestinal and renal electrolyte and water transport. Their function in intestine is well studied, but renal actions are less understood. Uroguanylin concentrations are increased in patients with chronic renal failure, nephrotic syndrome, or those on dialysis. Guanylate cyclase C (GC-C) is the receptor first described for these peptides. In guanylate cyclase C-deficient mice guanylin- and uroguanylin-induced renal natriuresis, kaliuresis, and diuresis are retained.. Effects of guanylin and uroguanylin on principal cells of human cortical collecting ducts (CCD) isolated from kidneys after tumor nephrectomy were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), slow whole-cell patch-clamp, and microfluorimetric analysis of intracellular Ca(2+) were used. Here we present first functional measurements of isolated human CCD.. Principal cells of CCD were identified by the amiloride-induced hyperpolarization of principal cells (-3.8 +/- 0.3 mV) (N= 52). Cells depolarized upon guanylin or uroguanylin (each 10 nmol/L) by 3.3 +/- 0.8 mV (N= 12) and 3.4 +/- 0.5 mV (N= 18), respectively, but were hyperpolarized by 8Br-cyclic guanosine monophosphate (cGMP) (100 micromol/L) (-3.0 +/- 0.2 mV) (N= 4). mRNA for GC-C was not detected in CCD. Effects of both peptides were inhibited by Ba(2+) (1 mmol/L) or phospholipase A(2) (PLA(2)) inhibition (AACOCF(3)) (5 micromol/L).. These findings suggest a new cGMP- and GC-C-independent but PLA(2)-dependent signaling pathway for these peptides in the kidney. Most likely guanylin and uroguanylin inhibit luminal K(+) channels of principal cells of human CCD via this pathway. This depolarization of principal cells consequently reduces the driving force of Na(+) and water reabsorption, explaining natriuresis and diuresis caused by these peptides.

Topics: Biological Transport; Calcium; DNA Primers; Electrolytes; Gastrointestinal Hormones; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; Humans; Kidney Neoplasms; Kidney Tubules, Collecting; Membrane Potentials; Natriuretic Peptides; Nephrectomy; Nephrons; Peptides; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Electrolyte and water homeostasis mostly depend on differentially regulated intestinal and renal transport. Guanylin and uroguanylin were proposed as first hormones linking intestinal with renal electrolyte and water transport, which is disturbed in pathophysiology. Guanylate cyclase C is the intestinal receptor for these peptides, but in guanylate cyclase C-deficient mice renal effects are retained. Unlike for the intestine the sites of renal actions and cellular mechanisms of guanylin peptides are still unclear.. After first data on proximal tubular effects in this study their effects are examined in detail in mouse cortical collecting duct (CCD). Effects of guanylin peptides on principal cells of isolated mouse CCD were studied by slow whole-cell patch-clamp analysis, reverse transcription-polymerase chain reaction (RT-PCR), and microfluorimetric measurements of intracellular Ca2+.. Guanylin peptides depolarized or hyperpolarized principal cells. Whereas 8-Br-cyclic guanosine monophosphate (8-Br-cGMP) hyperpolarized, 8-Br-cyclic adenosine monophosphate (8-Br-cAMP) depolarized principal cells. All effects of guanylin peptides were inhibited by Ba2+. Hyperpolarizations were blocked by clotrimazole or protein kinase G (PKG) inhibition, suggesting an involvement of basolateral Ca2+- and cGMP-dependent K+ channels. Effects remained in CCD isolated from guanylate cyclase C-deficient mice. Depolarizations were inhibited by arachidonic acid or inhibition of phospholipase A2 (PLA2), but not by protein kinase A (PKA) inhibition. Conclusion. These results suggest the existence of two signaling pathways for guanylin peptides in principal cells of mouse CCD. One pathway is cGMP- and PKG-dependent but not mediated by guanylate cyclase C, the second involves PLA2 and arachidonic acid. The first pathway most likely leads to an activation of the basolateral K+-conductance while the latter probably results in decreased activity of ROMK channels in the luminal membrane.

Topics: Animals; Arachidonic Acid; Biological Transport; Calcium; Cell Membrane; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gastrointestinal Hormones; Guanylate Cyclase; Kidney Cortex; Kidney Tubules, Collecting; Male; Mice; Mice, Inbred C57BL; Natriuretic Peptides; Phosphatidylinositol 3-Kinases; Phospholipases A; Phospholipases A2; Potassium; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

Guanylin (GN) and uroguanylin (UGN) are intestinally derived peptide hormones that are similar in structure and activity to the diarrhea-causing Escherichia coli heat-stable enterotoxins (STa). These secretagogues have been shown to affect fluid, Na+, K+, and Cl- transport in both the intestine and kidney, presumably by intracellular cyclic guanosine monophosphate (cGMP)-dependent signal transduction. However, the in vivo consequences of GN, UGN, and STa on renal function and their mechanism of action have yet to be rigorously tested.. We hypothesized that intravenous administration of GN, UGN, or STa would cause an increase in natriuresis in wild-type mice via cGMP and guanylyl cyclase-C (GC-C, Gucy2c), the only known receptor for these peptide-hormones, and that the peptide-induced natriuresis would be blunted in genetically altered mice devoid of GC-C receptors (GC-C(-/-) null).. In wild-type mice using a modified renal clearance model, GN, UGN, and STa elicited significant natriuresis, kaliuresis, and diuresis as well as increased urinary cGMP levels in a time- and dose-dependent fashion. Absolute and fractional urinary sodium excretion levels were greatest approximately 40 minutes following a bolus infusion with pharmacologic doses of these peptides. Unexpectedly, GC-C(-/-) null mice also responded to the GN peptides similarly to that observed in wild-type mice. Glomerular filtration rate (GFR), blood pressure, and plasma cGMP in the mice (wild-type or GC-C(-/-) null) did not significantly vary between the vehicle- and peptide-treatment groups. The effects of UGN may also influence long-term renal function due to down-regulation of the Na+/K+ ATPase gamma-subunit and the Cl- channel ClC-K2 by 60% and 75%, respectively, as assessed by differential display polymerase chain reaction (PCR) (DD-PCR) and Northern blot analysis of kidney mRNA from mice treated with UGN.. GN, UGN, and STa act on the mouse kidney, in part, through a cGMP-dependent, GC-C-independent mechanism, causing significant natriuresis by renal tubular processes. UGN may have further long-term effects on the kidney by altering the expression of such transport-associated proteins as Na+/K+ ATPase and ClC-K2.

Topics: Animals; Animals, Suckling; Bacterial Toxins; Blotting, Northern; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Injections, Intravenous; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Natriuresis; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger

Guanylin and uroguanylin are small, heat-stable peptides that were originally isolated from the small intestine and from urine, respectively. Functionally, it has been proposed that these peptides can regulate ion and water transport in various fluid-transporting epithelia. In the present study we evaluated the presence of mRNAs for human guanylin and uroguanylin in human inferior turbinate mucosa and nasal polyps.. The expression and localization of mRNAs for both peptides were investigated in inferior turbinate tissues and nasal polyps using reverse transcriptase polymerase chain reaction and in situ hybridization.. mRNAs for both peptides were detected in human turbinate mucosa and nasal polyps. In situ hybridization revealed that they were localized in the epithelial layer and submucosal glands of inferior turbinate mucosa and in the epithelial layer of nasal polyps. However, their expression was noted at low levels in the superficial epithelium of nasal polyps, compared with that of inferior turbinate mucosa.. These results indicate that guanylin and uroguanylin in the nasal mucosa may participate in normal physiological processes, but also suggest that altered expression of these genes in nasal polyps may cause impaired electrolyte and water transport across the epithelial cells.

Topics: Adult; Female; Gastrointestinal Hormones; Gene Expression Regulation; Humans; In Situ Hybridization; Male; Nasal Mucosa; Nasal Polyps; Natriuretic Peptides; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Turbinates

One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in under-developed countries. This geographic imbalance suggests an environmental contribution to the resistance of endemic populations to intestinal neoplasia. A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic bacteria associated with diarrheal disease. Here, a bacterial heat-stable enterotoxin was demonstrated to suppress colon cancer cell proliferation by a guanylyl cyclase C-mediated signaling cascade. The heat-stable enterotoxin suppressed proliferation by increasing intracellular cGMP, an effect mimicked by the cell-permeant analog 8-br-cGMP. The antiproliferative effects of the enterotoxin and 8-br-cGMP were reversed by L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as well as by removal of extracellular Ca(2+), or chelation of intracellular Ca(2+). In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca(2+) influx and inhibition of DNA synthesis in colon cancer cells. Induction of this previously unrecognized antiproliferative signaling pathway by bacterial enterotoxin could contribute to the resistance of endemic populations to intestinal neoplasia, and offers a paradigm for targeted prevention and therapy of primary and metastatic colorectal cancer.

Topics: Bacterial Toxins; Calcium; Cell Differentiation; Cell Division; Colonic Neoplasms; DNA; Dose-Response Relationship, Drug; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Immunity, Innate; Ligands; Membrane Potentials; Natriuretic Peptides; Patch-Clamp Techniques; Peptides; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Tumor Cells, Cultured

As the intestine is an essential organ for fish osmoregulation, the intestinal hormone guanylins may perform major functions, especially in euryhaline fish such as eels and salmonids. From the intestine of an eel, we identified cDNAs encoding three distinct guanylin-like peptides. Based on the sequence of mature peptide and sites of production, we named them guanylin, uroguanylin, and renoguanylin. Renoguanylin is a novel peptide that possesses the characteristics of both guanylin and uroguanylin and was abundantly expressed in the kidney. By immunohistochemistry, guanylin was localized exclusively in goblet cells, but not enterochromaffin cells, of the intestine. After transfer of eels from fresh water to seawater, mRNA expression of guanylin and uroguanylin did not change for 3 h, but it increased after 24 h. The increase was profound (2-6-fold) after adaptation to seawater. The expression of uroguanylin was also up-regulated in the kidney of seawater-adapted eels, but that of renoguanylin was not so prominent as other guanylins in both intestine and kidney. Collectively, the novel eel guanylin family appears to have important functions for seawater adaptation, particularly long-term adaptation. Eel guanylin may be secreted from goblet cells into the lumen with mucus in response to increased luminal osmolality and act on the epithelium to regulate water and salt absorption.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA Primers; DNA, Complementary; Eels; Gastrointestinal Hormones; Humans; Intestines; Kidney; Molecular Sequence Data; Natriuretic Peptides; Opossums; Organ Specificity; Peptide Hormones; Peptides; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Swine; Water-Electrolyte Balance

Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Complementary; Gastrointestinal Hormones; Intestinal Mucosa; Isoenzymes; Kidney; Molecular Sequence Data; Muridae; Myocardium; Natriuretic Peptides; Peptides; RNA, Messenger; Water Deprivation

Uroguanylin is a peptide hormone that regulates sodium excretion by the kidney when excess NaCl is consumed. A new study demonstrates that mice deficient in uroguanylin have blunted urinary sodium excretion responses to oral sodium loads in addition to elevated blood pressure (see related article beginning on page 1244). A physiological role for uroguanylin is discussed, linking the intestine and kidney in an endocrine axis for the maintenance of sodium balance.

Topics: Amino Acid Sequence; Animals; Disease Models, Animal; Gastrointestinal Hormones; Humans; Mice; Molecular Sequence Data; Natriuretic Peptides; Peptides; Sodium; Water-Electrolyte Balance

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.

Topics: Actins; Animals; DNA Primers; Gastrointestinal Hormones; Intestinal Mucosa; Natriuretic Peptides; Peptides; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride; Sodium, Dietary; Time Factors

Guanylin and uroguanylin are intestinal or urine peptides that stimulate epithelial electrolyte/fluid secretion by activating the cystic fibrosis gene product (CFTR).. Because CFTR is essentially involved in the electrolyte secretion of the pancreatic duct cells, the rat pancreas was investigated for expression and cell-specific localization of guanylin and uroguanylin; expression of major components of the guanylin signaling pathway, i.e., the guanylin/uroguanylin receptor guanylate cyclase C (GC-C), cGMP-dependent protein kinase II, and CFTR; and expression of the epithelial Cl-/HCO3- exchanger AE2.. Reverse transcriptase polymerase chain reaction analyses revealed high expression of guanylin, uroguanylin, and the functional coupling proteins in the rat pancreas. At the cellular level, guanylin and uroguanylin were localized by immunohistochemistry to the centroacinar cells and proximal duct cells of the exocrine pancreas. The guanylin/uroguanylin receptor GC-C, cGKII, CFTR, and AE2 were all found in the same segments of the ductal system, where they were confined to the apical membrane of centroacinar cells and proximal duct epithelial cells, a circumstance suggesting that both peptides may act through the ductal lumen.. In view of the well-known functional significance of guanylin and uroguanylin, the presence and cell-specific expression not only of the both peptides but also of their common functional coupling proteins implicates a regulatory function of these peptides in the electrolyte/fluid secretion within the rat exocrine pancreas.

Topics: Animals; Anion Transport Proteins; Antiporters; Chloride-Bicarbonate Antiporters; Cyclic GMP-Dependent Protein Kinase Type II; Cyclic GMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Electrolytes; Gastrointestinal Hormones; Gene Expression; Guanylate Cyclase; Immunohistochemistry; Male; Membrane Proteins; Natriuretic Peptides; Pancreas; Peptides; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; SLC4A Proteins

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.

Topics: Betaine; Gastrointestinal Hormones; HT29 Cells; Humans; Hypertonic Solutions; Natriuretic Peptides; Osmotic Pressure; Peptides; Protein Precursors; Protein Serine-Threonine Kinases; RNA, Messenger; Transcription, Genetic; Up-Regulation

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.

Topics: Animals; Antibodies, Monoclonal; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Epididymis; Female; Gastrointestinal Hormones; Gene Expression; Guanylate Cyclase; Immunohistochemistry; Ligands; Male; Natriuretic Peptides; Organ Specificity; Peptides; Phosphorylation; Rats; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.

Topics: Bacterial Toxins; Barium; Cells, Cultured; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Genistein; Guanylate Cyclase; Humans; Hydrogen-Ion Concentration; Kidney Tubules, Proximal; Natriuretic Peptides; Peptides; Pertussis Toxin; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Virulence Factors, Bordetella

The intestinal peptides, guanylin and uroguanylin, may have an important role in the endocrine control of renal function. Both peptides and their receptor, guanylyl cyclase C (GC-C), are also expressed within the kidney, suggesting that they may act locally in an autocrine/paracrine fashion. However, their physiological regulation within the kidney has not been studied. To begin to address this issue, we evaluated the distribution of uroguanylin and guanylin messenger RNA (mRNA) in the mouse nephron and the regulation of renal expression by changes in dietary salt/water intake. Expression was determined in 1) wild-type mice, 2) two strains of receptor-guanylyl cyclase-deficient mice (ANP-receptor-deficient, GC-A-/-, and GC-C-deficient mice); and 3) cultured renal epithelial (M-1) cells, by RT-PCR, Northern blotting and immunocytochemistry. Renal uroguanylin messenger RNA expression was higher than guanylin and had a different distribution pattern, with highest levels in the proximal tubules, whereas guanylin was mainly expressed in the collecting ducts. Uroguanylin expression was significantly lower in GC-C-/- mice than in GC-A-/- and wild-types, suggesting that absence of a receptor was able to down-regulate ligand expression. Salt-loading (1% NaCl in drinking water) increased uroguanylin-mRNA expression by >1.8-fold but had no effect on guanylin expression. Uroguanylin but not guanylin transcripts were detected in M-1 cells and increased in response to hypertonic media (+NaCl or mannitol). Our results indicate that high-salt intake increases uroguanylin but not guanylin expression in the mouse kidney. The synthesis of these peptides by tubular epithelium may contribute to the local control of renal function and its adaptation to dietary salt.

Topics: Animals; Blood Pressure; Cyclic GMP; Dose-Response Relationship, Drug; Drinking; Gastrointestinal Hormones; Guanylate Cyclase; Immunohistochemistry; Isoenzymes; Kidney; Male; Mice; Mice, Knockout; Natriuretic Peptides; Nephrons; Peptides; Rats; Receptors, Atrial Natriuretic Factor; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride; Sodium, Dietary; Tissue Distribution

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-/HCO3- 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 (delta F508) 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.

Topics: Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Cyclic GMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Electrolytes; Gastrointestinal Hormones; Gene Expression; Guanylate Cyclase; Humans; Natriuretic Peptides; Pancreas; Pancreatic Ducts; Pancreatic Juice; Patch-Clamp Techniques; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Signal Transduction

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.

Topics: Animals; Gastrointestinal Hormones; Gene Expression; Guinea Pigs; Humans; Natriuretic Peptides; Parotid Gland; Peptides; Rats; Rats, Wistar; Salivary Glands; Subcellular Fractions; Submandibular Gland; Water-Electrolyte Balance

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.. 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.. 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.. 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.

Topics: Animals; Antiporters; Carrier Proteins; Diarrhea; Female; Gastrointestinal Hormones; Guanylate Cyclase; Intestinal Mucosa; Kidney; Membrane Proteins; Mice; Mice, Inbred C57BL; Natriuretic Peptides; Osmotic Pressure; Peptides; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Sulfate Transporters

Topics: Amino Acid Sequence; Animals; Chromosome Mapping; Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Kidney; Molecular Sequence Data; Natriuresis; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sodium; Water-Electrolyte Balance

The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa.

Topics: Animals; Bacterial Toxins; Butyric Acid; Caco-2 Cells; Cell Line; Colon; COS Cells; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Natriuretic Peptides; Peptides; Transfection

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [125I]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Autoradiography; Binding Sites; Blotting, Western; Colon; Duodenum; Gastrointestinal Hormones; Guanylate Cyclase; Intestinal Mucosa; Ligands; Luminescent Measurements; Male; Natriuretic Peptides; Peptides; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; RNA, Messenger

The enteric peptides, guanylin and uroguanylin, are local regulators of intestinal secretion by activation of receptor-guanylate cyclase (R-GC) signaling molecules that produce cyclic GMP (cGMP) and stimulate the cystic fibrosis transmembrane conductance regulator-dependent secretion of Cl- and HCO3-. Our experiments demonstrate that mRNA transcripts for guanylin and uroguanylin are markedly reduced in colon polyps and adenocarcinomas. In contrast, a specific uroguanylin-R-GC, R-GCC, is expressed in polyps and adenocarcinomas at levels comparable with normal colon mucosa. Activation of R-GCC by uroguanylin in vitro inhibits the proliferation of T84 colon cells and elicits profound apoptosis in human colon cancer cells, T84. Therefore, down-regulation of gene expression and loss of the peptides may interfere with renewal and/or removal of the epithelial cells resulting in the formation of polyps, which can progress to malignant cancers of the colon and rectum. Oral replacement therapy with human uroguanylin was used to evaluate its effects on the formation of intestinal polyps in the Min/+ mouse model for colorectal cancer. Uroguanylin significantly reduces the number of polyps found in the intestine of Min/+ mice by approximately 50% of control. Our findings suggest that uroguanylin and guanylin regulate the turnover of epithelial cells within the intestinal mucosa via activation of a cGMP signaling mechanism that elicits apoptosis of target enterocytes. The intestinal R-GC signaling molecules for guanylin regulatory peptides are promising targets for prevention and/or therapeutic treatment of intestinal polyps and cancers by oral administration of human uroguanylin.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Apoptosis; Caco-2 Cells; Colonic Neoplasms; Cyclic GMP; Down-Regulation; Female; Gastrointestinal Hormones; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Molecular Sequence Data; Natriuretic Peptides; Peptides; Receptors, Cell Surface; RNA, Messenger; Tumor Cells, Cultured

Guanylyl cyclase (GC)-C, a single-transmembrane receptor protein for heat-stable enterotoxin, guanylin, and uroguanylin, and its N-terminal extracellular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant GC-C, containing the complete sequence, retained its binding affinity to heat-stable enterotoxin with a KD value (6.2 x 10(-10) M) and cyclase catalytic activity at a level similar to those of GC-C expressed in mammalian cell lines, such as COS-7. The N-terminal extracellular domain was prepared in a form which contained the hexahistidine tail at its C-terminus and was purified as a homogenous protein by Con A and Ni-chelating affinity chromatography from the culture medium of the insect cells. The purified N-terminal extracellular domain of GC-C exhibited the high (KD = 4 x 10(-10) M) and low (KD = 7 x 10(-8) M) affinity sites in binding to heat-stable enterotoxin. These results clearly indicate that the N-terminal extracellular domain of GC-C possesses the same biochemical characteristics as the complete GC-C protein even in the membrane-free form. Moreover, the extracellular domain is able to form an oligomer in a ligand-dependent manner, suggesting that the N-terminal extracellular domains interact with one another in binding to ligands.

Topics: Animals; Baculoviridae; Blotting, Western; Cell Line; Cell Membrane; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Gastrointestinal Hormones; Genetic Vectors; Guanylate Cyclase; Kinetics; Natriuretic Peptides; Peptide Fragments; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Recombinant Proteins; Spodoptera; Swine; Transfection

Guanylin and uroguanylin are small peptides containing two disulfide bonds that activate membrane guanylate cyclase-receptors in the intestine, kidney and other epithelia. Hybridization assays with a uroguanylin complementary DNA (cDNA) detected uroguanylin-like messenger RNAs (mRNAs) in the opossum spleen and testis, but these transcripts are larger than uroguanylin mRNAs. RT of RNA from spleen to produce cDNAs for amplification in the PCR followed by cloning and sequencing revealed a novel lymphoid-derived cDNA containing an open reading frame encoding a 109-amino acid polypeptide. This protein shares 84% and 40% of its residues with preprouroguanylin and preproguanylin, respectively. A 15-amino acid, uroguanylin-like peptide occurs at the COOH-terminus of the precursor polypeptide. However, this peptide is unique in having only three cysteine residues. We named the gene and its peptide product lymphoguanylin because the source of the first cDNA isolated was spleen and its mRNA is expressed in all of the lymphoid tissues tested. A 15-amino acid form of lymphoguanylin containing a single disulfide bond was synthesized that activates the guanylate cyclase receptors of human T84 intestinal and opossum kidney (OK) cells, although with less potency than uroguanylin and guanylin. Northern and/or RT-PCR assays detected lymphoguanylin mRNA transcripts in many tissues and organs of opossums, including those within the lymphoid/immune, cardiovascular/renal, reproductive, and central nervous organ systems. Lymphoguanylin joins guanylin and uroguanylin in a growing family of peptide agonists that activate transmembrane guanylate cyclase receptors, thus influencing target cell function via the intracellular second messenger, cGMP.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Cyclic GMP; DNA, Complementary; Gastrointestinal Hormones; Intestinal Mucosa; Intestines; Kidney; Lymphoid Tissue; Male; Molecular Sequence Data; Natriuretic Peptides; Opossums; Organ Specificity; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology; Spleen; Testis

The effects of rat guanylin, human guanylin, human uroguanylin and STa on net fluid and electrolyte transport in the closed jejunal loop were compared in anesthetized rats. STa administered into the lumen caused a concentration-dependent (10(-8) to 10(-6) M) inhibition of net fluid and NaCl absorption in the jejunal loop. Uroguanylin had a similar but weaker effect than STa. Both rat and human guanylin inhibited fluid and NaCl absorption only at 10(-6) M. Their order of potency was STa > human uroguanylin > rat guanylin = human guanylin. Changing the luminal pH from 5 to 8 failed to affect the action of guanylin on fluid absorption. Both STa and uroguanylin, but not guanylin, increased the luminal pH by stimulating bicarbonate secretion. Pretreatment of the jejunal loop with guanylin (10(-6) M) 5 min before the instillation of STa (10(-7) M) significantly reduced the inhibitory effect of STa on fluid absorption. It is concluded that guanylin and uroguanylin administered into the rat jejunal lumen have an STa-like action on fluid and electrolyte transport. Guanylin may act as an endogenous antagonist of STa in the rat jejunum and prevent excessive fluid loss by STa.

Topics: Animals; Bacterial Toxins; Biological Transport; Electrolytes; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Humans; Hydrogen-Ion Concentration; Jejunum; Male; Natriuretic Peptides; Peptides; Rats; Rats, Wistar; Solutions

Guanylin and uroguanylin are structurally related intestinal peptide hormones which were purified from a limited number of mammals and are capable of activating the particulate guanylate cyclase-C. Although the biological functions of guanylin and uroguanylin are not yet clarified in detail, they are involved in the regulation of the intestinal water and electrolyte balance. In order to verify the general importance of this hormone system in mammals, we cloned the corresponding cDNAs from pig. Here, we present the nucleotide sequences and the deduced amino acid sequences representing porcine guanylin and uroguanylin. The expression patterns of the corresponding genes, as shown by Northern hybridization and RT-PCR analysis, resemble those of the human homologues. Further, we demonstrate the bioactivity of both porcine peptide hormones by inducing the intracellular cGMP production in human T84 cells and by ion transport experiments using porcine intestinal mucosa in the Ussing chamber.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Cyclic GMP; DNA, Complementary; Gastrointestinal Hormones; Humans; Intestinal Mucosa; Ion Transport; Molecular Sequence Data; Natriuretic Peptides; Peptides; RNA, Messenger; Sequence Homology; Swine; Tumor Cells, Cultured

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; COS Cells; Enzyme Activation; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Mucosa; Kidney; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptide Fragments; Peptides; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Structure-Activity Relationship; Tissue Distribution

Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 microg/ml, N = 12) or uroguanylin (0.5 microg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 microg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (%TNa+, P<0.05) (from 78.46 +/- 0.86 to 64.62 +/- 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 +/- 0.01 to 0.15 +/- 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 +/- 0. 86 to 85.19 +/- 1.48, %TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins.

Topics: Animals; Atrial Natriuretic Factor; Drug Synergism; Gastrointestinal Hormones; Kidney; Natriuretic Peptides; Peptides; Rats; Rats, Wistar

Guanylate cyclases (GC) serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin), two disulfides (guanylin and uroguanylin) and three disulfides (E. coli stable toxin, ST). The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC) has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

Topics: Animals; Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Intestinal Mucosa; Kidney; Mice; Natriuretic Peptides; Opossums; Peptides; Rats; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Signal Transduction

Uroguanylin and guanylin are newly discovered endogenous heat-stable peptides that bind to and activate a membrane bound guanylyl cyclase signaling receptor (termed guanylyl cyclase C; GC-C). These peptides are not only found in blood but are secreted into the lumen of the intestine and effect a net secretion of electrolytes (Na+, K+, Cl-, HCO3-) and fluid into the intestine via a cyclic guanosine-3', 5'-monophosphate (cGMP) mechanism. GC-C is also the receptor for Escherichia coli heat-stable enterotoxin (STa) and activation by STa results in a diarrheal illness. Employing mouse renal in vivo models, we have demonstrated that uroguanylin, guanylin, and STa elicit natriuretic, kaliuretic, and diuretic effects. These biological responses are time- and dose-dependent. Maximum natriuretic and kaliuretic effects are observed within 30-40 min following infusion with pharmacological doses of the peptides in a sealed-urethra mouse model. Our mouse renal clearance model confirms these results and shows significant natriuresis following a constant infusion of uroguanylin for 30 min, while the glomerular filtration rate, plasma creatinine, urine osmolality, heart rate, and blood pressure remain constant. These data suggest the peptides act through tubular transport mechanisms. Consistent with a tubular mechanism, messenger RNA-differential display PCR of kidney RNA extracted from vehicle- and uroguanylin-treated mice show the message for the Na+/K+ ATPase gamma-subunit is down-regulated. Interestingly, GC-C knockout mice (Gucy2c -/-) also exhibit significant uroguanylin-induced natriuresis and kaliuresis in vivo, suggesting the presence of an alternate receptor signaling mechanism in the kidney. Thus, uroguanylin and guanylin seem to serve as intestinal and renal natriuretic peptide-hormones influencing salt and water transport in the kidney through GC-C dependent and independent pathways. Furthermore, our recent clinical probe study has revealed a 70-fold increase in levels of urinary uroguanylin in patients with congestive heart failure. In conclusion, our studies support the concept that uroguanylin and guanylin are endogenous effector peptides involved in regulating body salt and water homeostasis.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Gastrointestinal Hormones; Guanylate Cyclase; Intestinal Mucosa; Intestines; Kidney; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Natriuresis; Natriuretic Peptides; Peptides; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Urine

Guanylin and uroguanylin are intestinal peptides that stimulate guanylate cyclase C and cause chloride secretion. These peptides show topological instability due to two disulfide bonds. The disulfide bonds were reduced and S-carboxymethylated to cleave the bonds and obtain stable and sole derivatives. We established a new and reliable RIA system for the stable derivatives from both peptides. With the use of this system, the response of the peptides to salt loading of the rat small intestine was evaluated. The lumen of the small intestines of Sprague-Dawley rats was perfused in vivo with Krebs-Ringer solution containing different concentrations of salt or mannitol. Mature guanylin, proguanylin, and mature uroguanylin were found in the perfusate in the basal state. The highest salt loading (200 mM NaCl for 20 min) increased the guanylin secretion about threefold (1.9 +/- 0.2 vs. 5.4 +/- 0.5 pmol/min), with the effect lasting for 60 min. The uroguanylin secretion was less affected. Hyperosmolar mannitol also caused a significant but smaller increase of guanylin secretion. Increased guanylin could lead to increased salt and water secretion of the intestine; thus members of the guanylin family have potential roles in the regulation of water and salt metabolism in the small intestine.

Topics: Amino Acid Sequence; Animals; Chlorides; Chromatography, High Pressure Liquid; Cross Reactions; Diuretics, Osmotic; Dose-Response Relationship, Drug; Gastrointestinal Hormones; Homeostasis; Intestine, Small; Male; Mannitol; Methylation; Molecular Sequence Data; Natriuretic Peptides; Peptide Fragments; Peptides; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Sodium Chloride; Specific Pathogen-Free Organisms; Water-Electrolyte Balance

Uroguanylin (UGN) and guanylin (GN) are the endogenous intestinal ligands for guanylyl cyclase C (GC-C). We examined the circadian expression of UGN, GN, and GC-C in the jejunum, ileum, and proximal colon of young adult rats by Northern blot analyses. These assays revealed that UGN is more abundant in the proximal small intestine, whereas GN and GC-C are more abundant in the proximal colon. mRNA levels showed significant circadian variation for UGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-fold peak/trough difference), and GC-C (3- to 5-fold peak/trough difference). The maximal abundance occurred in the dark period for all three mRNAs, although peak UGN and GN expression occurred later in the dark period in the jejunum relative to the ileum and colon. Immunoblot analyses using monospecific polyclonal antibodies against UGN and GN prohormones confirmed the regional and circadian variation detected by Northern assays. Thus the expression of these genes is regulated not only by histological position but also by circadian time.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blotting, Northern; Blotting, Western; Circadian Rhythm; Colon; Enzyme Activators; Gastrointestinal Hormones; Gene Expression; Guanylate Cyclase; Ileum; Intestines; Jejunum; Male; Molecular Sequence Data; Natriuretic Peptides; Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger

The solution structures of the two compounds of human uroguanylin (I and II), which were generated during disulfide bond forming reaction, were found to be topological isomers by 1H-nuclear magnetic resonance spectroscopy. These isomers are interconvertible in aqueous media at rates which vary with the pH and temperature of the solution. Because compound I is active in the cGMP producing assay, but compound II is not, this interconversion may be useful for evaluating the activity of human uroguanylin both in vivo and in vitro.

Topics: Amino Acid Sequence; Animals; Gastrointestinal Hormones; Humans; Isomerism; Models, Molecular; Molecular Sequence Data; Natriuretic Peptides; Nuclear Magnetic Resonance, Biomolecular; Peptides; Peptides, Cyclic; Protein Conformation; Rats; Sequence Alignment; Solutions

Guanylin/GCAP-I and uroguanylin/GCAP-II are two structurally related peptides which play an important role in the regulation of water/electrolyte balance within the gut. In order to enable the investigation and comparison of both peptide hormones at the genomic level, we decided to clone the corresponding genes. The human gene for guanylin/GCAP-I and its 5'-flanking region have been described recently. Here, we report the three exon/two intron structure of the human uroguanylin/GCAP-II gene and its localization on chromosome 1 p35-34, as determined by radiation hybrid mapping. Together with data obtained for the guanylin/GCAP-I gene we show that these genes are localized in the same chromosomal area with other guanlyl cyclase-activating peptides like ANP etc. Northern hybridization revealed that the expression of the uroguanylin/GCAP-II gene is highest in the intestinal mucosa, especially in the ileum and colon. By means of polymerase chain reaction (PCR), an expression was also observed in the stomach where no guanylin/GCAP-I expression is detectable. Using immunohistochemical methods, uroguanylin/GCAP-II immunoreactive material was distinctly localized in D-type gastric and intestinal endocrine cells. Although the comparable data on the genomic organisation of both peptide hormones verify their high degree of relationship, this finding indicates a special task of uroguanylin/GCAP-II within the stomach, such as regulatory functions in gastric secretion. The redundant expression of the GCAP/GC-C system in the small and large intestine, however, is as yet unclear.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Chromosome Mapping; Chromosomes, Human, Pair 1; Gastric Mucosa; Gastrointestinal Hormones; Gene Expression Regulation; Humans; Immunohistochemistry; Molecular Sequence Data; Natriuretic Peptides; Peptides; Polymerase Chain Reaction; Sequence Analysis, DNA; Somatostatin; Somatostatin-Secreting Cells

Uroguanylin and guanylin are isolated mainly from the gastrointestinal tract and are activators of guanylyl cyclase C receptor (GC-C), which mediates the production of intracellular cyclic guanosine 3',5'-monophosphate (cyclic GMP). The bronchodilator effects of agents that raise cyclic GMP levels, such as atrial natriuretic peptide, have been reported, and uroguanylin mRNA has recently been detected in extra-gastrointestinal tissues, including the lung, suggesting their role in pulmonary activity. In the first step of this study, we examined the relaxant effects of uroguanylin and guanylin on isolated tracheal smooth muscle of guinea-pigs, and measured tissue cyclic GMP levels by means of enzymeimmunoassay. Uroguanylin produced concentration-dependent relaxant effects on resting tone and significant elevated cyclic GMP levels. Guanylin produced the same, but less potent, effects. In this study, we first investigated the effects of uroguanylin and guanylin on antigen-induced bronchoconstriction and airway microvascular leakage in actively sensitized guinea-pigs. Anesthetized male guinea-pigs, ventilated via a tracheal cannula, were placed in a plethysmograph to measure pulmonary mechanics for 10 min after challenging with 1 mg/kg of ovalbumin. Evans blue dye was then extravasated into their airway tissues to measure microvascular leakage. Intravenous pretreatment with uroguanylin significantly inhibited ovalbumin-induced bronchoconstriction and microvascular leakage in a dose-dependent manner. These inhibitory effects were mimicked by 8-bromoguanosine 3', 5'-cyclic monophosphate. This study is the first to show that uroguanylin not only had a potent bronchodilatory effect but also inhibited microvascular leakage. These results encouraged us to continue the above experimental and clinical studies in bronchial asthma.

Topics: Airway Resistance; Animals; Blood Pressure; Bronchoconstriction; Capillary Permeability; Cyclic GMP; Dose-Response Relationship, Drug; Gastrointestinal Hormones; Guinea Pigs; Immunoenzyme Techniques; In Vitro Techniques; Lung Compliance; Male; Muscle Relaxation; Muscle, Smooth; Natriuretic Peptides; Ovalbumin; Peptides; Trachea

Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 microM) increased glomerular filtration rate from 0.77 +/- 0.07 to 1.34 +/- 0.3 ml . g-1 . min-1 at the highest concentration. A maximal increase in Na+ excretion was achieved at 0. 66 microM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 +/- 1.7 to 58.8 +/- 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested (P < 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 microM) decreased fractional Na+ reabsorption from 73. 9 +/- 2.4 to 64.5 +/- 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 microM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.

Topics: Amino Acid Sequence; Animals; Diuresis; Gastrointestinal Hormones; Glomerular Filtration Rate; Homeostasis; Humans; Kidney; Male; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptides; Perfusion; Potassium; Rats; Rats, Wistar; Second Messenger Systems; Sequence Alignment; Sequence Homology, Amino Acid; Sodium; Time Factors; Urodynamics

The peptide hormone uroguanylin stimulates chloride secretion via activation of intestinal guanylyl cyclase C (GC-C). It is characterized by two disulfide bonds in a 1-3/2-4 pattern that causes the existence of two topological stereoisomers of which only one induces intracellular cGMP elevation. To obtain an unambiguous structure-function relationship of the isomers, we determined the solution structure of the separated uroguanylin isoforms using NMR spectroscopy. Both isomers adopt well-defined structures that correspond to those of the isomers of the related peptide guanylin. Furthermore, the structure of the GC-C-activating uroguanylin isomer A closely resembles the structure of the agonistic Escherichia coli heat-stable enterotoxin. Compared with guanylin isomers, the conformational interconversion of uroguanylin isomers is retarded significantly. As judged from chromatography and NMR spectroscopy, both uroguanylin isoforms are stable at low temperatures, but are subject to a slow pH-dependent mutual isomerization at 37 degrees C with an equilibrium isomer ratio of approximately 1:1. The conformational exchange is most likely under the sterical control of the carboxy-terminal leucine. These results imply that GC-C is activated by ligands exhibiting the molecular framework corresponding to the structure of uroguanylin isomer A.

Topics: Amino Acid Sequence; Bacterial Toxins; Enterotoxins; Enzyme Activation; Epithelial Cells; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Molecular Sequence Data; Natriuretic Peptides; Nuclear Magnetic Resonance, Biomolecular; Peptides; Protein Conformation; Protein Isoforms; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Stimulation, Chemical; Structure-Activity Relationship; Thermodynamics

Guanylin, a peptide purified from rat jejunum, is thought to regulate water and electrolyte balance in the intestine. We show here, using a combination of Northern blots, Western blots, and functional assays, that guanylin and its receptor (GCC) are not distributed in parallel within the rat intestine. To investigate the possibility that there might be a second intestinal peptide that serves as a ligand for GCC, we assayed tissue extracts for the ability to stimulate cyclic GMP synthesis in a GCC-expression cell line. Duodenal extracts display a peak of biological activity that is not present in colon and that does not comigrate with guanylin or proguanylin. The activity co-purifies with a novel peptide (TIATDECELCINVACTGC) that has high homology with uroguanylin, a peptide initially purified from human and opossum urine. A rat uroguanylin cDNA clone was found to encode a propeptide whose C-terminus corresponds to our purified peptide. Northern blots with probes generated from this clone reveal that prouroguanylin mRNA is strongly expressed in proximal small intestine, but virtually absent from colon, corroborating our biochemical measurements. Taken together, these studies demonstrate an intestinal origin for uroguanylin, and show that within the intestine its distribution is complementary to that of guanylin.

Topics: Amino Acid Sequence; Animals; Base Sequence; Chromatography, High Pressure Liquid; Colon; DNA, Complementary; Duodenum; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Molecular Sequence Data; Natriuretic Peptides; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sequence Analysis, DNA; Tissue Distribution

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.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Cell Line; Cyclic GMP; Enterotoxins; Escherichia coli; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Hydrogen-Ion Concentration; Intestinal Mucosa; Kinetics; Membrane Potentials; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptide Fragments; Peptides; Radioligand Assay; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

Uroguanylin and guanylin are intestinal peptides that activate a receptor-guanylate cyclase, which is also a receptor for Escherichia coli heat-stable enterotoxin (STa). These peptides may have a role in the body's regulation of fluid and electrolytes.. STa, bioactive guanylin, and bioactive uroguanylin were evaluated for effects in: 1) the suckling mouse intestinal fluid secretion assay; 2) an in vitro suckling mouse intestinal loop assay; 3) an intestinal receptor autoradiography assay; 4) a control or agonist-stimulated assay for cGMP response in T84 cells; and 5) an in vivo renal function assay in mice.. In vivo, orally administered uroguanylin and STa but not guanylin, stimulated intestinal fluid secretion. All three peptides activated intestinal guanylate cyclase and had common intestinal receptors. In vitro, after pretreatment with chymotrypsin, only uroguanylin and STa retained agoinst activity. Chymostatin preserved guanylin activity. STa and uroguanylin induced diuresis, natriuresis, and kaliuresis. Guanylin was less potent than uroguanylin and STa.. The results suggest that the endogenous intestinal peptides, uroguanylin and guanylin, regulate water and electrolyte homeostasis both through local effects on intestinal epithelia and endocrine effects on the kidney.

Topics: Animals; Animals, Suckling; Bacterial Toxins; Cells, Cultured; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Gastrointestinal Hormones; Intestinal Mucosa; Intestinal Secretions; Intestines; Kidney; Mice; Mice, Inbred ICR; Natriuretic Agents; Natriuretic Peptides; Peptides

Guanylin and uroguanylin are peptides that activate receptor guanylate cyclases (GCs) and elicit increased intestinal secretion. Bacteria that cause traveler's diarrhea produce heat-stable toxins (STs) that mimic this action. Investigation of the distribution and identity of receptor GCs in the gastrointestinal tract of rats revealed that receptors were localized to epithelial cells in stomach and intestine. Clusters of cells in gastric mucosa and enterocytes lining the intestine exhibited specific binding of 125I-labeled ST. Ligated loops of stomach and intestine treated with intraluminal ST had significant increases in guanosine 3',5'-cyclic monophosphate (cGMP), with duodenum exhibiting the greatest response. Expression of guanylate cyclase C (GCC) mRNA and a truncated, GCC-like mRNA was found in both stomach and intestine. Both mRNAs were isolated as cDNAs encoding the GC catalytic domain. The 0.9-kilobase (kb) cDNA is 99.8% identical to GCC, whereas the truncated, 0.75-kb GCC-like cDNA has a 159-nucleotide deletion and is 96.6% identical to GCC at the protein level. Uroguanylin and guanylin mRNAs were detected in stomach and intestine. Uroguanylin mRNA was most abundant in small intestine, whereas guanylin mRNA was highest in large intestine. Thus the stomach and intestine are targets for regulation of transport by guanylin and uroguanylin via cGMP.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Binding Sites; Colon; DNA, Complementary; Enterotoxins; Escherichia coli Proteins; Female; Gastric Mucosa; Gastrointestinal Hormones; Guanylate Cyclase; Intestinal Mucosa; Intestine, Small; Male; Molecular Sequence Data; Natriuretic Peptides; Organ Specificity; Peptides; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction

Uroguanylin and guanylin, endogenous ligands of the guanylate cyclase C receptor, are presumed to mediate fluid and electrolyte secretion in the intestine. The aim of this study was to characterize the expression patterns of uroguanylin and guanylin messenger RNA (mRNA) in the mouse intestine.. A mouse uroguanylin complementary DNA was amplified from a partial genomic clone, and Northern analyses and in situ hybridization were performed to localize guanylin and uroguanylin mRNA along the duodenal-colonic and crypt-villus axes.. Uroguanylin mRNA was expressed throughout the mouse intestine and also in the kidney. Signal intensity was greatest in the small intestine for uroguanylin and in the distal small intestine and colon for guanylin. In situ hybridization showed uroguanylin mRNA localized predominantly in intestinal villi and the corticomedullary junction of the kidney, whereas guanylin mRNA was localized in both crypts and villi in the small intestine and to superficial epithelial cells in the colon.. Mouse uroguanylin mRNA expression is discrete from guanylin expression in the intestine. The patterns of distribution in the intestine and the known pH optima of these ligands suggest a complementary role for these secretagogues.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cloning, Molecular; DNA, Complementary; Gastrointestinal Hormones; In Situ Hybridization; Intestinal Mucosa; Mice; Molecular Sequence Data; Natriuretic Peptides; Peptides; RNA, Messenger

Recently, the peptides guanylin and uroguanylin were identified as endogenous ligands of the membrane-bound guanylate cyclase C (GC-C) that is mainly expressed in the intestinal epithelium. In the present study, bioactive guanylin and uroguanylin have been prepared by solid-phase methodology using Fmoc/HBTU chemistry. The two disulfide bonds with relative 1/3 and 2/4 connectivity have been introduced selectively by air oxidation of thiol groups and iodine treatment of Cys(Acm) residues. Using this strategy, several sequential derivatives were prepared. Temperature-dependent HPLC characterization of the bioactive products revealed that guanylin-related peptides exist as a mixture of two compounds. The isoforms are interconverted within approximately 90 min, which prevents their separate characterization. This effect was not detected for uroguanylin-like peptides. Synthetic peptides were tested for their potential to activate GC-C in cultured human colon carcinoma cells (T84), known to express high levels of GC-C. The results obtained show that both disulfide bonds are necessary for GC-C activation. The presence of the amino-terminally neighboring residues of Cys104 for guanylin and Cys100 for uroguanylin has been found to be essential for GC-C stimulation. Unexpectedly, a hybrid peptide obtained from substitution of the central tripeptide AYA of guanylin by the tripeptide VNV of uroguanylin was not bioactive.

Topics: Amino Acid Sequence; Animals; Chlorides; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cyclic GMP; Enzyme Activation; Female; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Ion Transport; Molecular Sequence Data; Natriuretic Peptides; Peptides; Protein Conformation; Rats; Rats, Wistar; Tumor Cells, Cultured

Uroguanylin is an endogenous ligand of the intestinal receptor guanylate cyclase-C (GC-C). Both uroguanylin and the related peptide ligand guanylin bind to GC-C and stimulate an increase in cyclic GMP, inducing chloride secretion via the cystic fibrosis transmembrane conductance regulator. We describe the cloning of the complete mouse uroguanylin gene (Guca1b) and show that Guca1b is tightly linked to the mouse guanylin gene on chromosome 4. The two genes are structurally similar, being composed of three short exons; the uroguanylin gene spans 2.4 kb and the guanylin gene spans 1.7 kb. Uroguanylin mRNA is most prominent in proximal small intestine, whereas guanylin mRNA is predominantly expressed in distal small intestine and colon. The upstream promoter sequence of the mouse uroguanylin gene contains a canonical TATA element at the site of transcription initiation and consensus binding sites for several known transcription factors, including HNF-1 and Sp1 within the first 1 kb. Although the gene structure and coding sequences of uroguanylin and guanylin are similar, the 5' flanking sequences and patterns of expression of these two genes in the intestine are different. It is likely that uroguanylin and guanylin represent gene duplications that have evolved to allow overlapping and complementary patterns of expression in the intestine.

Topics: Animals; Base Sequence; Binding Sites; Chromosome Mapping; Cloning, Molecular; Crosses, Genetic; DNA Primers; DNA, Complementary; Female; Gastrointestinal Hormones; Gene Expression; Genetic Linkage; Humans; Intestinal Mucosa; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Muridae; Natriuretic Peptides; Peptides; RNA, Messenger; TATA Box; Tissue Distribution

Guanylin and uroguanylin are peptides that stimulate membrane guanylate cyclases (GC) and regulate intestinal and renal function via cGMP. Complementary DNAs were isolated encoding opossum preproguanylin and a 279-amino acid portion of a receptor-guanylate cyclase expressed in opossum kidney (OK) cells (GC-OK). The tissue expression of messenger RNA transcripts for these signaling molecules were then compared. Northern and/or reverse transcription-PCR assays revealed that guanylin, uroguanylin, and GC-OK messenger RNAs are expressed in tissues within the digestive, renal, central nervous, reproductive, and lymphoid organ systems. Receptor autoradiography localized the receptors for uroguanylin and guanylin to renal proximal tubules and seminiferous tubules of testis. Synthetic guanylin and uroguanylin peptides activated the receptor-GCs in opossum kidney cortex and in cultured OK cells eliciting increased intracellular cGMP. Expression of agonist and receptor-GC signaling molecules provides a pathway for paracrine and/or autocrine regulation of cellular functions via cGMP in the digestive, renal, central nervous, reproductive, and lymphoid/immune organ systems. Uroguanylin also links the intestine and kidney in a potential endocrine axis that activates tubular receptor-GCs and influences renal function.

Topics: Amino Acid Sequence; Animals; Base Sequence; Central Nervous System; Digestive System; DNA, Complementary; Gastrointestinal Hormones; Genitalia; Kidney; Lymphatic System; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptides; Protein Precursors; RNA, Messenger; Signal Transduction

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.

Topics: Amino Acid Sequence; Animals; Biological Assay; Cell Line; Chromatography, High Pressure Liquid; Colon; Cyclic GMP; Duodenum; Gastrointestinal Hormones; Intestinal Mucosa; Intestine, Small; Molecular Sequence Data; Natriuretic Peptides; Peptides; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic; Urine

Uroguanylin is a small peptide isolated from opossum urine that activates membrane guanylate cyclases. We report the isolation by molecular cloning of cDNAs encoding the 109 amino acid preprouroguanylin containing the active uroguanylin peptide at its C-terminus. Preprouroguanylin mRNAs of 1.2 kb were detected throughout the small and large intestine and in the atria and ventricles of heart, but not in kidney, stomach or liver. Transfection of COS-1 cells with the uroguanylin cDNA resulted in prouroguanylin secretion. Both uroguanylin and prouroguanylin were isolated from opossum plasma. Thus, uroguanylin is made by the intestine and heart and circulates as a bioactive form of uroguanylin and the inactive prouroguanylin.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Line; Chlorocebus aethiops; Cloning, Molecular; DNA, Complementary; Gastrointestinal Hormones; Gene Expression; Intestinal Mucosa; Intestine, Large; Intestine, Small; Molecular Sequence Data; Myocardium; Natriuretic Peptides; Opossums; Organ Specificity; Peptide Biosynthesis; Peptides; Protein Precursors; Recombinant Proteins; RNA, Messenger; Sequence Homology, Amino Acid; Transfection

Uroguanylin, a member of the guanylin peptide family, is an endogenous activator of intestinal guanylate cyclase (GC-C). A cDNA encoding a precursor for human uroguanylin was cloned from a human colon cDNA library and sequenced. The precursor was 112 amino acids long and included a signal peptide at the N-terminus and the human uroguanylin sequence at the C-terminus. RNA blot analysis and the reverse transcription-polymerase chain reaction (RT-PCR) showed that human uroguanylin mRNA is expressed in the stomach and intestine. Uroguanylin, as well as guanylin, may be a potent physiological regulator of intestinal fluid and electrolyte transport.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA Primers; DNA, Complementary; Gastrointestinal Hormones; Gene Expression; Gene Library; Humans; Molecular Sequence Data; Natriuretic Peptides; Oligonucleotides, Antisense; Peptide Biosynthesis; Peptides; Polymerase Chain Reaction; Protein Precursors; Protein Sorting Signals; RNA, Messenger; Sequence Homology, Amino Acid

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.

Topics: Amino Acid Sequence; Animals; Biological Assay; Cell Line; Chymotrypsin; Colon; Cyclic GMP; Gastrointestinal Hormones; Intestinal Mucosa; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptide Fragments; Peptides

Pathogenic strains of enteric bacteria secrete small heat-stable toxins (STs) that activate membrane guanylyl cyclase receptors found in the intestine. The intestinal peptide agonists, guanylin and uroguanylin, are structurally related to STs. Receptors for 125I-ST were found throughout the entire length of the intestinal tract of all the birds examined. These receptors were restricted to intestinal epithelial cells covering villi and forming intestinal glands and were not observed in other strata of the gut wall. The most intense labeling of receptors by 125I-ST occurred in the region of the microvillus border of individual enterocytes. There appeared to be a decrease in receptor density distally along the length of the small intestine, although labeling of receptors by 125I-ST was observed throughout the small intestine and colon. Cellular cGMP accumulation responses to Escherichia coli ST and rat guanylin in the domestic turkey and duck were greater in the proximal small intestine compared to the distal small intestine or colon. Brush border membranes (BBM) isolated from the mucosa of proximal small intestine of turkeys exhibited agonist-stimulated guanylyl cyclase activity. The rank order potency for enzyme activation was E. coli ST > uroguanylin > guanylin. Competitive radioligand binding assays using 125I-ST and turkey intestine BBM revealed a similar rank order affinity for the receptors that was exemplified by the Kd values of ST 2.5 nM, uroguanylin 80 nM and guanylin 2.6 microM. It may be concluded that functional receptors for the endogenous peptides, guanylin and uroguanylin, occur in the apical membranes of enterocytes throughout the avian intestine. The receptor-guanylyl cyclase(s) of proximal small intestine were preferentially activated by uroguanylin relative to guanylin, but both endogenous peptides were less potent than their molecular mimic, E. coli ST.

Topics: Amino Acid Sequence; Animals; Autoradiography; Biological Assay; Birds; Drug Stability; Enterotoxins; Escherichia coli; Gastrointestinal Hormones; Guanylate Cyclase; Hot Temperature; Intestinal Mucosa; Molecular Sequence Data; Natriuretic Peptides; Peptides; Rabbits; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Tissue Distribution

Guanylin, a peptide homologue of the bacterial heat-stable enterotoxins (ST), is an endogenous activator of guanylate cyclase C (GC-C). We have initiated a search for other members of the guanylin peptide family and in the current study describe a "guanylin-like peptide" from human urine. Bioactivity was monitored by determining the effect of urine extracts on T84 cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. Purification yielded two bioactive peaks of peptides that, when sequenced by NH2-terminal analysis, possessed 15 and 16 amino acids. The sequence of the smaller peptide represented an NH2-terminal truncation of the larger peptide. We have termed the larger peptide human uroguanylin; it has the following amino acid sequence: NDDCELCVNVACTGCL. Human uroguanylin shares amino acid sequence homology with guanylin and ST. Synthetic uroguanylin increased cGMP levels in T84 cells, competed with 125I-labeled ST for receptors, and stimulated Cl- secretion as reflected by an increased short-circuit current. Thus we report the isolation from human urine of a unique peptide, uroguanylin, that behaves in a manner similar to guanylin and appears to be a new member of this peptide family.

Topics: Adult; Amino Acid Sequence; Animals; Cell Line; Chlorides; Colon; Cyclic GMP; Escherichia coli; Gastrointestinal Hormones; Humans; In Vitro Techniques; Intestinal Mucosa; Male; Mass Spectrometry; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptides; Radioligand Assay; Rats; Sequence Homology, Amino Acid

The intestinal hormone guanylin and bacterial heat-stable enterotoxins (STs) are members of a peptide family that activates intestinal membrane guanylate cyclase. Two different peptides that activate the human intestinal T84 cell guanylate cyclase have been purified from urine and intestinal mucosa of opossums (Didelphis virginiana). The highly acidic peptide, QEDCELCINVACTGC, was named uroguanylin because it was isolated from urine and shares 53% identity with guanylin. A second peptide, SHTCEICAFAACAGC, was purified from urine and intestinal mucosa. This alanine-rich peptide was 47% identical to uroguanylin and 73% identical to human guanylin, suggesting that it may be an opossum homologue of guanylin. Synthetic uroguanylin-(2-15) (i.e., EDCELCINVACTGC) was 10-fold more potent than synthetic rat guanylin, but both peptides were less potent than Escherichia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 125I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepithelial Cl- secretion was stimulated by 1 microM uroguanylin, indicated by an increase in the short circuit current of T84 cells. Thus, uroguanylin is another paracrine hormone in the emerging peptide family that activates intestinal membrane guanylate cyclase. The second peptide may be the opossum form of guanylin, or perhaps, it is still another member of this peptide family. The presence of uroguanylin and guanylin in urine and receptors in proximal tubules suggests that these peptides may also originate from renal tissue and may regulate kidney function.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Biological Transport; Electric Conductivity; Enterotoxins; Enzyme Activation; Escherichia coli Proteins; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Infant, Newborn; Molecular Sequence Data; Natriuretic Peptides; Opossums; Peptides; Rats; Receptors, Cell Surface; Sequence Alignment; Sequence Homology, Amino Acid