Effects of guanylin and uroguanylin on rat jejunal fluid and electrolyte transport: comparison with heat-stable enterotoxin

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.     

Guanylin and related peptides.

Guanylin and uroguanylin are short peptides homologous to heat-stable enterotoxins of Escherichia coli and other enteric bacteria. Guanylin and uroguanylin are synthetized from the respective prepropeptides mainly in gastrointestinal mucosa and are secreted both into intestinal lumen and into the blood. Luminally secreted peptides stimulate chloride and bicarbonate secretion in the intestine through the mechanism involving guanylate cyclase C receptor, cyclic GMP, protein kinase G and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Bacterial enterotoxins, which have greater potency than endogenous peptides, induce excessive fluid secretion into intestinal lumen leading to secretory diarhea. Uroguanylin is expressed mainly in enterochromaffin cells of duodenum and proximal small intestine whereas guanylin is abundant in goblet cells of colonic epithelium. Uroguanylin and guanylin increase urinary sodium and potassium excretion both as circulating hormones and as paracrine mediators produced within the kidney. Uroguanylin functions as "intestinal natriuretic hormone" which is secreted in response to oral sodium loading and maintains sodium balance during postprandial period. Plasma and urinary concentrations of guanylin and uroguanylin increase in renal failure and heart failure. Guanylin peptides possess antiproliferative activity in intestinal cells culture and their expression decreases in colonic carcinoma indicating that their deficiency may contribute to the pathogenesis of this disease.     

STa peptide analogs for probing guanylyl cyclase C

Guanylyl cyclase C (GC-C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18-residue heat-stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12-residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC-C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.     

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension

Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO₃ secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.     

Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology.

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.     

Interaction of atrial natriuretic peptide, urodilatin, guanylin and uroguanylin in the isolated perfused rat kidney

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.     

Guanylin peptide family: history, interactions with ANP, and new pharmacological perspectives

The guanylin family of peptides has 3 subclasses of peptides containing either 3 intramolecular disulfide bonds found in bacterial heat-stable enterotoxins (ST), or 2 disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity. These hormones are synthesized in the intestine and released both luminally and into the circulation, and are also produced within the kidney. Stimulation of renal target cells by guanylin peptides in vivo or ex vivo elicits a long-lived diuresis, natriuresis, and kaliuresis by both cGMP-dependent and independent mechanisms. Uroguanylin may act as a hormone in a novel endocrine axis linking the digestive system and kidney as well as a paracrine system intrarenally to increase sodium excretion in the postprandial period. This highly integrated and redundant mechanism allows the organism to maintain sodium balance by eliminating excess sodium in the urine. In addition, small concentrations of the atrial natriuretic peptide (ANP) can synergize with low concentrations of both guanylin or uroguanylin, which do not induce natriuresis per se, to promote significant natriuresis. Interestingly, the activation of the particulate guanylate cyclase receptors by natriuretic peptides can promote relaxation of animal and human penile erectile tissue and increase intracavernosal pressure to induce penile erection. These peptides can be prototypes for new drugs to treat erectile dysfunction, especially in patients with endothelial and nitrergic dysfunction, such as in diabetes.     

A novel guanylin family (guanylin,uroguanylin, and renoguanylin) in eels: possible osmoregulatory hormones in intestine and kidney

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.     

Colonocyte basolateral membranes contain Escherichia coli heat-stable enterotoxin receptors

Heat-stable enterotoxin (ST(a)) elaborated by E. coli is a major cause of diarrhea. The transmembrane protein guanylyl cyclase C (GC-C) is the acknowledged receptor for ST(a) and for the mammalian peptides guanylin and uroguanylin. Binding to GC-C results in generation of cGMP, activation of type II cGMP-dependent protein kinase, phosphorylation of CFTR and increased chloride and bicarbonate secretion. We had previously shown that ST(a) receptors (GC-C) are found on the brush border membranes of small intestinal enterocytes and of colonocytes. However, since it has subsequently been shown that the endogenous ligands for these receptors, guanylin and uroguanylin, circulate in blood, we proposed the existence of ST(a) binding sites on the basolateral membranes (BLM) of colonocytes. Specific binding of 125I-ST(a) to rat colonocyte BLM was seen. The kinetics of binding to the BLM were similar to binding to BBM. The nature of the BLM receptor is unknown. This suggests that circulating guanylin and uroguanylin, analogues of ST(a), may also function via the basolateral surface.     

The Heat-Stable Enterotoxin-Guanylin Receptor is Expressed in Rat Hepatocytes and in a Rat Hepatoma (H-35) Cell Line

Abstract

Background/Aims: Guanylyl cyclase C (GC-C) is an intestinal transmembrane receptor which binds both guanylin, an endogenous ligand, and Escherichia coli heat-stable enterotoxin (STa) resulting in 5′-cyclic guanosine monophosphate (cGMP) accumulation and chloride secretion. In the adult rat, there is a high basal level of GC-C expression in the intestine, but not in the liver. Increased expression of GC-C in the rat liver has been demonstrated during the perinatal period as well as with liver regeneration and during an acute phase response. The aim of this study was to identify and utilize cell culture models to further characterize the expression of GC-C in the liver. Methods: STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression by Northern analysis were determined in primary cultures of rat hepatocytes and H-35 cells, a rat hepatoma cell line, following treatment with dexamethasone and/or interleukin-6 (IL-6). Results: In rat hepatocytes treated with the combination of dexamethasone and IL-6, there was an increase in STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression as compared to untreated cells. In H-35 cells treated with dexamethasone alone, there was an increase in STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression as compared to untreated cells. Conclusion: Primary cultures of rat hepatocytes and H-35 cells can be utilized to further study upregulation of GC-C in the hepatocyte. The expression of this receptor in hepatocytes, combined with the recent demonstration of circulating guanylin, is consistent with a functional role for GC-C in the liver.     

Circadian regulation of uroguanylin and guanylin in the rat intestine

Uroguanylin (UGN) and guanylin (GN) are theendogenous intestinal ligands for guanylyl cyclase C (GC-C). Weexamined the circadian expression of UGN, GN, and GC-C in the jejunum,ileum, and proximal colon of young adult rats by Northern blotanalyses. These assays revealed that UGN is more abundant in theproximal small intestine, whereas GN and GC-C are more abundant in theproximal colon. mRNA levels showed significant circadian variation forUGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-foldpeak/trough difference), and GC-C (3- to 5-fold peak/troughdifference). The maximal abundance occurred in the dark period for allthree mRNAs, although peak UGN and GN expression occurred later in thedark period in the jejunum relative to the ileum and colon. Immunoblotanalyses using monospecific polyclonal antibodies against UGN and GNprohormones confirmed the regional and circadian variation detected byNorthern assays. Thus the expression of these genes is regulated notonly by histological position but also by circadian time.

     

Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides.

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.     

Receptor guanylyl cyclase C (GC-C): regulation and signal transduction

Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.     

Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine

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.     

Insulin and heregulin-beta1 upregulate guanylyl cyclase C expression in rat hepatocytes: reversal by phosphodiesterase-3 inhibition

Guanylyl cyclase C (GC-C) is the receptor for the hormones guanylin and uroguanylin. Although primarily expressed in the rat intestine, GC-C is also expressed in the liver during neonatal or regenerative growth or during the acute phase response. Little is known about the hepatic regulation of GC-C expression. The influence of various hepatic growth or acute phase regulators on GC-C expression was evaluated by immunoblot analysis of protein from primary rat hepatocytes grown in a serum-free medium. Insulin and heregulin-beta1 strongly stimulated GC-C expression by 24 h of cell culture. Several different hormones and agents suppressed this action, including transforming growth factor beta (TGF-beta), as well as inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase) and phosphodiesterase 3 (PDE-3, an insulin- and PI-3-kinase-dependent enzyme). The compartmental downregulation of cAMP levels by PDE-3 may be a critical step in the hormonal action that culminates in GC-C synthesis.     

Isolation and expression of a guanylate cyclase-coupled heat stable enterotoxin receptor cDNA from a human colonic cell line.

Heat stable enterotoxins (STs) are low molecular-weight peptides secreted by enterotoxigenic bacteria. One type of these enterotoxins (STa) induces intestinal secretion leading to acute diarrhea by binding to a membrane form of guanylate cyclase. We have isolated a cDNA from a human colonic cell line, T84, encoding for a guanylate cyclase-coupled enterotoxin receptor (STaR). The predicted amino acid sequence of the human STa receptor is 81% identical with the previously cloned enterotoxin receptor (GC-C) from rat intestine. COS-7 cells transiently transfected with the cloned cDNA expressed specific concentration-dependent response to STa as measured by cyclic GMP accumulation and is about 20 times more sensitive to the stimulation by STa than has been shown for GC-C.