The evolutionary divergence of receptor guanylyl cyclase C has implications for preclinical models for receptor-directed therapeutics.

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.

The Natriuretic Peptide System: A Single Entity, Pleiotropic Effects.

In the modern scientific landscape, natriuretic peptides are a complex and interesting network of molecules playing pleiotropic effects on many organs and tissues, ensuring the maintenance of homeostasis mainly in the cardiovascular system and regulating the water-salt balance. The characterization of their receptors, the understanding of the molecular mechanisms through which they exert their action, and the discovery of new peptides in the last period have made it possible to increasingly feature the physiological and pathophysiological role of the members of this family, also allowing to hypothesize the possible settings for using these molecules for therapeutic purposes. This literature review traces the history of the discovery and characterization of the key players among the natriuretic peptides, the scientific trials performed to ascertain their physiological role, and the applications of this knowledge in the clinical field, leaving a glimpse of new and exciting possibilities for their use in the treatment of diseases.

Evolution of the membrane/particulate guanylyl cyclase: From physicochemical sensors to hormone receptors.

Guanylyl cyclase (GC) is an enzyme that produces 3',5'-cyclic guanosine monophosphate (cGMP), one of the two canonical cyclic nucleotides used as a second messenger for intracellular signal transduction. The GCs are classified into two groups, particulate/membrane GCs (pGC) and soluble/cytosolic GCs (sGC). In relation to the endocrine system, pGCs include hormone receptors for natriuretic peptides (GC-A and GC-B) and guanylin peptides (GC-C), while sGC is a receptor for nitric oxide and carbon monoxide. Comparing the functions of pGCs in eukaryotes, it is apparent that pGCs perceive various environmental factors such as light, temperature, and various external chemical signals in addition to endocrine hormones, and transmit the information into the cell using the intracellular signaling cascade initiated by cGMP, e.g., cGMP-dependent protein kinases, cGMP-sensitive cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases. Among vertebrate pGCs, GC-E and GC-F are localized on retinal epithelia and are involved in modifying signal transduction from the photoreceptor, rhodopsin. GC-D and GC-G are localized in olfactory epithelia and serve as sensors at the extracellular domain for external chemical signals such as odorants and pheromones. GC-G also responds to guanylin peptides in the urine, which alters sensitivity to other chemicals. In addition, guanylin peptides that are secreted into the intestinal lumen, a pseudo-external environment, act on the GC-C on the apical membrane for regulation of epithelial transport. In this context, GC-C and GC-G appear to be in transition from exocrine pheromone receptor to endocrine hormone receptor. The pGCs also exist in various deuterostome and protostome invertebrates, and act as receptors for environmental, exocrine and endocrine factors including hormones. Tracing the evolutionary history of pGCs, it appears that pGCs first appeared as a sensor for physicochemical signals in the environment, and then evolved to function as hormone receptors. In this review, the author proposes an evolutionary history of pGCs that highlights the emerging role of the GC/cGMP system for signal transduction in hormone action.

Molecular mechanisms underlying guanylin-induced transcellular Cl- secretion into the intestinal lumen of seawater-acclimated eels.

Guanylin (GN) stimulates Cl- secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl- transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl- secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3- from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl- and Na+, which was blocked by serosal DNDS. These results suggest that Cl- is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+-HCO3- cotransporter (NBC) but not by Na+-K+-2Cl- cotransporter 1 (NKCC1), and Cl- is secreted by unknown DPC-sensitive Cl- channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42- transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl- secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl- secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

Palmitic acid induces guanylin gene expression through the Toll-like receptor 4/nuclear factor-κB pathway in rat macrophages.

Recently, we showed that double-transgenic rats overexpressing guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), specifically in macrophages demonstrate an antiobesity phenotype and low-expression levels of proinflammatory cytokines in the mesenteric fat even when fed a high-fat diet. Here, we examined the levels and mechanism of Gn and GC-C transcription following saturated fatty acid and lipopolysaccharide (LPS), an activator of Toll-like receptor 4 (TLR4), exposure by using the NR8383 macrophage cell line. In addition, the levels of guanylin and cGMP were increased by addition of either palmitic acid or LPS. Next, we investigated the interaction of the gene transcription and nuclear factor-κB (NF-κB) by using an NF-κB inhibitor and chromatin immunoprecipitation assay. We showed that palmitic acid induced Gn gene expression via TLR4 and NF-κB. Moreover, we demonstrated that NF-κB binding to the Gn promoter was responsible for the induction of gene transcription by palmitic acid or LPS. Our results indicate that saturated fatty acids such as palmitic acid activate Gn gene expression via the NF-κB pathway, raising the possibility that the activated Gn-GC-C system may contribute to the inhibition of high-fat diet-induced proinflammatory cytokines in macrophages.

Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Absence of Receptor Guanylyl Cyclase C Enhances Ileal Damage and Reduces Cytokine and Antimicrobial Peptide Production during Oral Salmonella enterica Serovar Typhimurium Infection.

Nontyphoidal Salmonella disease contributes toward significant morbidity and mortality across the world. Host factors, including gamma interferon, tumor necrosis factor alpha, and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we investigated the roles of receptor guanylyl cyclase C (GC-C), which is predominantly expressed in the intestine and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C (Gucy2c-/-) displayed accelerated mortality compared with that for wild-type mice following infection via the oral route, even though both groups possessed comparable systemic Salmonella infection burdens. Survival following intraperitoneal infection remained similar in both groups, indicating that GC-C offered protection via a gut-mediated response. The serum cortisol level was higher in Gucy2c-/- mice than wild-type (Gucy2c+/+) mice, and an increase in infection-induced thymic atrophy with a loss of immature CD4+ CD8+ double-positive thymocytes was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting, and distortion of the villus architecture, was seen in Gucy2c-/- mice concomitantly with a larger number of ileal tissue-associated bacteria. Transcription of key mediators of Salmonella-induced inflammation (interleukin-22/Reg3ß) was altered in Gucy2c-/- mice in comparison to that in Gucy2c+/+ mice. A reduction in fecal lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c-/- mice. Gucy2c-/- mice cohoused with wild-type mice continued to show reduced amounts of lactobacilli and increased susceptibility to infection. Our study, therefore, suggests that the receptor GC-C confers a survival advantage during gut-mediated Salmonella enterica serovar Typhimurium pathogenesis, presumably by regulating Salmonella effector mechanisms and maintaining a beneficial microbiome.

Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin.

Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers' diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STaL9A/N12S, STaL9A/A14H, STaN12S/A14T, and STaL9A/N12S/A14H We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLTR192G/L211A) for the toxoid fusions 3×STaL9A/N12S-mnLTR192G/L211A, 3×STaL9A/A14H-mnLTR192G/L211A, 3×STaN12S/A14T-mnLTR192G/L211A, and 3×STaL9A/N12S/A14H-mnLTR192G/L211A; examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity in vitro and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.

Renoguanylin stimulates apical CFTR translocation and decreases HCO3- secretion through PKA activity in the Gulf toadfish (Opsanus beta).

The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (Opsanus beta). Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to the apical membrane, which is negated by PKA inhibition. Moreover, RGN decreased HCO3- secretion, likely by limiting apical HCO3-/Cl- exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO3- secretion.

Theoretical structural characterization of lymphoguanylin: A potential candidate for the development of drugs to treat gastrointestinal disorders.

Guanylin peptides (GPs) are small cysteine-rich peptide hormones involved in salt absorption, regulation of fluids and electrolyte homeostasis. This family presents four members: guanylin (GN), uroguanylin (UGN), lymphoguanylin (LGN) and renoguanylin (RGN). GPs have been used as templates for the development of drugs for the treatment of gastrointestinal disorders. Currently, LGN is the only GP with only one disulfide bridge, making it a remarkable member of this family and a potential drug template; however, there is no structural information about this peptide. In fact, LGN is predicted to be highly disordered and flexible, making it difficult to obtain structural information using in vitro methods. Therefore, this study applied a series of 1µs molecular dynamics simulations in order to understand the structural behavior of LGN, comparing it to the C115Y variant of GN, which shows the same Cys to Tyr modification. LGN showed to be more flexible than GN C115Y. While the negatively charged N-terminal, despite its repellent behavior, seems to be involved mainly in pH-dependent activity, the hydrophobic core showed to be the determinant factor in LGN's flexibility, which could be essential in its activity. These findings may be determinant in the development of new medicines to help in the treatment of gastrointestinal disorders. Moreover, our investigation of LGN structure clarified some issues in the structure-activity relationship of this peptide, providing new knowledge of guanylin peptides and clarifying the differences between GN C115Y and LGN.

Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.

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.

Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl(-) channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney.

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease.

OBJECTIVE: Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn's disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. MATERIAL AND METHODS: Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3',5'-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. CONCLUSIONS: GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Guanylin peptides regulate electrolyte and fluid transport in the Gulf toadfish (Opsanus beta) posterior intestine.

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.

Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine.

Guanylin (GN) inhibited water absorption and short-circuit current (Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na(+)-K(+)-2Cl(-) cotransporter (NKCC), which is a key player for the Na(+)-K(+)-Cl(-) transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20-30% water absorption still remained. The remainder may be due to the Cl(-)/HCO3 (-) exchanger and Na(+)-Cl(-) cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2ß and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCß genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2ß was MI > PI, and that of NCCß was MI << PI. The present study showed that GN not only inhibits the bumetanide-sensitive Na(+)-K(+)-Cl(-) transport system governed by NKCC2ß, but also regulates unknown ion transporters different from GN-insensitive SLC26A6 and NCC. A candidate is cystic fibrosis transmembrane conductance regulator Cl(-) channel, as demonstrated in mammals, but its expression is low in eel intestine, and its role may be minor, as indicated by the small effect of its inhibitors.

Reduced Plasma Guanylin Levels Following Enterotoxigenic Escherichia coli-Induced Diarrhea.

The intestinal peptide hormones guanylin (GN) and uroguanylin (UGN) interact with the epithelial cell receptor guanylate cyclase C to regulate fluid homeostasis. Some enterotoxigenic Escherichia coli (ETEC) produce heat-stable enterotoxin (ST), which induces diarrhea by mimicking GN and UGN. Plasma concentrations of prohormones of GN (proGN) and UGN (proUGN) are reportedly decreased during chronic diarrheal diseases. Here we investigate whether prohormone concentrations also drop during acute diarrhea caused by ST-producing ETEC strains TW10722 and TW11681. Twenty-one volunteers were experimentally infected with ETEC. Blood (n = 21) and urine (n = 9) specimens were obtained immediately before and 1, 2, 3, and 7 days after ETEC ingestion. Concentrations of proGN and proUGN were measured by ELISA. Urine electrolyte concentrations were measured by photometry and mass spectrometry. Ten volunteers developed diarrhea (D group), and eleven did not (ND group). In the D group, plasma proGN, but not proUGN, concentrations were substantially reduced on days 2 and 3, coinciding with one day after diarrhea onset. No changes were seen in the ND group. ETEC diarrhea also seemed to affect diuresis, the zinc/creatinine ratio, and sodium and chloride secretion levels in urine. ETEC-induced diarrhea causes a reduction in plasma proGN and could potentially be a useful marker for intestinal isotonic fluid loss.

Effect of guanylin peptides on pancreas steatosis and function in experimental diet-induced obesity and after bariatric surgery.

Introduction: Obesity contributes to ectopic fat deposition in non-adipose organs, including the pancreas. Pancreas steatosis associates with inflammation and ß-cell dysfunction, contributing to the onset of insulin resistance and type 2 diabetes. An improvement of pancreatic steatosis and indices of insulin resistance is observed following bariatric surgery, but the underlying mechanisms remain unknown. We sought to analyze whether guanylin (GUCA2A) and uroguanylin (GUCA2B), two gut hormones involved in the regulation of satiety, food preference and adiposity, are involved in the amelioration of pancreas fat accumulation after bariatric surgery. Methods: Pancreas steatosis, inflammation, islet number and area were measured in male Wistar rats with diet-induced obesity (n=125) subjected to surgical (sham operation and sleeve gastrectomy) or dietary (pair-fed to the amount of food eaten by gastrectomized animals) interventions. The tissue distribution of guanylate cyclase C (GUCY2C) and the expression of the guanylin system were evaluated in rat pancreata by real-time PCR, Western-blot and immunohistochemistry. The effect of guanylin and uroguanylin on factors involved in insulin secretion and lipogenesis was determined in vitro in RIN-m5F ß-cells exposed to lipotoxic conditions. Results: Sleeve gastrectomy reduced pancreas steatosis and inflammation and improved insulin sensitivity and synthesis. An upregulation of GUCA2A and GUCY2C, but not GUCA2B, was observed in pancreata from rats with diet-induced obesity one month after sleeve gastrectomy. Interestingly, both guanylin and uroguanylin diminished the lipotoxicity in palmitate-treated RIN-m5F ß-cells, evidenced by lower steatosis and downregulated lipogenic factors Srebf1, Mogat2 and Dgat1. Both guanylin peptides reduced insulin synthesis (Ins1 and Ins2) and release from RIN-m5F ß-cells, but only guanylin upregulated Wnt4, a factor that controls ß-cell proliferation and function. Discussion: Together, sleeve gastrectomy reduced pancreatic steatosis and improved ß-cell function. Several mechanisms, including the modulation of inflammation and lipogenesis as well as the upregulation of GUCA2A in the pancreas, might explain this beneficial effect of bariatric surgery.

Guanylate cyclase-C Signaling Axis as a theragnostic target in colorectal cancer: a systematic review of literature.

Introduction: Colorectal cancer (CRC) is a devastating disease that affects millions of people worldwide. Recent research has highlighted the crucial role of the guanylate cyclase-C (GC-C) signaling axis in CRC, from the early stages of tumorigenesis to disease progression. GC-C is activated by endogenous peptides guanylin (GU) and uroguanylin (UG), which are critical in maintaining intestinal fluid homeostasis. However, it has been found that these peptides may also contribute to the development of CRC. This systematic review focuses on the latest research on the GC-C signaling axis in CRC. Methods: According to the aim of the study, a systematic literature search was conducted on Medline and PubMed databases. Ultimately, a total of 40 articles were gathered for the systematic review. Results: Our systematic literature search revealed that alterations in GC-C signaling compartments in CRC tissue have demonstrated potential as diagnostic, prognostic, and therapeutic markers. This research highlights a potential treatment for CRC by targeting the GC-C signaling axis. Promising results from recent studies have explored the use of this signaling axis to develop new vaccines and chimeric antigen receptors that may be used in future clinical trials. Conclusion: The findings presented in this review provide compelling evidence that targeting the GC-C signaling axis may be an advantageous approach for treating CRC.

A ß-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer.

BACKGROUND & AIMS: Sporadic colorectal cancers arise from initiating mutations in APC, producing oncogenic ß-catenin/TCF-dependent transcriptional reprogramming. Similarly, the tumor suppressor axis regulated by the intestinal epithelial receptor GUCY2C is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by ß-catenin/TCF signaling. METHODS: We performed RNA sequencing analysis of 4 unique conditional human colon cancer cell models of ß-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, chromatin immunoprecipitation sequencing, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) knockout, and CRISPR epigenome editing, which were cross-validated with human tissue chromatin immunoprecipitation sequencing datasets, to identify functional gene enhancers mediating GUCY2C ligand loss. RESULTS: RNA sequencing analyses reveal the GUCY2C hormones as 2 of the most sensitive targets of ß-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant ß-catenin/TCF signaling. CONCLUSIONS: These studies reveal DNA elements regulating corepression of GUCY2C ligand transcription by ß-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could reestablish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUCY2C axis and oppose transformation.