Heat-stable Enterotoxin Receptor Research Articles

Abstract IA019: Clinical trials of GUCY2C (GCC) agonists for colorectal cancer prevention

Abstract Guanylyl cyclase C (GUCY2C), expressed on intestinal epithelial cells, is the receptor for heat-stable enterotoxins (STs) produced by diarrheagenic bacteria. Fluid secretion reflects ST activation of GUCY2C, which produces cyclic (c)GMP activating ion channels. Beyond toxins, GUCY2C binds uroguanylin in small intestine and guanylin in colorectum, which also stimulate secretion. This mechanism is the basis for the oral agonists linaclotide (ST analog) and plecanatide (uroguanylin analog) to treat constipation. Guanylin is the most commonly lost gene product, while GUCY2C expression is retained, in colorectal tumors. Eliminating GUCY2C promotes tumor formation, while oral GUCY2C agonists or transgenic guanylin reduce intestinal tumors, in mice. These data support a hypothesis in which loss of guanylin silencing GUCY2C is a key step in intestinal transformation which can be prevented by oral GUCY2C agonists. However, GUCY2C agonists are formulated for duodenal activity, without bioavailability in the colorectum. Here, we explored whether this pharmacokinetic (PK) barrier could be abrogated with high doses (HD) of linaclotide. Indeed, HD linaclotide induced a cGMP response in mucosal biopsies obtained by colonoscopy following oral polyethylene glycol (PEG) preparation. This cGMP response was validated by VASP phosphorylation and suppression of crypt proliferation. Similarly, HD linaclotide induced cGMP in colon biopsies obtained by sigmoidoscopy following oral PEG. However, HD linaclotide failed to induce cGMP in colon biopsies obtained by sigmoidoscopy i n the absence of oral PEG . Thus, oral PEG abrogated the PK barrier, delivering linaclotide to the colorectum. Unfortunately, dolcanatide, a proteolysis-resistant analog of plecanatide, also failed to overcome the colorectal PK barrier, although it produced duodenal secretion and diarrhea. Thus, guanylin loss silencing GUCY2C appears to be a key step in intestinal transformation. Preclinical models suggest that oral GUCY2C agonists stimulate GUCY2C signaling, opposing tumorigenesis. However, translation of these observations appears to be constrained by a PK barrier, reflecting agonists formulated for duodenal activity, without colorectal bioavailability. Exploiting GUCY2C for chemoprevention awaits agonists formulated for the colorectum. We are exploring the ability of GUCY2C agonists to activate cGMP in duodenum (within the PK barrier), as a prelude to trials in patients with FAP at risk for duodenal tumors. Citation Format: Scott A. Waldman. Clinical trials of GUCY2C (GCC) agonists for colorectal cancer prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA019.

The Heat-Stable Enterotoxin Receptor, Guanylyl Cyclase C, as a Pharmacological Target in Colorectal Cancer Immunotherapy: A Bench-to-Bedside Current Report.

Cancer immunotherapy is becoming a routine treatment modality in the oncology clinic, in spite of the fact that it is a relatively nascent field. The challenge in developing effective immunotherapeutics is the identification of target molecules that promote anti-tumor efficacy across the patient population while sparing healthy tissue from damaging autoimmunity. The intestinally restricted receptor guanylyl cyclase C (GUCY2C) is a target that has been investigated for the treatment of colorectal cancer and numerous animal, and clinical studies have demonstrated both efficacy and safety. Here, we describe the current state of GUCY2C-directed cancer immunotherapy and the future directions of this work.

Guanylyl cyclase C and guanylin reduce fat droplet accumulation in cattle mesenteric adipose tissue

Guanylyl cyclase C (GC-C) is a member of a family of enzymes that metabolize GTP to cGMP and was first identified as a receptor for heat-stable enterotoxin. Guanylin (GNY) has since been identified as an endogenous ligand for GC-C in the intestine of several mammalian species. The GNY/GC-C system regulates ion transportation and pH in the mucosa. Recently, it was reported that GC-C and GNY are involved in lipid metabolism in rat mesenteric adipose tissue macrophages. To examine the role of GC-C and GNY in lipid metabolism in cattle, we used a bovine mesenteric adipocyte primary culture system and a coculture system for bovine adipocytes and GNY-/GC-C-expressing macrophages. Fat droplets were observed to accumulate in bovine mesenteric adipocytes cultured alone, whereas few fat droplets accumulated in adipocytes indirectly cocultured with macrophages. We also observed that GC-C was present in bovine mesenteric adipose tissue, and that fat droplet accumulation decreased after in vitro GNY administration. Expressions of mRNAs encoding lipogenic factors decreased significantly in adipocytes after either coculture or GNY administration. These results suggest that the GNY/GC-C system is part of the control system for lipid accumulation in bovine mesenteric adipose tissue.

GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor))

Review on GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)), with data on DNA, on the protein encoded, and where the gene is implicated.

The multiple roles of guanylate cyclase C, a heat stable enterotoxin receptor

Guanylate cyclase C (GC-C) is a transmembrane receptor that is expressed primarily on intestinal epithelial cells. Activation of this receptor by its endogenous peptide ligands initiates cyclic guanosine monophosphate-dependent (cGMP) salt and water movement in the intestine. GC-C is targeted by the enterotoxigenic Escherichia coli heat-stable enterotoxin STa, which deregulates this pathway and causes secretory diarrhea. This review discusses current work on the physiological function of GC-C in the intestine. Familial GC-C mutations demonstrate that epithelial cGMP signaling is critical to electrolyte and fluid balance in the neonatal intestine. Chronic deregulation of GC-C activity in early life increases susceptibility to a number of disorders, including obstruction and inflammatory bowel disease. Murine models indicate that GC-C regulates the composition of intestinal commensal microflora and that it suppresses bacterial infection and modulates colonic injury and inflammation. Therapeutic GC-C ligands are used to successfully treat constipation-predominant irritable bowel syndrome and recent studies show that extracellular cGMP is an important mechanism of reducing abdominal pain associated with this disorder. Originally identified as a target of E. coli enterotoxin STa, GC-C is an important regulator of physiological salt and water homeostasis and may directly impact a wide range of intestinal disorders.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Pharmacological characterization of receptor guanylyl cyclase reporter cell lines

Receptor guanylyl cyclases are implicated in a growing number of pathophysiologies and, therefore, represent an important target class for drug development. We report here the generation and pharmacological characterization of three particulate guanylyl cyclase (pGC) reporter cell lines. Plasmid constructs encoding the natriuretic peptide receptors GC-A and GC-B, and the heat-stable enterotoxin receptor GC-C, were stably transfected in a parental reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cGMP. In our reporter cell lines pGC activity can be monitored in living cells in real-time . By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we show that our reporter assay monitors pGC activity with very high sensitivity. In contrast to previous findings, we could detect significant stimulation of GC-A and GC-B by each of the natriuretic peptides ANP, BNP and CNP. In addition, the clearance receptor ligand Cys-ANF(4–18) and the ANP receptor antagonist Arg-ANF(6–18) were characterized as partial GC-A agonists. The results imply that our novel pGC reporter cell lines are well suited for the characterization of receptor pharmacology and may be used for natural ligand characterization of guanylyl cyclase orphan receptors.

GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor))

GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor))

Familial Diarrhea Syndrome Caused by an Activating GUCY2C Mutation

BackgroundFamilial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis.MethodsWe used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells.ResultsWe identified a heterozygous missense mutation (c.2519G→T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members.ConclusionsIncreased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C–CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest [Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

Distribution and characterization of the Escherichia coli heat-stable enterotoxin (STa) receptor throughout the intestinal tract of newborn camels (Camelus dromedaries)

Heat-stable enterotoxin (STa) secretion from Enterotoxigenic Escherichia coli (ETEC) is crucial for the pathogenesis of diarrhea in both animal and human. The goal of this study was to investigate the distribution of the STa-specific receptors in the newborn camel's enterocytes and brush border membrane vesicles (BBMVs). Flow cytometric analysis was used to investigate the density of STa-receptors on enterocytes and BBMVs prepared from anterior jejunum, posterior jejunum, ileum, and colon. Strong density and affinity of STa-receptors was present on enterocytes and BBMVs of the ileum compared to that in the other intestinal segments. It was concluded that the ileum is the major target for STa action in newborn camels.

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Enterotoxin preconditioning restores calcium-sensing receptor-mediated cytostasis in colon cancer cells

Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.

Guanylyl Cyclase C Regulates the Intestinal Crypt‐Villus Axis

Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs) and the endogenous ligands guanylin and uroguanylin, is specifically expressed in apical membranes of intestinal epithelial cells where it regulates fluid and electrolyte secretion. However, a novel paradigm is emerging in which GCC signaling regulates the proliferation of human colon carcinoma cells. Here, the role of GCC in regulating enterocyte dynamics along the crypt-villus axis was examined. GCC target-inactivated (GCC−/−) mice exhibit elongated crypts from the duodenum to the colon, populated by more rapidly cycling progenitor cells. Further, enterocytes exhibited increased rates of migration and apoptosis, but decreased differentiation, along the crypt-villus axis of GCC−/− mice. Moreover, activation of GCC reduced proliferation of cells in human normal colonic mucosa obtained from patients and inhibited cell cycle progression of NCM460 human colonic crypt cells. Indeed, GCC delayed G1/S cell cycle transition which was mediated by cGMP-dependent protein kinase. These data demonstrate that GCC contributes to the homeostatic transition from proliferation to differentiation along the intestinal crypt-villus axis. Since expression of the endogenous ligands is frequently lost during colorectal carcinogenesis, GCC may represent a novel intestinal tumor suppressor.

Lack of guanylyl cyclase C, the receptor for Escherichia coli heat‐stable enterotoxin, results in reduced polyp formation and increased apoptosis in the multiple intestinal neoplasia (Min) mouse model

Guanylyl cyclase C (GC-C), a transmembrane receptor for bacterial heat-stable enterotoxin and the mammalian peptides guanylin and uroguanylin, mediates intestinal ion secretion and affects intestinal cell growth via cyclic GMP signaling. In intestinal tumors, GC-C expression is maintained while guanylin and uroguanylin expression is lost, suggesting a role for GC-C activation in tumor formation or growth. We show by in situ hybridization that GC-C expression is retained in adenomas from multiple intestinal neoplasia (Apc(Min/+)) mice. In order to determine the in vivo role of GC-C in intestinal tumorigenesis, we generated Apc(Min/+) mice homozygous for a targeted deletion of the gene encoding GC-C and hypothesized that these mice would have increased tumor multiplicity and size compared to wild-type Apc(Min/+) mice on the same genetic background. In contrast, the absence of GC-C resulted in a reduction of median polyp number by 55%. There was no change in the median diameter of polyps, suggesting no effect on tumor growth. Somatic loss of the wild-type Apc allele, an initiating event in intestinal tumorigenesis, also occurred in polyps from GC-C-deficient Apc(Min/+) mice. We have found increased levels of apoptosis as well as increased caspase-3 and caspase-7 gene expression in the intestines of GC-C-deficient Apc(Min/+) mice compared with Apc(Min/+) mice. We propose that these alterations are a possible compensatory mechanism by which loss of GC-C signaling also affects tumorigenesis.

Guanylyl cyclase C (GC-C) regulates the dynamics of the proliferating compartment of intestinal crypts

Background GC-C, the receptor for bacterial heat-stable enterotoxins (STs) and the endogenous ligands guanylin and uroguanylin, regulates volume homeostasis in intestine. Beyond fluid balance, GC-C inhibits colon cancer cell proliferation, although a role of this receptor in regulating normal enterocyte dynamics remains undefined. Methods Regulation of proliferation was examined in vitro and ex vivo employing NCM460 human colon crypt cells and human colonic mucosa cells from patients, respectively. Regulation of enterocyte dynamics in vivo was examined employing null mice for GC-C expression (GC-C−/−). Results Activation of GC-C by ST induced cGMP accumulation and reduced cell cycle progression in NCM460 cells. A cell-permeant analog of cGMP, 8-br-cGMP, mimicked the effect of ST and induced a transient cell cycle delay at the G1/S transition. GC-C−/− mice exhibit increased length of the crypt-villus axis in small intestine and the crypt-lumen axis in colon. Elongation of this axis reflected expansion of the crypt proliferating compartment, populated by more rapidly cycling and migrating progenitor cells. Moreover, ST and 8-br-cGMP inhibited the proliferation of normal human colonic mucosa cells from patients. Conclusions As expression of guanylin or uroguanylin is frequently lost in colorectal tumors, the expansion of the crypt proliferating compartment in GC-C−/− mice suggests a role for this receptor as a tumor suppressor contributing to intestinal carcinogenesis. Clinical Pharmacology & Therapeutics (2005) 77, P22–P22; doi: 10.1016/j.clpt.2004.11.084

Glycosylation of the receptor guanylate cyclase C: role in ligand binding and catalytic activity.

GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 degrees C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N -acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.

Bile acids stimulate expression of intestinal differentiation markers in gastroesophageal junction (GEJ) adenocarcinoma cells

Reflux of acid and bile acids, and the subsequent development of intestinal metaplasia of the esophagus (Barrett's esophagus) or gastric cardia are risk factors for the development of adenocarcinoma at the GEJ. However, the molecular mechanisms translating reflux exposure to epithelial cell transformation in the upper gastrointestinal tract remain undefined. Here, quantitative RT-PCR revealed increased expression of guanylyl cyclase C (GC-C), the receptor for bacterial heat-stable enterotoxins (STa) and a marker of differentiated enterocytes, in adenocarcinomas of the esophagus and stomach, but not in squamous carcinomas of the esophagus, gastrointestinal stromal tumors, or normal esophageal or gastric mucosae. Similarly, the human GEJ adenocarcinoma cell line OE19 expressed GC-C mRNA and accumulated cyclic GMP in response to STa. Interestingly, exposure of OE19 to the bile acid deoxycholate (DCA) stimulated GC-C promoter activity, mRNA expression, protein expression (Western blot), and function (STa-induced cyclic GMP accumulation). Moreover, treatment with DCA increased expression of the homeodomain proteins Cdx-2 (mRNA and protein) and Oct1 (protein), upstream transcription factors important in regulating intestine-specific expression of GC-C. Of significance, hydrophilic bile acids such as cholate, taurodeoxycholate and ursodeoxycholate were ineffective compared to the hydrophobic bile acids chenodeoxycholic acid and DCA in inducing GC-C and Cdx-2 mRNA expression. These observations suggest that transformation at the GEJ is initiated, in part, by induction of the transcriptional program mediating enterocyte differentiation, including Oct1, Cdx-2, and GC-C, upon exposure to specific bile acids. Further, they suggest that suppression of hydrophobic bile acid production, for example by treatment with ursodeoxycholate, which does not activate the intestinal transcription program in epithelial cells, might be effective chemoprevention for GEJ tumorigenesis. Clinical Pharmacology & Therapeutics (2004) 75, P10–P10; doi: 10.1016/j.clpt.2003.11.038

The heat‐stable enterotoxin receptor, guanylyl cyclase C (GC‐C), is a molecular marker for upper and lower gastrointestinal malignancies

Clinical Pharmacology & Therapeutics (2003) 73, P76–P76; doi:

A Novel PDZ Protein Regulates the Activity of Guanylyl Cyclase C, the Heat-stable Enterotoxin Receptor

Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin.

Guanylin, Uroguanylin, and Heat-stable Euterotoxin Activate Guanylate Cyclase C and/or a Pertussis Toxin-sensitive G Protein in Human Proximal Tubule Cells

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.