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, SHTCEICAFAA-CAGC, 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 Escherchia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 12'I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepi-

Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.Pathogenic strains of E. coli and other bacteria produce a family of heat-stable enterotoxins (STs) that activate intestinal guanylate cyclase. STs are acidic peptides that contain 18 or 19 amino acids with six cysteines and three disulfide bridges that are required for full expression of bioactivity (6). The increase of intestinal epithelial cyclic GMP elicited by STs is thought to cause a decrease in water and sodium absorption and an increase in chloride secretion (7,8). These changes in intestinal fluid and electrolyte transport then act to cause secretory diarrhea. In developing countries, the diarrhea resulting from STs causes many deaths, particularly in the infant population (9). STs are also considered a major cause of traveler's diarrhea in developed countries (10). They have also been reported to be a leading cause of morbidity and death in domestic animals (11).In the present study, we designed a bioassay to search for a potential endogenous ligand that activates the intestinal guanylate cyclase. This bioassay is based on the demonstration that T84 cells in culture respond to ST in a selective and sensitive manner with graded increases of intracellular cyclic GMP. This bioassay revealed that the intestine as well as the kidney possessed an active material. Purification of this material from the rat intestine was accomplished and the structure was determined to be a 15-amino acid peptide with 4 cysteines t...

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.

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 f luid 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 inf luenced 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 pHdependent fashion, the activity of receptors that control intestinal salt and water transport via cGMP.Guanylin and uroguanylin are structurally related peptides that were isolated from intestinal mucosa and urine (1-5). A receptor for guanylin and uroguanylin that has been identified at the molecular level is a transmembrane form of guanylate cyclase, termed GC-C (6). This membrane protein was originally discovered as an intestinal receptor for the heat-stable toxin (ST) peptides, which are secreted intraluminally by enteric bacteria that cause traveler's diarrhea (7). Bacterial ST peptides are related in primary structure to uroguanylin and guanylin, thus acting as molecular mimics of the enteric peptide hormones (reviewed in refs. 8 and 9). Membrane receptor-guanylate cyclases are found on the luminal surface of enterocytes throughout the small and large intestine and in other epithelia (10-13). Binding of peptide agonists to an extracellular domain of the receptor activates the intracellular catalytic domain producing the second messenger cGMP within target enterocytes (1-6). Intracellular cGMP stimulates transepithelial chloride secretion by regulating the phosphorylation state and chloride channel activit...

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 ^!25I-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 ^l25I-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 K_d values of ST 2.5 nM, uroguanylin 80 nM and guanylin 2.6 µM. 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.