Guanylin mRNA Research Articles

The Paracrine Hormone for the GUCY2C Tumor Suppressor, Guanylin, Is Universally Lost in Colorectal Cancer

Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer. Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium. Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100- to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients <50 years old (P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient = 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors. Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells. Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy.

Highlights of This Issue

There is uncertainty about the benefits of using genome-wide sequencing to implement cancer-preventive strategies at the population level, with some projections suggesting little benefit. Sieh and colleagues used data for known breast cancer susceptibility variants to assess the benefits and harms of targeting populations at the highest genomic risk of breast cancer. The authors found that targeting those in the top 25% of the breast cancer risk distribution would include approximately half of all future breast cancer cases. This study indicates that preventive strategies based on genome sequencing will bring greater gains in disease prevention than previously projected.Prostate cancer is thought to be the most heritable cancer. Hjelmborg and colleagues performed a large prospective study within the Nordic Twin Study of Cancer cohort to examine the heritability of prostate cancer. The cumulative risk for twins whose co-twin was diagnosed with prostate cancer was greater for monozygotic twins than for dizygotic twins across all ages. Genetic differences contributed substantially to variation in both the risk and the liability of developing prostate cancer. These results support that there are significant genetic contributions to the risk of developing prostate cancer.Receptor-defined breast cancer proportions vary across Africa. To evaluate this closely in South Africa, Dickens and colleagues utilized data from South Africa's national cancer registry and from Namibia's cancer hospital to evaluate estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) statuses in breast cancer patients. The authors found that the ER+/PR+HER2− subtype was the most common, followed by the triple-negative subtype, the ER+/PR+HER2+ subtype, and the ER−PR−HER2+ subtype. ER+ breast cancer dominates in all Southern African races and improving survival should be achievable through earlier diagnosis and appropriate treatment.GUCY2C is an intestine-specific tumor suppressor that controls epithelial homeostasis, but the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This likely reflects silencing of GUCY2C through loss of its paracrine hormone, guanylin. Wilson and colleagues quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer. Guanylin mRNA was reduced 100- to 1,000-fold in 85% of tumors compared to matched normal adjacent mucosa. Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy.

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

BackgroundGuanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.MethodsWe utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C+/+ and GC-C−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10−/−, GC-C+/+IL-10−/− and GC-C−/−IL-10−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.ResultsRelative to GC-C+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C−/−IL-10−/− animals was significantly more severe relative to GC-C+/+IL-10−/− mice. Unlike GC-C+/+IL-10−/− controls, colon pathology in GC-C−/−IL-10−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C−/−IL-10−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.ConclusionsThe GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.

Cloning and Expression of Guanylin‐like Peptides in Teleost Fish

Homologues of the guanylin peptide family are expressed in teleost fish. Using the eel as a model euryhaline species, cDNAs for three peptides, guanylin, uroguanylin, and renoguanylin, were cloned and found to be expressed within both renal and intestinal epithelia. Seawater (SW) acclimation resulted in upregulation of uroguanylin mRNA expression in the intestinal epithelia of both immature "yellow" and sexually mature "silver" eels. SW acclimation also resulted in an increase in guanylin mRNA in silver eel intestine, whereas there was no change in renoguanylin mRNA expression under any condition. No changes in expression were found in the kidney following SW acclimation.

Expression of guanylin and uroguanylin mRNA in human nasal mucosa and nasal polyps

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

Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: a potential role for a novel signaling pathway in the epididymis.

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

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.

Cloning and Expression of Guanylin from the European Eel (Anguilla anguilla)

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5′ spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory “yellow” and the more sexually mature, migratory “silver” eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.

Structure and activity of uroguanylin and guanylin from the intestine and urine of rats.

Uroguanylin and guanylin are related peptides that activate common guanylate cyclase signaling molecules in the intestine and kidney. Uroguanylin was isolated from urine and duodenum but was not detected in extracts from the colon of rats. Guanylin was identified in extracts from small and large intestine but was not detected in urine. Uroguanylin and guanylin have distinct biochemical and chromatographic properties that facilitated the separation, purification, and identification of these peptides. Northern assays revealed that mRNA transcripts for uroguanylin were more abundant in small intestine compared with large intestine, whereas guanylin mRNA levels were greater in large intestine relative to small intestine. Synthetic rat uroguanylin and guanylin had similar potencies in the activation of receptors in T84 intestinal cells. Production of uroguanylin and guanylin in the mucosa of duodenum is consistent with the postulate that both peptides influence the activity of an intracellular guanosine 3',5'-cyclic monophosphate signaling pathway that regulates the transepithelial secretion of chloride and bicarbonate in the intestinal epithelium.

The Uroguanylin Gene (Guca1b) Is Linked to Guanylin (Guca2) on Mouse Chromosome 4

Uroguanylin is an endogenous ligand of the intestinal receptor guanylate cyclase-C (GC-C). Both uroguanylin and the related peptide ligand guanylin bind to GC-C and stimulate an increase in cyclic GMP, inducing chloride secretion via the cystic fibrosis transmembrane conductance regulator. We describe the cloning of the complete mouse uroguanylin gene (Guca1b) and show thatGuca1bis tightly linked to the mouse guanylin gene on chromosome 4. The two genes are structurally similar, being composed of three short exons; the uroguanylin gene spans 2.4 kb and the guanylin gene spans 1.7 kb. Uroguanylin mRNA is most prominent in proximal small intestine, whereas guanylin mRNA is predominantly expressed in distal small intestine and colon. The upstream promoter sequence of the mouse uroguanylin gene contains a canonical TATA element at the site of transcription initiation and consensus binding sites for several known transcription factors, including HNF-1 and Sp1 within the first 1 kb. Although the gene structure and coding sequences of uroguanylin and guanylin are similar, the 5′ flanking sequences and patterns of expression of these two genes in the intestine are different. It is likely that uroguanylin and guanylin represent gene duplications that have evolved to allow overlapping and complementary patterns of expression in the intestine.

Uroguanylin and guanylin: Distinct but overlapping patterns of messenger RNA expression in mouse intestine

BACKGROUND & AIMS: Uroguanylin and guanylin, endogenous ligands of the guanylate cyclase C receptor, are presumed to mediate fluid and electrolyte secretion in the intestine. The aim of this study was to characterize the expression patterns of uroguanylin and guanylin messenger RNA (mRNA) in the mouse intestine. METHODS: A mouse uroguanylin complementary DNA was amplified from a partial genomic clone, and Northern analyses and in situ hybridization were performed to localize guanylin and uroguanylin mRNA along the duodenal-colonic and crypt-villus axes. RESULTS: Uroguanylin mRNA was expressed throughout the mouse intestine and also in the kidney. Signal intensity was greatest in the small intestine for uroguanylin and in the distal small intestine and colon for guanylin. In situ hybridization showed uroguanylin mRNA localized predominantly in intestinal villi and the corticomedullary junction of the kidney, whereas guanylin mRNA was localized in both crypts and villi in the small intestine and to superficial epithelial cells in the colon. CONCLUSIONS: Mouse uroguanylin mRNA expression is discrete from guanylin expression in the intestine. The patterns of distribution in the intestine and the known pH optima of these ligands suggest a complementary role for these secretagogues. (Gastroenterology 1997 Sep;113(3):1000-6)

Tissue distribution and plasma concentration of human guanylin.

Guanylin, a peptide homologue of the bacterial heat-stable enterotoxins, is an endogenous activator of guanylate cyclase C (GC-C). We determined the tissue content and plasma concentration of human guanylin, and its cellular source in the intestine. Human guanylin is distributed widely from the duodenum to the rectum, the highest content being in the ileum and proximal colon. The plasma concentration of immunoreactive guanylin in the normal individuals tested was 30.3 +/- 3.7 fmol/ml (mean +/- SE) and that in patients with chronic renal failure was elevated with increasing serum creatinine concentration. Guanylin immunoreactivity was detected in the villus epithelial cells in the small intestine and these guanylin-containing cells were increased in number along the cephalocaudal axis of the gut. Guanylin was also present in Paneth cells in the small intestine and superficial epithelial cells in the large intestine. Guanylin mRNA was detected in the intestine by the reverse transcription-polymerase chain reaction. Guanylin may have paracrine action on neighboring enterocytes, activating intestinal guanylate cyclase and thereby regulating intestinal fluid as well as electrolyte transport through the second messenger, cyclic GMP.

Immunohistochemical Localization of Guanylin in the Rat Small Intestine and Colon

Guanylin is an endogenous mammalian ligand which binds to guanylate cyclase C(GC-C), the Escherichia coli heat-stable enterotoxin receptor. This interaction results in intestinal Cl− and fluid secretion, which is largely, if not exclusively, mediated through the cystic fibrosis transmembrane regulator (CFTR). Using in situ hybridization, we have previously localized guanylin mRNA to villus epithelial cells of the rat small intestine and to superficial epithelial cells of the rat colon. In the present study, we demonstrate immunoreactive guanylin in a subpopulation of goblet cells in the rat jejunum and ileum. In the colon, there was immunostaining of superficial epithelial cells and goblet cells. The immunohistochemical localization of guanylin parallels the observed distribution of guanylin mRNA. Localization of guanylin in goblet cells leads us to speculate that an in vivo function of guanylin regulated, CFTR-mediated Cl− secretion is to hydrate intestinal mucin.

Mapping of guanylin to murine chromosome 4 and human chromosome 1p34–p35

Guanylin is a 15-amino-acid peptide similar in structure and in function to ST{sub a}, the heat stable enterotoxin of enterotoxigenic Escherichia coli (4). Both guanylin and ST{sub a} bind guanylyl cyclase-C (GC-C), resulting in increased levels of intracellular cGMP and induction of Cl- secretion (4) via the cystic fibrosis transmembrane regulator (CFM) (2). Guanylin is a highly regulated intestinal gene that is differentially expressed along the duodenal-to-colonic and villus-to-crypt axes. Guanylin mRNA abundance is maximal in the distal small intestine and proximal colon, where the mRNA is detected mainly in differentiated villus epithelial cells and superficial colonic epithelial cells, respectively. The murine guanylin gene (Guca2) has been isolated and sequenced; the gene is 1.7 kb and consists of 3 exons. We report here the mapping of Guca2 to mouse chromosome 4 by linkage analysis and to human chromosome region 1p34-p35 using fluorescence in situ hybridization (FISH). 20 refs., 2 figs.

Genomic Sequence of the Murine Guanylin Gene

Guanylin, a 15-amino-acid peptide, is an endogenous ligand of the intestinal receptor guanylate cyclase-C. After binding to this receptor, guanylin increases the intracellular concentration of cyclic GMP and induces chloride secretion. We have isolated a genomic clone containing the entire murine guanylin gene. The guanylin gene is composed of three exons that span 1700 bp. The first 133 nucleotides of upstream promoter sequence lack the canonical TATA, CAAT, and SP1 elements. Guanylin transcription is nearly exclusively limited to the intestine, and the presence of guanylin mRNA is greatest in the distal colon and ileum. Therefore, characterization of the guanylin promoter is likely to provide another paradigm for intestine-specific gene regulation.

Guanylin Activates Rat Colonic Particulate Guanylate Cyclase

The polypeptide guanylin is an endogenous activator of small intestinal guanylate cyclase. In rat, guanylin mRNA is found predominantly in intestinal tissues,with its highest abundance in the colon. To date, the effect of guanylin on rat colonic particulate guanylate cyclase, however, has not been examined. It was, therefore, of interest to determine whether the addition of guanylin to intact rat colonocytes, or directly to isolated crude colonic membranes, stimulated guanylate cyclase activity. These studies demonstrated that: 1) rat guanylin, in a concentration-dependent manner, rapidly (within min), but transiently, stimulated particulate guanylate cyclase activity when added to intact colonocytes; 2) guanylin also stimulated guanylate cyclase activity when added directly to isolated colonic membranes; and 3) this latter effect of guanylin on guanylate cyclase activity was increased by ATP or ADP and markedly accentuated by ATPγS. Taken together, these results demonstrate that guanylin rapidly stimulates rat colonic particulate guanylate cyclase activity and, moreover, that this effect can be modulated by adenine nucleotides.

Guanylin mRNA Is Expressed in Villous Enterocytes of the Rat Small Intestine and Superficial Epithelia of the Rat Colon

Guanylin, an endogenous ligand for the Escherichia coli heat-stable enterotoxin receptor, is a recently characterized intestinal peptide. To understand the possible physiologic function of guanylin, we examined the cellular location of guanylin mRNA expression in the rat intestine. Intestinal cells were sequentially isolated from villous tip to crypt in rat jejunum and ileum. Northern blots of total RNA identified a single 0.65 kb guanylin transcript predominantly in the villous cell fractions. In situ hybridization studies demonstrated maximal signal intensity in villous cells in rat ileum and surface epithelial cells in the colon. In the ileum, the signal was nonuniform in distribution in the surface epithelial cells, with focal areas of intense signal in clusters of columnar absorptive cells. In both colon and ileum, signal intensity was near background level in deep crypt cells, lamina propria, and muscularis.

Peptide-regulated guanylate cyclase pathways in rat colon: in situ localization of GCA, GCC, and guanylin mRNA

Guanylate cyclases play a role in both physiological and pathological secretion in the mammalian intestine. Agents that raise guanosine 3',5'-cyclic monophosphate (cGMP) levels, such as atrial natriuretic peptide (ANP), guanylin (an endogenous intestinal peptide), or Escherichia coli heat-stable enterotoxin type a (STa; a bacterial toxin), enhance electrolyte secretion and the accumulation of luminal fluid. Although secretion in all parts of intestine is sensitive to changes in cGMP metabolism, an increasing body of evidence suggests that these responses are particularly important in proximal colon. To date, three peptide-sensitive membrane-bound guanylate cyclases [types A, B, and C (GCA, GCB, and GCC, respectively)] have been cloned from mammalian tissues. GCA responds to ANP, GCB to C-type natriuretic peptide, and GCC to guanylin and STa. Expression of these receptor/cyclase genes has not previously been investigated at the cellular level in the colon. Nucleotide probes specific for GCA, GCB, GCC, and guanylin were generated by polymerase chain reaction. These probes were used to evaluate colonic cyclase and guanylin mRNA expression in the rat. GCB mRNA is not detectable in this tissue either by in situ hybridization or by Northern blot analysis. In contrast, GCA, GCC, and guanylin mRNAs are all conspicuously expressed. With the in situ hybridization technique, GCA mRNA expression is seen in cells in the lamina propria. GCC mRNA expression is seen in epithelial cells throughout colonic crypts, and also, although at a slightly lower level, in cells of the surface epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of guanylin. Its existence in various mammalian tissues.

Guanylin (PNTCEICAYAACTGC) is a peptide recently isolated from the intestine, the actions of which appear to be mimicked by bacterial heat-stable enterotoxins (Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. K., Duffin, K. L., and Smith, C. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 947-951). A cDNA clone encoding the peptide was isolated from a rat intestinal cDNA library using a degenerate oligonucleotide probe. The mRNA (approximately 0.8-0.9 kilobase) encoding the peptide contained an open reading frame of 115 amino acids, including an amino-terminal signal peptide. The carboxyl-terminal region of the predicted polypeptide contained a sequence identical to guanylin, but the 15-amino acid peptide likely represents an artifact of previous acetic acid extraction methods, since an aspartate residue precedes the amino-terminal proline. A lysine-lysine dipeptide bond is one likely processing site of pro-guanylin and would generate a 60-amino acid mature peptide. Other potential cleavage sites exist at single lysine and arginine residues, which could result in peptides ranging from 22 to 56 amino acids. Transfection of COS-7 cells with the guanylin cDNA resulted in the expression of a secreted protein of M(r) 10,000. The expressed proguanylin failed to elevate cyclic GMP concentrations in human colonic T84 cells, but acetic acid treatment of pro-guanylin activated it and resulted in large elevations of cyclic GMP. Guanylin mRNA was prevalent in rat intestine but was also found in low abundance in adrenal gland, kidney, and uterus/oviduct. Guanylyl cyclase C, the apparent guanylin receptor, was found in abundant amounts in the intestine by Northern analysis, and by the polymerase chain reaction or cDNA cloning it was also found in adrenal gland, airway epithelial cells, brain, and olfactory and tracheal mucosa. Therefore, the ligand and apparent receptor (guanylyl cyclase C) both originate from mammalian genes, and are expressed in various mammalian tissues.