Guanylate cyclase-C agonists as peripherally acting treatments of chronic visceral pain
Authors: Stuart M. Brierley, Luke Grundy, Joel Castro, Andrea M. Harrington, Gerhard Hannig, Michael Camilleri
Journal: Trends in Pharmacological Sciences (2021)

Linaclotide and plecanatide are synthetic peptide agonists of guanylate cyclase-C (GC-C), a transmembrane receptor that is predominantly located on intestinal epithelial cells. These medications have very low oral bioavailability, which, when combined with the expression profile of GC-C, provides a mechanism of action 'targeted to the gut'.Linaclotide has been shown to inhibit colonic nociceptors and reduce peripheral drive from the colon, resulting in reduced numbers of activated dorsal horn neurons within the spinal cord and reduced pain responses to noxious colorectal distension. These effects are greatest in animal models of chronic visceral hypersensitivity that drive the 'bottom-up' and/or 'top-down' sensitization relevant to irritable bowel syndrome (IBS).Activation of GC-C results in the generation and release of cyclic guanosine-3′,5′-monophosphate (cGMP) from intestinal epithelial cells. When released into the submucosal space through the basolateral membrane, extracellular cGMP acts as a neuromodulator to inhibit pain-sensing nerve fibers innervating the colon. cGMP has also been demonstrated to inhibit pain-sensing nerves from human donors.In all human Phase III clinical studies conducted to date in patients with irritable bowel syndrome with constipation (IBS-C) or chronic idiopathic constipation (CIC), linaclotide significantly improved abdominal pain, discomfort, and bloating.Recent preclinical studies also provide the prospect that linaclotide may offer a novel therapeutic option, not only for the treatment of chronic abdominal pain in IBS-C, but also comorbid bladder dysfunction and endometriosis-associated pain. Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population. Over the past decade, preclinical and clinical studies have revealed a variety of novel mechanisms relating to the visceral analgesic effects of guanylate cyclase-C (GC-C) agonists. Here we discuss the mechanisms by which GC-C agonists target the GC-C/cyclic guanosine-3′,5′-monophosphate (cGMP) pathway, resulting in visceral analgesia as well as clinically relevant relief of abdominal pain and other sensations in IBS patients. Due to the preponderance of evidence we focus on linaclotide, a 14-amino acid GC-C agonist with very low oral bioavailability that acts within the gut. Collectively, the weight of experimental and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics. Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population. Over the past decade, preclinical and clinical studies have revealed a variety of novel mechanisms relating to the visceral analgesic effects of guanylate cyclase-C (GC-C) agonists. Here we discuss the mechanisms by which GC-C agonists target the GC-C/cyclic guanosine-3′,5′-monophosphate (cGMP) pathway, resulting in visceral analgesia as well as clinically relevant relief of abdominal pain and other sensations in IBS patients. Due to the preponderance of evidence we focus on linaclotide, a 14-amino acid GC-C agonist with very low oral bioavailability that acts within the gut. Collectively, the weight of experimental and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics. Irritable bowel syndrome (IBS) (see Glossary) is a common chronic functional gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population [1.Enck P. et al.Irritable bowel syndrome.Nat. Rev. Dis. Primers. 2016; 2: 16014Crossref PubMed Scopus (474) Google Scholar]. Current diagnosis of IBS is based on symptoms enshrined in the multiple iterations of Rome criteria and exclusion of pathophysiology based on standard clinical testing [2.Ford A.C. et al.Irritable bowel syndrome.Lancet. 2020; 396: 1675-1688Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar]. Subclassifications of IBS are determined by the nature of stool irregularities, such as those experiencing constipation (IBS-C), diarrhea (IBS-D), or alternating constipation and diarrhea (IBS-M) [2.Ford A.C. et al.Irritable bowel syndrome.Lancet. 2020; 396: 1675-1688Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar]. Additionally, chronic idiopathic constipation (CIC) affects ~14% of the global population. Symptoms associated with CIC diagnosis include reduced bowel movements, hard stools, straining during defecation, as well as bloating and discomfort. Thus, CIC patients have some symptoms that overlap with those of IBS-C. Given the clinical burden of these conditions, efficacious therapies are necessary. There are multiple efficacious treatments for bowel dysfunction, as illustrated in systematic reviews and network meta-analyses in patients with chronic constipation [3.Luthra P. et al.Efficacy of drugs in chronic idiopathic constipation: a systematic review and network meta-analysis.Lancet Gastroenterol. Hepatol. 2019; 4: 831-844Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar,4.Nelson A.D. et al.Comparison of efficacy of pharmacological treatments for chronic idiopathic constipation: a systematic review and network meta-analysis.Gut. 2017; 66: 1611-1622Crossref PubMed Scopus (82) Google Scholar] or IBS-C [5.Black C.J. et al.Efficacy of secretagogues in patients with irritable bowel syndrome with constipation: systematic review and network meta-analysis.Gastroenterology. 2018; 155: 1753-1763Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar]. However, there is a major unmet need in the management of symptoms such as pain and bloating. Although there are many candidate approaches in development as visceral analgesics [6.Camilleri M. Toward an effective peripheral visceral analgesic: responding to the national opioid crisis.Am. J. Physiol. Gastrointest. Liver Physiol. 2018; 314: G637-G646Crossref PubMed Scopus (13) Google Scholar], one of the more attractive pharmacological approaches involves the peripherally acting guanylate cyclase-C (GC-C) agonists, linaclotide and plecanatide. These agonists 'tap' into the intrinsic mechanisms controlling intestinal fluid homeostasis, which are regulated by the endogenous hormones uroguanylin and guanylin (Box 1). Importantly, linaclotide and plecanatide have limited bioavailability (Box 2) and hence have clinical safety as well as being bereft of central adverse effects, unlike traditional analgesics such as μ-opioids or centrally acting neuromodulators and most available cannabinoid agents [6.Camilleri M. Toward an effective peripheral visceral analgesic: responding to the national opioid crisis.Am. J. Physiol. Gastrointest. Liver Physiol. 2018; 314: G637-G646Crossref PubMed Scopus (13) Google Scholar].Box 1Uroguanylin and guanylin: endogenous GC-C agonists that increase intracellular cGMPUroguanylin and guanylin are endogenous hormones of the guanylin peptide family of cGMP-regulating peptides, which regulate fluid secretion in response to meals, aiding in the digestive process. Uroguanylin is a 16-amino acid peptide expressed by tuft-like epithelial cells, which are primarily located within the proximal small intestine, with fewer cells in stomach, distal small intestine, and colorectum [61.Brenna O. et al.Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.Cell Tissue Res. 2016; 365: 331-341Crossref PubMed Scopus (35) Google Scholar]. Uroguanylin activates GC-C, which is abundantly expressed in intestinal epithelial cells [19.Castro J. et al.Linaclotide inhibits colonic nociceptors and relieves abdominal pain via guanylate cyclase-C and extracellular cyclic guanosine 3',5'-monophosphate.Gastroenterology. 2013; 145: 1334-1346Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,22.Grundy L. et al.Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction.JCI Insight. 2018; 3e121841Crossref PubMed Scopus (32) Google Scholar], with greatest potency in the slightly acidic (pH 6) environments of the duodenum and proximal jejunum. In contrast, guanylin is a 15-amino acid peptide that is predominantly expressed in the colorectum by goblet cells. Guanylin activates GC-C in neutral to slightly basic pH environments [61.Brenna O. et al.Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.Cell Tissue Res. 2016; 365: 331-341Crossref PubMed Scopus (35) Google Scholar]. Upon binding by the natural hormones uroguanylin and guanylin, the intrinsic guanylate cyclase activity of GC-C rapidly elevates the intracellular concentration of the second messenger cGMP via the enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cyclic GMP (cGMP) [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar, 8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar, 9.Kuhn M. Molecular physiology of membrane guanylyl cyclase receptors.Physiol. Rev. 2016; 96: 751-804Crossref PubMed Scopus (195) Google Scholar]. cGMP is involved in the regulation of a broad range of physiological processes, including the activation of cGMP-dependent protein kinase II (PKG-II) and a direct interaction with cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases [9.Kuhn M. Molecular physiology of membrane guanylyl cyclase receptors.Physiol. Rev. 2016; 96: 751-804Crossref PubMed Scopus (195) Google Scholar,10.Sindic A. Current understanding of guanylin peptides actions.ISRN Nephrol. 2013; 2013813648Crossref PubMed Google Scholar]. The role of cGMP in PKG-II phosphorylation and protein kinase A (PKA) activation is important for intestinal secretion, as they regulate the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), which is colocalized with PKG-II at the apical surface of intestinal epithelial cells. Increased activity of CFTR leads to the efflux of Cl¯ and HCO3¯ ions into the intestinal lumen and promotes water secretion into the gastrointestinal tract [8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar,62.Foulke-Abel J. et al.Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.Gastroenterology. 2016; 150: 638-649Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar]. Furthermore, cGMP-activated PKG-II inhibits the activity of the sodium/hydrogen exchanger 3 protein (NHE3), blocking absorption of sodium ions from the intestinal lumen and inhibiting fluid absorption [62.Foulke-Abel J. et al.Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.Gastroenterology. 2016; 150: 638-649Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar]. The important role of GC-C in intestinal fluid homeostasis is confirmed in studies using GC-C gene knockout (gucy2c–/–) mice. Furthermore, in humans, gain of function mutations in GC-C are associated with chronic diarrhea [63.Muller T. et al.Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C.Gut. 2016; 65: 1306-1313Crossref PubMed Scopus (54) Google Scholar], whilst loss of function mutations in GC-C have been associated with meconium ileus, which is characterized by neonatal intestinal obstruction [64.Smith A. et al.Meconium ileus in a Lebanese family secondary to mutations in the GUCY2C gene.Eur. J. Hum. Genet. 2015; 23: 990-992Crossref PubMed Scopus (21) Google Scholar]. The mechanisms by which GC-C regulates intestinal secretion are reviewed in-depth elsewhere ([65.Waldman S.A. Camilleri M. Guanylate cyclase-C as a therapeutic target in gastrointestinal disorders.Gut. 2018; 67: 1543-1552Crossref PubMed Scopus (49) Google Scholar], which contains references to the original discoveries).Box 2Chemistry and molecular properties of linaclotide and plecanatideLinaclotide is a synthetic, selective 14-amino acid peptide agonist of GC-C, composed of naturally occurring amino acids, and that shares some sequence similarities with guanylin and uroguanylin. Linaclotide's structure was designed with three disulfide bonds (Cys1–Cys6, Cys2–Cys10, Cys5–Cys13) to stabilize its molecular structure, enhance its resistance to degradation, and lock it into a constitutively active conformation [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar,18.Silos-Santiago I. et al.Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.Pain. 2013; 154: 1820-1830Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Linaclotide exhibits high affinity and pH-independent binding to GC-C on monolayers of human colonic carcinoma T84 cells that occurs, unlike guanylin and uroguanylin, across the broad range of physiological conditions (pH 5–8) found in the gastrointestinal tract [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar,8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar]. Linaclotide also binds to rat intestinal epithelial cells in a pH-independent manner [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. When combined, these beneficial actions enhance the ability of linaclotide to bind to GC-C and mediate the pharmacological actions of linaclotide [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar,18.Silos-Santiago I. et al.Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.Pain. 2013; 154: 1820-1830Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Upon binding, GC-C rapidly elevates the intracellular concentration of cGMP via the conversion of cytoplasmic GTP to cGMP [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar, 8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar, 9.Kuhn M. Molecular physiology of membrane guanylyl cyclase receptors.Physiol. Rev. 2016; 96: 751-804Crossref PubMed Scopus (195) Google Scholar]. Notably, linaclotide has a higher potency of activation of cGMP production compared with guanylin/uroguanylin when studied in vitro in T84 cells [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar,8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar]. Linaclotide is relatively stable in gastric fluid, showing minimal metabolism during a 3-h in vitro incubation with simulated gastric fluid [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. The single pharmacologically active metabolite of linaclotide, MM-419447 (Des-Tyr14), is produced following the proteolytic cleavage of its C terminal tyrosine by incubation with carboxylpeptidase A [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. This process occurs more rapidly in duodenal and jejunal loops than in ileal loops [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. MM-419447 binds to GC-C in T84 cells or rat intestinal epithelial cells in a pH-independent and equipotent manner to linaclotide [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar,8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar,11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar].Orally administered peptides are generally poorly absorbed across the intestinal epithelium due to their size and hydrophilicity [66.Muttenthaler M. et al.Trends in peptide drug discovery.Nat. Rev. Drug Discov. 2021; 20: 309-325Crossref PubMed Scopus (207) Google Scholar], rendering them unable to passively traverse the lipid bilayer of the luminal cell membrane of enterocytes. Consistent with this, linaclotide has a low permeability coefficient, as determined in Caco-2 cells [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. Furthermore, the absolute oral bioavailability of linaclotide and MM-419447 [evaluated in rats and mice administered a dose of up to 10 mg/kg of linaclotide (ca. 1000–2000 times the therapeutic dose in humans)] was ≤0.1% [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. The minimal absorption of linaclotide into the systemic circulation holds true in clinical studies. Patients orally given 290 μg of linaclotide once daily for 7 seven days showed no quantifiable concentrations of linaclotide or MM-419447 in plasma samples [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar]. Stool samples from healthy volunteers showed recovery of the active peptide was almost exclusively in the form of the metabolite MM-419447, representing 3–5% of the orally administered linaclotide dose [11.Busby R.W. et al.Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.J. Pharmacol. Exp. Ther. 2013; 344: 196-206Crossref PubMed Scopus (103) Google Scholar].Plecanatide is a 16-amino acid peptide analog of uroguanylin, in which aspartic acid is replaced with glutamic acid in the third position of the N terminus [38.Shailubhai K. et al.Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.World J. Gastrointest. Pharmacol. Ther. 2015; 6: 213-222Crossref PubMed Google Scholar]. Accordingly, plecanatide has the same mechanism of action as uroguanylin and has been shown to stimulate cGMP production in vitro in T84 cells [38.Shailubhai K. et al.Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.World J. Gastrointest. Pharmacol. Ther. 2015; 6: 213-222Crossref PubMed Google Scholar]. Plecanatide preferentially binds GC-C with high affinity in the slightly acidic environments (pH 5–6) of the duodenum [67.Shailubhai K. et al.Plecanatide, an oral guanylate cyclase C agonist acting locally in the gastrointestinal tract, is safe and well-tolerated in single doses.Dig. Dis. Sci. 2013; 58: 2580-2586Crossref PubMed Scopus (74) Google Scholar], making it somewhat distinct from linaclotide, which binds throughout the gastrointestinal tract in a pH-independent, but concentration-dependent manner. Similar to linaclotide, plecanatide is not absorbed systemically, eliminating the potential for off-target effects [38.Shailubhai K. et al.Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.World J. Gastrointest. Pharmacol. Ther. 2015; 6: 213-222Crossref PubMed Google Scholar]. Uroguanylin and guanylin are endogenous hormones of the guanylin peptide family of cGMP-regulating peptides, which regulate fluid secretion in response to meals, aiding in the digestive process. Uroguanylin is a 16-amino acid peptide expressed by tuft-like epithelial cells, which are primarily located within the proximal small intestine, with fewer cells in stomach, distal small intestine, and colorectum [61.Brenna O. et al.Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.Cell Tissue Res. 2016; 365: 331-341Crossref PubMed Scopus (35) Google Scholar]. Uroguanylin activates GC-C, which is abundantly expressed in intestinal epithelial cells [19.Castro J. et al.Linaclotide inhibits colonic nociceptors and relieves abdominal pain via guanylate cyclase-C and extracellular cyclic guanosine 3',5'-monophosphate.Gastroenterology. 2013; 145: 1334-1346Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,22.Grundy L. et al.Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction.JCI Insight. 2018; 3e121841Crossref PubMed Scopus (32) Google Scholar], with greatest potency in the slightly acidic (pH 6) environments of the duodenum and proximal jejunum. In contrast, guanylin is a 15-amino acid peptide that is predominantly expressed in the colorectum by goblet cells. Guanylin activates GC-C in neutral to slightly basic pH environments [61.Brenna O. et al.Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.Cell Tissue Res. 2016; 365: 331-341Crossref PubMed Scopus (35) Google Scholar]. Upon binding by the natural hormones uroguanylin and guanylin, the intrinsic guanylate cyclase activity of GC-C rapidly elevates the intracellular concentration of the second messenger cGMP via the enzymatic conversion of cytoplasmic guanosine triphosphate (GTP) to cyclic GMP (cGMP) [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar, 8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar, 9.Kuhn M. Molecular physiology of membrane guanylyl cyclase receptors.Physiol. Rev. 2016; 96: 751-804Crossref PubMed Scopus (195) Google Scholar]. cGMP is involved in the regulation of a broad range of physiological processes, including the activation of cGMP-dependent protein kinase II (PKG-II) and a direct interaction with cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases [9.Kuhn M. Molecular physiology of membrane guanylyl cyclase receptors.Physiol. Rev. 2016; 96: 751-804Crossref PubMed Scopus (195) Google Scholar,10.Sindic A. Current understanding of guanylin peptides actions.ISRN Nephrol. 2013; 2013813648Crossref PubMed Google Scholar]. The role of cGMP in PKG-II phosphorylation and protein kinase A (PKA) activation is important for intestinal secretion, as they regulate the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), which is colocalized with PKG-II at the apical surface of intestinal epithelial cells. Increased activity of CFTR leads to the efflux of Cl¯ and HCO3¯ ions into the intestinal lumen and promotes water secretion into the gastrointestinal tract [8.Busby R.W. et al.Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.Eur. J. Pharmacol. 2010; 649: 328-335Crossref PubMed Scopus (163) Google Scholar,62.Foulke-Abel J. et al.Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.Gastroenterology. 2016; 150: 638-649Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar]. Furthermore, cGMP-activated PKG-II inhibits the activity of the sodium/hydrogen exchanger 3 protein (NHE3), blocking absorption of sodium ions from the intestinal lumen and inhibiting fluid absorption [62.Foulke-Abel J. et al.Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.Gastroenterology. 2016; 150: 638-649Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar]. The important role of GC-C in intestinal fluid homeostasis is confirmed in studies using GC-C gene knockout (gucy2c–/–) mice. Furthermore, in humans, gain of function mutations in GC-C are associated with chronic diarrhea [63.Muller T. et al.Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C.Gut. 2016; 65: 1306-1313Crossref PubMed Scopus (54) Google Scholar], whilst loss of function mutations in GC-C have been associated with meconium ileus, which is characterized by neonatal intestinal obstruction [64.Smith A. et al.Meconium ileus in a Lebanese family secondary to mutations in the GUCY2C gene.Eur. J. Hum. Genet. 2015; 23: 990-992Crossref PubMed Scopus (21) Google Scholar]. The mechanisms by which GC-C regulates intestinal secretion are reviewed in-depth elsewhere ([65.Waldman S.A. Camilleri M. Guanylate cyclase-C as a therapeutic target in gastrointestinal disorders.Gut. 2018; 67: 1543-1552Crossref PubMed Scopus (49) Google Scholar], which contains references to the original discoveries). Linaclotide is a synthetic, selective 14-amino acid peptide agonist of GC-C, composed of naturally occurring amino acids, and that shares some sequence similarities with guanylin and uroguanylin. Linaclotide's structure was designed with three disulfide bonds (Cys1–Cys6, Cys2–Cys10, Cys5–Cys13) to stabilize its molecular structure, enhance its resistance to degradation, and lock it into a constitutively active conformation [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopus (140) Google Scholar,18.Silos-Santiago I. et al.Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.Pain. 2013; 154: 1820-1830Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. Linaclotide exhibits high affinity and pH-independent binding to GC-C on monolayers of human colonic carcinoma T84 cells that occurs, unlike guanylin and uroguanylin, across the broad range of physiological conditions (pH 5–8) found in the gastrointestinal tract [7.Bryant A.P. et al.Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.Life Sci. 2010; 86: 760-765Crossref PubMed Scopu