Guanylate Cyclase-C Receptor and Ligand Expression in Colonic Mucosa in Chronic Constipation

Abstract

Introduction: Linaclotide is a synthetic oral peptide that binds guanylate cyclase C (GC-C) receptors in the intestinal epithelium and has demonstrated efficacy in chronic idiopathic constipation (CC). Guanylin and uroguanylin stimulate GC-C receptors which increases intracellular levels of cGMP resulting in intestinal fluid secretion and acceleration of transit time. Cyclic GMP (cGMP) transporter proteins function as an ATP-driven export pump for cyclic AMP and cGMP. The expression of GC-C receptor and ligand, uroguanylin, and guanylin, and the cGMP transporters in the human colon has not been well studied. Aims: 1) To compare the mRNA expression and protein levels of the GC-C receptor, guanylin and uroguanylin, cGMP transporter proteins, multidrug resistance protein 4 and 5 (MRP4 and MRP5) in the colonic mucosa of female patients with CC and healthy controls and, 2) To determine if there is an association with overall GI symptoms. Methods: Women between the ages of 18-55 were recruited mainly from advertisement with: 1) Rome III positive CC (30.7±11.8 years), and 2) healthy controls (34.6±10.1 years) (n=12 in each group). Subjects completed GI symptom questionnaires including overall GI symptom severity using a 21-point numeric rating scale (0-20). All subjects underwent a flexible sigmoidoscopy with random biopsies taken at 30 cm from the anal verge. RT-PCR, ELISA assays and Western blots were performed to measure mRNA expression and protein levels, respectively. Student t-tests or non-parametric Wilcoxon rank sum tests were used for group comparisons. Spearman correlation was also used. A p value of <0.05 was considered significant. Results: There were no significant group differences in mRNA expression of the GC-C receptor, GCC receptor agonists (guanylin and uroguanylin) and the cGMP transporter proteins. Similar findings were found with respect to protein levels, except for MRP4, which was significantly higher in CC patients (0.043 pg/μg) vs. controls (0.034 pg/μg) (p=0.01). The CC patients had a moderate GI symptom severity (mean 10.45±5.11 [0-20]). There was a significant negative correlation between guanylin protein levels and current overall GI symptom severity in CC patients (r= -0.701, p=0.024). Conclusion: Findings suggest that the GC-C signaling pathway plays a role in the pathogenesis and symptomatology in CC. Further studies in CC are needed to determine: 1) if mucosal guanylin levels are predictive of severity or treatment response to a GC-C agonist, and 2) the significance of increased expression of MRP4, e.g., if mucosal levels are predictive of the response to a constipation medication given that MRP4 is a drug transporter protein and confers resistance to a variety of drugs. Disclosure - Lin Chang- Consultant and Grant Support: Ironwood, Consultant: Forest. This research was supported by an industry grant from Ironwood Pharmaceuticals