Guanylin, Uroguanylin and Guanylate Cyclase-C Are Expressed in the Gastrointestinal Tract of Horses

Abstract

Guanylate cyclase-C (GC-C) is a multifunctional receptor encoded by the GUCY2C gene, representing an attractive target for therapy in several gastrointestinal diseases in humans. Little is known about this system in horses. We investigated for the first time the gene expression of guanylin, uroguanylin and GC-C receptors in different horse's gastrointestinal tracts. Tissue samples from stomach, duodenum, jejunum, ileum, head and body of cecum, left and right dorsal colon, left and right ventral colon, pelvic flexure, transverse colon, descending colon and rectum were collected from adult horses within 1 h post mortem. For each sample, total RNA was extracted from 100 mg of ground tissue, and qRT-PCR performed on GUCA2a, GUCA2b and GUCY2 transcripts on a CFX96 Touch instrument. Data analysis was carried out with Bio-Rad CFX Manager software, and genes of interest normalized relative to the abundance of the two reference genes (SDHA, HPRT). Additionally, the protein expression levels of GC-C receptor were analyzed through western blotting. A common pattern of expression throughout the gastrointestinal lumen for all three investigated transcripts was found. The expression of GUCA2a, GUCA2b and GUCY2 genes was higher in jejunum, ileum, descending colon and rectum. The levels of expression of GC-C protein confirmed these data. The findings of this study might open new scenarios for the therapeutic approach to enteric diseases of horse using selective agonists of GC-C.

Keywords: gastrointestinal tract; guanylate cyclase-C; guanylin; horse; uroguanylin.

FIGURE 1

Gene expression throughout the gastrointestinal tract: the x-axis shows the different intestinal segments; the relative expression in log2 normalized for the two reference genes is indicated below the graph. The stomach, tissue with the lowest expression of all three genes, was set as calibrator. In the y-axis is indicated the relative expression in log2 scale.

FIGURE 2

Bar charts representing the relative expression value of GUCA2A (A) GUCA2B (B) and GUCYC2 (C): the x-axis shows six different intestinal tracts; the relative expression for the six horses for each of the 3 genes tested, normalized for the two reference genes, is also indicated below the graph. The left ventral colon, tissue with the lowest expression, was set as calibrator. In the y-axis is indicated the relative expression value with standard deviation. Bars marked with an asterisk indicate significant statistical difference (^∗p < 0.05 and ^∗∗p < 0.001).

FIGURE 3

Protein expression of GC-C receptor in horse intestine. Detection of the levels of GUCY2C in stomach (1), duodenum (2), jejunum (3), ileum (4), body of cecum (5), left and right dorsal colon (6 and 7, respectively), left ventral colon (8), pelvic flexure (9), transverse colon (10), descending colon (11) and rectum (12). The densitometric analysis derives from six separate blots and a representative immunoblot is shown. Equal protein loading was verified by using an anti β-actin antibody. The detection was executed by an ECL western blotting analysis system. Data points marked with an asterisk indicate statistically significant differences with respect to the stomach, tissue with the lowest levels of GC receptor (^∗p < 0.05).

FIGURE 4

Percent identity matrix of GC-C amino acid sequences from different mammals using Clustal Omega.

FIGURE 5

Comparison of complexes formed between either horse (A) or human GC-C (B) and linaclotide obtained by molecular docking. Cyclase and kinase catalytic domains are highlighted as blue and red ribbon, and corresponding binding regions are traced by linaclotide and GTP, respectively. Predicted transmembrane domains are highlighted in orange. Superimposition of the catalytic domains of human and horse guanylate cyclase complexed with linaclotide (C).