Title

 

Authors

R. Vicini¹, B. Balestra¹, C. Cremon², L. Zecchi², R. De Giorgio², O. Pastoris¹, V. Stanghellini², R. Corinaldesi², G. Barbara², M. Tonini^1†
 
1Dept. of Legal Medicine, Forensic Sciences and Pharmaco-Toxicology, University of Pavia
²Dept. of Clinical Medicine and Center for Applied Biomedical Research, University of Bologna

 

Abstract

Mucosal mediators obtained from colonic biopsies of patients with Irritable Bowel Syndrome (IBS) impact on gut enteric and extrinsic sensory neural pathways in recipient animals or human colonic tissues. While previous studies showed the importance of histamine, serotonin and proteases on these neural responses,the contribution of other potentially relevant mediators has not yet been fully elucidated. Based on these observations, we assessed the impact of a wide range of possible mediators on cholinergic motor responses evoked by electrical field stimulation in guinea pig ileal myenteric plexus preparations. Mucosal biopsies were obtained from the descending colon of 37 patients with IBS and 11 healthy controls (HC). Mediators spontaneously released by colonic biopsies was collected in Hanks medium and their impact was tested on cholinergic twitch contractions from guinea pig longitudinal muscle myenteric plexus preparations in vitro. To identify the role of different mediators, the following antagonists were assessed: 1) the selective prostanoid D₂ receptor antagonist BW A868C (3 µM); 2) the selective histamine H₁, H₂ and H₃ receptor antagonists mepyramine, ranitidine and thioperamide (1 µM); 3) the serotonin 5-HT_1/2, 5-HT₃ and 5-HT₄ receptor antagonists ketanserin, ondansetron and RS-39604 (1 µM); 4) the serine protease inhibitor nafamostat (FUT-175) (1 µM); 5) the selective tachykinin receptor antagonists NK_1-3, RP67580 (0.1 µM), MEN11420 (1 µM), and SR142801 (0.1 µM); 6) the selective antagonists at transient receptor potential cation channel, subfamily V, member 1 (TRPV1), HC-030031 and capsazepine (1 µM); 7) the purinergic P_2X antagonist pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS) tetrasodium salt (10 µM). Seventy-nine percent of IBS supernatants enhanced cholinergic twitch contractions compared to 58% evoked by control supernatants. This enhancement was significantly higher after the administration of IBS than HC supernatants (35% vs 18%; P<0.05), irrespective of bowel habits. The enhancement of twitch height evoked by IBS supernatants was significantly reduced by 66% (P<0.05, n=6) by the P_2X receptor antagonist PPADS (10 µM); by 64% (p<0.01, n=9) by the prostanoid receptor antagonist BW A868C (3 µM); by 40% (P<0.05, n=10) and 50% (P<0.05, n=5) by the TRPV1 antagonists HC-030031 (1 µM) and capsazepine (1µM) respectively. The effect of the serine protease inhibitor FUT 175 (1 µM) was not significant (P>0.05, n=7), whereas blockade of serotonin 5-HT_1/2,3,4 (n=5), histamine H_1-3 (n=5), tachykinin NK_1-3 (n=8) receptors was ineffective. Mediators from colonic mucosa of IBS patients excite enteric cholinergic motor neuron in vitroand this effect was mediated by activation of purinergic P_2X receptors, prostanoid receptors, and TRPV1. Our results provide novel mechanistic insight into the mechanism underlying altered motor function in IBS.