107 Stimulation of Spinal Afferents Evokes Slowly-Activating Excitatory Responses in Enteric Neurons in Parallel with Release of Mast Cell Proteases in Guinea Pig Ileum and Colon

Abstract

Introduction: Patients with IBD show higher thresholds to slow ramp distension of the rectum, as opposed to patients with a diarrhea-predominant IBS (IBS-D). Recently we have found elevated serine protease (SerP) activities in both IBS-D and ulcerative colitis (UC) fecal materials. In mice, intracolonic (IC) infusion of fecal IBS-D supernatants evokes a proteinase-activated receptor (PAR)-2 mediated colonic hypersensitivity to distension. Objectives: Our aims were to evaluate the influence of fecal supernatant from UC patients on visceral sensitivity in mice and to test the involvement of PAR-4, and its activator cathepsin G (cat-G) as possible mediators in this nociceptive response. Methods: Fecal samples from UC patients and healthy subjects were dissolved in saline, centrifuged and filtered. Under anaesthesia electrodes were inserted in the abdominal muscle to record abdominal cramps in C57/BL6 male mice. Four days after surgery mice were IC infused with 0.3 mL fecal supernatant from UC patients or controls, and colorectal distensions (CRD) were performed using a balloon inflated from 0 to 0.12 mL each steps lasting 10 sec with 5 min intervals. To evaluate the role of PAR-4, micewere treatedwith a PAR-4 antagonist (P4pal-10, pepducin, 3x0.25 mg/kg) IP before and during UC supernatant infusion. The participation of cat-G was assessed by incubating the UC supernatant with a mixture of antiproteases (aprotinin and SBTI, 0.22 and 1.70 mg/mL respectively) or with a cat-G inhibitor (0.057 mg/mL). The effect of PAR-4 activation or cat-G was tested by IC infusion of 100 ug PAR-4 agonist (PAR4AP, 0.667mg/mL) or 0.025 UN cat-G (0.167UN/mL). Results: IC infusion of UC supernatants decreased the intensity of abdominal EMG response by 71%, 49% and 35% for 0.04, 0.06 and 0.08 mL of CRD (p<0.05) compared to supernatant from healthy subjects. Blockade of PAR-4 or incubation of UC supernatant with cat-G inhibitor not only suppressed these effects but triggered colonic hypersensitivity. The total inhibition of SerP activities by aprotinin + SBTI suppressed all nociceptive effects. PAR-4-AP IC infusion decreased visceral sensitivity by 70% and 43% for distension volumes of 0.04 and 0.06 mL (p<0.05). Infusion of cat-G resulted in a significant decrease for all volumes of distension from 0.04 to 0.12 mL. Conclusions: Our study demonstrates that, despite an elevated SerP activity, UC fecal supernatant decreases colorectal sensitivity in mice. This visceral hyposensitivity results from PAR4 activation by luminal cat-G, counterbalancing the pronociceptive effect of simultaneous activation of other PARs by other serine-proteases contained in the fecal supernatant.