## 4 Discussion  
In this study, the mechanism underlying the trypsin-mediated depolarization of pacemaker potentials in murine small intestinal ICCs was examined. Trypsin, a PAR2 agonist, dose-dependently depolarized the pacemaker potentials and decreased their amplitudes in the murine small intestinal ICCs (Figure 1). In addition to trypsin, this study examined the effects of AC55541, another PAR2 agonist, on pacemaker potentials. Consistent with the properties of trypsin, AC55541 dose-dependently depolarized the pacemaker potentials and decreased their amplitude in murine small intestinal ICCs (Figure 2). Trypsin-induced responses were mediated by the additive effects of NK1 and NK2 receptors (Figure 3). Additionally, trypsin-induced responses were dependent on extracellular Ca\({}^{2+}\) and the downstream G protein, PLC, PKC, and IP\({}_{3}\) signaling pathways. Treatment with GDP-\(\beta\)-S or pre-incubation in Ca\({}^{2+}\)-free solution inhibited trypsin-induced responses (Figures 4 and 5). Moreover, trypsin did not depolarize the pacemaker potentials in the presence of U-73122, staurosporine, Go6976, or xestospongin C (Figure 6). Additionally, HC067047 did not inhibit trypsin-induced responses (Figure 7). Therefore, the trypsin/PAR2 signaling pathway modulated GI motility by regulating the activity of ICCs in the murine small intestine. Figure 7: Effect of HC067047, a TRPV4 inhibitor, on trypsin-induced pacemaker potential depolarization in murine small intestinal cells of Cajal. (**A**) Trypsin depolarized pacemaker potentials in the presence of HC067047. (**B**,**C**) Responses to trypsin in the presence of HC067047 are summarized. Bars represent the mean \(\pm\) standard error of mean. CTRL, control. Currently, more than 500 types of proteases have been identified in all mammals. proteases are reported to be involved in complex physiological processes, including ovulation, intestinal digestion, inflammation, immunity, and wound healing [41]. Trypsin is the major digestive enzyme secreted in the anterior portion of the intestine. Trypsinogen (proform of trypsin) is synthesized by pancreatic acinar cells, secreted into the duodenum, and activated by enterokinases. PARs are a novel subclass of the seven transmembrane-spanning GPCRs [1]. Among the four known types of PARs [2], PAR1, PAR3, and PAR4 are activated by thrombin, while PAR2 is activated by trypsin [3]. The expression of PARs is detected in various tissues, including those of the pancreas, liver, kidney, prostate, ovary, and GI tract and several cells, such as the smooth muscle cells, neurons, fibroblasts, epithelial cells, and neutrophils [42]. PAR2 is involved in various pathophysiological processes of several organs, including the skin [43], cardiovascular system [44; 45], pulmonary system [46], and GI tract [2]. In particular, PAR2 is expressed throughout the GI tract, especially in the intestinal cells, nerve cells, and myocytes. This indicates the importance of PAR2 in regulating GI functions and the pathogenesis of GI diseases [47]. PAR2 agonists regulate GI motility by modulating the transport of ions in intestinal epithelial cells [48]. However, the regulatory effects of PAR2 on GI motility vary depending on the location. Although PAR2 promotes contraction in the gastric body [12], contraction and relaxation occur repeatedly in the gastric fundus [14]. PAR2 inhibits spontaneous contractions in the stomach and colon [49]. The results of mouse studies revealed that PAR1 and PAR2 promote GI transit through the L-type Ca\({}^{2+}\) channel pathway [50]. Additionally, PAR2 agonists promoted the contraction of murine intestinal smooth muscle, which was dependent on the sensory neural pathways and the NK1 and NK2 receptors [51]. In this study, trypsin-induced cellular responses were dependent on the additive effects of NK1 and NK2 receptors (Figure 3). Generally, GI motility is highly regulated by cholinergic mechanisms. Hence, this study examined the role of the cholinergic system in trypsin-induced pacemaker potential depolarization.