## 3 Results
### Effects of AC55541 on Pacemaker Potentials in Murine Small Intestinal ICCs  
Next, the effect of AC55541 on pacemaker potentials in murine small intestinal ICCs was examined. The ICCs were incubated with the PAR2 agonist AC55541 [24]. AC55541 (10-50 uM) dose-dependently depolarized the pacemaker potentials and decreased their amplitude (Figure 2A-C). The mean degrees of depolarization in the presence of 10, 30, and 50 uM ACC55541 were 3.6 \(\pm\) 0.5 (\(p<0.01\)), 11.9 \(\pm\) 0.9 (\(p<0.01\)), and 24.5 \(\pm\) 1.8 mV (\(p<0.01\)), respectively (Figure 2D, \(n=12\)), while the mean amplitudes were 23.8 \(\pm\) 1.5, 11.2 \(\pm\) 0.8 (\(p<0.01\)), and 2.9 \(\pm\) 0.8 mV (\(p<0.01\)) (Figure 2E, \(n=12\)), respectively. This indicated that trypsin activates PAR2 on murine small intestinal ICCs. Involvement of Neurokinin Receptors in Trypsin-Induced Pacemaker Potential Depolarization in Murine Small Intestinal ICCs  
To examine the role of cholinergic pathways in trypsin-induced pacemaker potential depolarization in murine small intestinal ICCs, the trypsin-induced responses were compared in the presence and absence of the muscarinic receptor antagonist atropine. Atropine did not affect the pacemaker potentials in murine small intestinal ICCs. Trypsin depolarized the pacemaker potentials in murine small intestinal ICCs in the presence of atropine(Figure 3A). This suggested that the trypsin-induced pacemaker potential depolarization was not regulated by the muscarinic cholinergic system. Next, the roles of neurokinin receptors in trypsin-induced pacemaker potential depolarization in murine small intestinal ICCs were examined. ICCs were treated with the specific neurokinin receptor antagonists SR-140333 (NK1 receptor inhibitor; 1 \(\upmu\)M) and SR-48968 (NK2 receptor inhibitor; 1 \(\upmu\)M) alone or in combination [36]. In ICCs treated with SR-140333 or SR-48968, trypsin slightly depolarized the pacemaker potentials and decreased their amplitude (Figure 3B,C). However, treatments with the combination of SR-140333 and SR-48968 completely inhibited the trypsin-induced responses (Figure 3D), which suggested that NK1 and NK2 receptors exert an additive effect. The mean degrees of depolarization in the presence of atropine, SR-140333, SR-48968, and SR-140444 + SR-48968 were 24.1 \(\pm\) 1.7, 11.5 \(\pm\) 1.0 (\(p\) < 0.01), 11.6 \(\pm\) 0.9 (\(p\) < 0.01), and 1.1 \(\pm\) 0.2 mV (\(p\) < 0.01), respectively (Figure 3E, \(n\) = 21), while the mean amplitudes were 2.7 \(\pm\) 0.5 (\(p\) < 0.01), 15.0 \(\pm\) 1.0 (\(p\) < 0.01), 22.4 \(\pm\) 1.8, and 24.3 \(\pm\) 1.7 mV (Figure 3F, \(n\) = 21), respectively. These results suggest that both NK1 and NK2 receptors mediate trypsin-induced pacemaker potential depolarization in murine small intestinal ICCs. Involvement of G Proteins in Trypsin-Induced Pacemaker Potential Depolarization in Murine Small Intestinal ICCs  
To examine the roles of G proteins in trypsin-induced pacemaker potential depolarization, ICCs were treated with GDP-\(\beta\)-S, which permanently inactivates G protein-binding proteins [33; 34]. Trypsin (1 \(\upmu\)M) slightly depolarized pacemaker potentials and increased their amplitude after the intracellular application of GDP-\(\beta\)-S (1 mM) (Figure 4A). The mean degree of depolarization and mean amplitude in the presence of GDP-\(\beta\)-S were 3.4 \(\pm\) 0.5 (\(p\) < 0.01) (Figure 4B, \(n\) = 6) and 22.8 \(\pm\) 1.3 mV (\(p\) < 0.01) (Figure 4C, \(n\) = 6), respectively. These results suggest that G proteins are required for trypsin-induced pacemaker potential depolarization in murine small intestinal ICCs. Figure 1: Effects of trypsin on the pacemaker potentials in murine small intestinal interstitial cells of Cajal. (**–**) Trypsin (0.1–1 \(\upmu\)M) dose-dependently depolarized pacemaker potentials and decreased their amplitudes. Responses to trypsin are summarized in (**D,E). Bars represent mean \(\pm\) standard error of mean. **\(p\) < 0.01 compared with the control. CTRL, control.