## 3 Results
### Effects of AC55541 on Pacemaker Potentials in Murine Small Intestinal ICCs
Figure 3: Effect of cholinergic muscarinic or tachykinin receptor antagonists on trypsin-induced pacemaker potential depolarization in murine small intestinal interstitial cells of Cajal. (**A**) Atropine (cholinergic muscarinic receptor antagonist) did not affect trypsin-induced pacemaker potential depolarization. (**B**) SR-140333 (tachykinin NK1 receptor antagonist) slightly inhibited trypsin-induced pacemaker potential depolarization. (**C**) SR-48968 (tachykinin NK2 receptor antagonist) slightly inhibited trypsin-induced pacemaker potential depolarization. (**D**) The combination of SR-140333 and SR-48968 completely inhibited trypsin-induced pacemaker potential depolarization. Responses to trypsin in the presence of atropine, SR-140333, SR-48968, and SR-140333 + SR-48968 are summarized in (**E**,**F**). Bars represent mean \(\pm\) standard of mean. **\(p<0.01\) compared with the non-treated controls. CTRL, control. Atro., atropine. SR-1, SR-140333. SR-4, SR-48968. Figure 2: Effects of AC55541 on the pacemaker potentials in murine small intestinal interstitial cells of Cajal. (**A**–C**) AC55541 (10–50 uM) dose-dependently depolarized pacemaker potentials and decreased their amplitudes. Responses to AC55541 are summarized in (**D**,**E**). Bars represent mean \(\pm\) standard of mean. **\(p<0.01\) compared with the control. CTRL, control. Involvement of Extracellular and Intracellular Calcium Ions in Trypsin-Induced Pacemaker Potential Depolarization in Murine Small Intestinal ICCs  
Intracellular and extracellular Ca\({}^{2+}\) are involved in modulating intestinal motility [37]. To investigate the role of Ca\({}^{2+}\) in trypsin-induced pacemaker potential depolarization, trypsin-induced depolarization was measured under Ca\({}^{2+}\)-free conditions or in the presence of thapsigargin, an inhibitor of Ca\({}^{2+}\)-ATPase in the endoplasmic reticulum. Pre-treatment with external Ca\({}^{2+}\)-free solution inhibited pacemaker potentials and trypsin-induced pacemaker potential depolarization (Figure 5A). However, pre-treatment with thapsigargin inhibited the pacemaker potential but not the trypsin-induced pacemaker potential depolarization (Figure 5B). The mean degrees of depolarization under external Ca\({}^{2+}\)-free conditions and in the presence of thapsigargin were 12.5 \(\pm\) 1.0 (_p_ \(<\) 0.01) and 24.3 \(\pm\) 1.5 mV (Figure 5C, \(n\) = 8), respectively, while the mean amplitudes were 1.5 \(\pm\) 0.5 and 1.4 \(\pm\) 0.4 mV (Figure 5D, \(n\) = 7), respectively. These results suggest that trypsin-induced pacemaker potential depolarization requires extracellular Ca\({}^{2+}\) in murine small intestinal ICCs. Involvement of PLC, PKC, and Inositol Triphosphate (IP\({}_{3}\)) Pathways in Trypsin-Induced Pacemaker Potential Depolarization in Murine Small Intestinal ICCs  
Next, the role of the PLC, PKC, and IP\({}_{3}\) pathways in trypsin-induced pacemaker potential depolarization was examined using U-73122, staurosporine, Go6976, rottlerin, and xestospongin C. In the presence of U-73122 (5 uM) (Figure 6A), staurosporine (5 nM) (Figure 6B), Go6976 (1 uM) (Figure 6C), and xestospongin C (1 uM) (Figure 6E), trypsin did not depolarize the pacemaker potentials. However, trypsin depolarized the pacemaker potentials in the presence of rottlerin (5 uM) (Figure 6D). The mean degrees of depolarization in the presence of U-73122, staurosporine, Go6976, rottlerin, and xestospongin C were 2.5 \(\pm\) 0.6 (_p_ \(<\) 0.01, \(n\) = 7), 0.8 \(\pm\) 0.4 (_p_ \(<\) 0.01, \(n\) = 8), 0.6 \(\pm\) 0.5 (_p_ \(<\) 0.01, \(n\) = 9), 23.7 \(\pm\) 0.8 (_n_ = 8), and 5.5 \(\pm\) 0.6 mV (_p_ \(<\) 0.01, \(n\) = 6) (Figure 6F), respectively, while the mean amplitudes were 1.6 \(\pm\) 0.4 (_n_ = 7), 24.3 \(\pm\) 1.6 (_p_ \(<\) 0.01, \(n\) = 8), 24.0 \(\pm\) 0.7 (_p_ \(<\) 0.01, \(n\) = 9), 1.5 \(\pm\) 0.5 (_n_ = 8), and 1.7 \(\pm\) 0.4 mV (_n_ = 6) (Figure 6G), respectively. These results suggest that the PLC, PKC, and IP\({}_{3}\) pathways are involved in trypsin-induced pacemaker potential depolarization in murine small intestinal ICCs.