## 4 Discussion
Treatment with the muscarinic receptor antagonist atropine did not affect the pacemaker's potentials. In the presence of atropine, trypsin depolarized the pacemaker's potentials (Figure 3A), which suggested that the muscarinic cholinergic system was not involved in trypsin-mediated pacemaker potential depolarization. [PERSON] and [PERSON] [51] demonstrated that treatment with NK1 and NK2 receptor antagonists decreased trypsin-induced contractions, indicating the involvement of neurokinin receptors in trypsin-induced responses. Therefore, the role of neurokinin receptors in trypsin-induced pacemaker potential depolarization in murine small intestinal ICCs was examined in this study. ICCs were treated with SR-140333 (specific neurokinin receptor NK1 antagonist) and SR-48968 (NK2 antagonist) alone or in combination. In the presence of SR-140333 or SR-48968, trypsin slightly depolarized the pacemaker potentials and decreased their amplitude (Figure 3B,C). However, treatment with the combination of SR-140333 and SR-48968 completely inhibited trypsin-induced responses (Figure 3D). These findings suggest that trypsin-induced responses are dependent on the additive effects of NK1 and NK2 receptors. Tachykinin NK receptors are distributed in the enteric nerves, smooth muscles, and ICCs. Thus, NK receptors may modulate GI motility by regulating the activity of GI cells [52]. [PERSON] et al. [53] suggested that substance P may modulate intestinal motility by stimulating NK 1 receptors of the ICCs. The findings of this study indicated that both NK1 and NK2 receptors mediate trypsin-induced responses. It is known that the NK receptor is involved in trypsin-induced small intestinal smooth muscle contraction [51]. Therefore, considering the results in this paper, it may be thought that when trypsin depolarizes the pacemaker potential of ICC, this stimulation excites smooth muscle and eventually causes small intestine contraction. It can also be seen that NK receptors are involved in all these processes. Furthermore, trypsin did not depolarize the pacemaker potentials in the presence of U-73122, staurosporine, Go6976, or xestospongin C. However, trypsin depolarized the pacemaker potentials in the presence of rottlerin (Figure 6). Trypsin (a PAR-2 selective serine protease) stimulates[3H]inositol phosphate formation and Ca\({}^{2+}\) mobilization through PLC isoforms coupled to the heterotrimeric G proteins G\(\alpha\)q/11 [2]. Additionally, GPCRs coupled to G\(\alpha\)q/11 activate p42/44 MAP kinase through PKC [54]. [PERSON] et al. [55] suggested that trypsin-induced increases in the Na\({}^{+}\) and Cl\({}^{-}\) short-circuit currents and transepithelial resistance were inhibited in human bronchial and nasal epithelial cells upon treatment with the PLC inhibitor U-73122. Therefore, the effects of trypsin may be regulated by PLC. Consistently, this study demonstrated that treatment with U-73122 completely inhibited the trypsin-induced responses. In the presence of staurosporine (a broad-spectrum PKC inhibitor) and Go6976 (a calcium-dependent PKC \(\alpha/\beta\) inhibitor), trypsin did not depolarize the pacemaker's potentials (Figure 6B,C). However, trypsin depolarized the pacemaker potentials in the presence of rottlerin (a calcium-independent PKC \(\delta\) inhibitor) (Figure 6D). These findings indicate that PKC-dependent mechanisms were involved in trypsin-induced responses and that Ca\({}^{2+}\) regulation was critical for these mechanisms. In various tissues, PAR2 coupled with PLC hydrolyzes PIP\({}_{2}\) into IP\({}_{3}\) and diacylglycerol (DAG). IP\({}_{3}\) increases the levels of [Ca\({}^{2+}\)], whereas DAG activates PKC [6; 11]. In this study, trypsin did not depolarize the pacemaker potentials in the presence of xestospongin C, an IP\({}_{3}\) receptor blocker (Figure 6E). This indicated that IP\({}_{3}\) receptors mediate trypsin-induced responses. Additionally, the members of the TRP ion channels, including TRPV1, TRPV4, and TRPA1, are the major downstream targets of PAR2 [36; 37].