## 3 Results  
### JYGCT Inhibits the Pacemaker Potential of the ICC in the Murine Small Intestine  
We performed whole-cell patch clamp experiments using the cultured ICC to examine the pacemaker potential of these cells. We noted that, under these conditions, the ICC spontaneously generated a pacemaker potential with a mean frequency of 11.3 \(\pm\) 0.9 cycles/min and a mean amplitude of 24.8 \(\pm\) 1.3 mV. When JYGCT (1-10 mg/mL) was applied, the pacemaker potential decreased (Figure 1A). The mean frequency was 9.3 \(\pm\) 0.5 cycles/min (\(p\) \(<\) 0.01) at 1 mg/mL, 5.6 \(\pm\) 0.5 cycles/min (\(p\) \(<\) 0.0001) at 5 mg/mL, and 1.5 \(\pm\) 0.4 cycles/min (\(p\) \(<\) 0.0001) at 10 mg/mL (Figure 1E); the mean amplitude was 23.9 \(\pm\) 1.0 mV, 2.4 \(\pm\) 0.5 mV (\(p\) \(<\) 0.0001), and 2.1 \(\pm\) 0.8 mV (\(p\) \(<\) 0.0001) (Figure 1F). Thus, these results suggest that JYGCT inhibits the pacemaker potential of the ICC. ATP-Sensitive K+ Channels Are Involved in the JYGCT-Induced Pacemaker Potential Inhibition in the ICC  
To investigate the involvement of the K+ channels, we used several K+ channel blockers. In the presence of TEA, JYGCT inhibited the pacemaker potential in the ICC (Figure 2A). In addition, JYGCT inhibited the pacemaker potential when co-treated with 4-aminopyridine or apamin (Figure 2B,C). Moreover, glibenclamide (an ATP-sensitive K+ (KATP) channel blocker) did not affect the pacemaker potential and, under these conditions, JYGCT also showed no effect on the pacemaker potential (Figure 2D); further, glibenclamide reversed the effects of JYGCT (Figure 2E). In the presence of 5 mg/mL JYGCT, the mean frequency and amplitude were 5.6 \(\pm\) 0.6 cycles/min and 2.6 \(\pm\) 0.4 mV for TEA, 5.3 \(\pm\) 0.5 cycles/min and 2.6 \(\pm\) 0.4 mV for 4-aminopyridine, 5.5 \(\pm\) 0.6 cycles/min and 2.5 \(\pm\) 0.4 mV for apamin, and 12.1 \(\pm\) 0.9 cycles/min (\(p\) \(<\) 0.0001) and 22.7 \(\pm\) 1.0 mV (\(p\) \(<\) 0.0001) for glibenclamide (Figure 2F,G). These results suggest that JYGCT activates the KATP channels in the ICC. Figure 2: Effect of various K+ channel blockers on the inhibition of pacemaker potential by JYGCT in the ICC. (**A**) Pretreatment with TEA had no effect; (**B**) 4-aminopyridine did not affect the inhibition by JYGCT; (**C**) A pamin did not affect the pacemaker potential inhibition induced by JYGCT; (**D**) Glibenclamide blocked the pacemaker potential inhibition by JYGCT; (**E**) The suppression of pacemaker potential by JYGCT was restored by glibenclamide; (**F**,**G**) Summary of the inhibitory frequency and amplitude effects of pretreatment with TEA, 4-aminopyridine, apamin or glibenclamide. Bars represents the mean \(\pm\) SE. *****  
Transient Receptor Potential Vanilloid 1 (TRPV1) Channels Are Involved in the JYGCT-Induced Pacemaker Potential Inhibition in the ICC  
Many studies show that TRPV1 is involved in the physiological functions of the GI tract [19; 20; 21]; therefore, we investigated the relevance of the TRPV1 channel. We incubated cells with capsaicinepine or SB452533, a TRPV1 receptor antagonist, for 5 min. Pretreatment with capsaicinepine or SB452533 blocked the JYGCT-induced effects (Figure 3A,B). The frequency of the pacemaker potential with JYGCT and capsaicinepine or SB452533 was 11.4 \(\pm\) 0.9 cycles/min (\(p\) < 0.0001) or 11.9 \(\pm\) 1.0 cycles/min (\(p\) < 0.0001), with a corresponding amplitude of 22.7 \(\pm\) 1.1 mV (\(p\) < 0.0001) or 22.8 \(\pm\) 1.7 mV (\(p\) < 0.0001) (Figure 3C,D). Thus, these results suggest that the TRPV1 channels regulate the JYGCT-induced responses. Cyclic Adenosine Monophosphate (cAMP) Is Involved in the JYGCT-Induced Pacemaker Potential Inhibition in the ICC  
The cAMP and guanosine monophosphate (cAMP)-dependent pathway was checked by SQ-22536, an inhibitor of adenylate cyclase, and ODQ, an inhibitor of guanylate cyclase. In the presence of SQ-22536 (10 \(\upmu\)M), JYGCT did not inhibit the pacemaker potential of the ICC (Figure 4A).