The effect of Xenin25 on spontaneous circular muscle contractions of rat distal colon in vitro

Abstract

Xenin25 has a variety of physiological functions in the Gastrointestinal (GI) tract, including ion transport and motility. However, the motility responses in the colon induced by Xenin25 remain poorly understood. Therefore, the effect of Xenin25 on the spontaneous circular muscle contractions of the rat distal colon was investigated using organ bath chambers and immunohistochemistry. Xenin25 induced the inhibition followed by postinhibitory spontaneous contractions with a higher frequency in the rat distal colon. This inhibitory effect of Xenin25 was significantly suppressed by TTX but not by atropine. The inhibitory time (the duration of inhibition) caused by Xenin25 was shortened by the NTSR1 antagonist SR48692, the NK1R antagonist CP96345, the VPAC2 receptor antagonist PG99-465, the nitric oxide-sensitive guanylate-cyclase inhibitor ODQ, and the Ca²⁺ -dependent K⁺ channel blocker apamin. The higher frequency of postinhibitory spontaneous contractions induced by Xenin25 was also attenuated by ODQ and apamin. SP-, NOS-, and VIP-immunoreactive neurons were detected in the myenteric plexus (MP) of the rat distal colon. Small subsets of the SP-positive neurons were also Calbindin positive. Most of the VIP-positive neurons were also NOS positive, and small subsets of the NK1R-positive neurons were also VIP positive. Based on the present results, we propose the following mechanism. Xenin25 activates neuronal NTSR1 on the SP neurons of IPANs, and transmitters from the VIP and apamin-sensitive NO neurons synergistically inhibit the spontaneous circular muscle contractions via NK1R. Subsequently, the postinhibitory spontaneous contractions are induced by the offset of apamin-sensitive NO neuron activation via the interstitial cells of Cajal. In addition, Xenin25 also activates the muscular NTSR1 to induce relaxation. Thus, Xenin25 is considered to be an important modulator of post prandial circular muscle contraction of distal colon since the release of Xenin25 from enteroendocrine cells is stimulated by food intake.

Keywords: Xenin25; enteric nervous system; intestinal motility; smooth muscle cell.

FIGURE 1

Effects of 10⁻⁷ M Xenin25 on the spontaneous circular muscle contractions of the rat distal colon. Xenin25 induced the inhibition followed by postinhibitory spontaneous contractions with a higher frequency in the rat distal colon. The tension of postinhibitory spontaneous contractions gradually recovered but was not stronger than that of preinhibitory spontaneous contractions, and the frequency of postinhibitory spontaneous contractions was higher than that of preinhibitory spontaneous contractions. Xenin25 was applied at the end point of the preinhibitory spontaneous circular muscle contraction (↓). The inhibition time (sec) was quantified as the period between the last peak before the application of Xenin25 and the first peak of the postinhibitory spontaneous contraction. The frequency was quantified as the contractions per minute (cpm), and the tension (mN) was quantified as the amplitude from baseline.

FIGURE 2

Dose–response curves of Xenin25 on the spontaneous circular muscle contractions in the rat distal colon. The inhibition time (sec) and the change in the frequency of postinhibitory spontaneous contractions were increased in a concentration‐dependent manner, but the change in the tension of postinhibitory spontaneous contractions was decreased adversely. The EC₅₀ of the inhibition time was 8.0 × 10⁻⁸ M Xenin25, the EC₅₀ of the mean change in frequency was 8.8 × 10⁻⁸ M Xenin25, and the IC₅₀ of the mean change in tension was 8.4 × 10⁻⁸ M Xenin25. The values are presented as the mean ± SE (n = 4 ~ 5). The concentration–response curves were analyzed by nonlinear regression curve fitting using GraphPad Prism (v. 9.0; GraphPad Software, San Diego, CA, USA).

FIGURE 3

Effects of Na⁺ channel inhibitors on the responses to Xenin25. (a) Inhibition time: The application of 10⁻⁶ M TTX (n = 4) or 10⁻⁶ M TTX combined with 10⁻⁶ M A809367 (n = 8) significantly decreased the inhibition time to the control (10⁻⁸ M Xenin25) (p < 0.005), but there was no significant difference in the inhibition time observed in response to these two treatments (p > 0.05). (b) Changes in frequency: The application of 10⁻⁶ M TTX (n = 4) or 10⁻⁶ M TTX combined with 10⁻⁶ M A809367 (n = 5) significantly decreased the change in the frequency compared to the control treatment (p < 0.005), but there was no significant difference in the changes in the frequency observed in response to these two treatments (p > 0.05). C. Changes in tension: The application of 10⁻⁶ M TTX (n = 4) or 10⁻⁶ M TTX combined with 10⁻⁶ M A809367 (n = 6) had no effects on the changes in tension induced by 10⁻⁸ M Xenin25 (p > 0.05).

FIGURE 4

Effects of the selective neural inhibitors on the motility response to 10⁻⁷ M Xenin25. Every inhibitor was applied 15 min before the application of 10⁻⁷ M Xenin25. A: Guanylate cyclase (NO receptor) inhibitor ODQ (10⁻⁵~10⁻⁷ M), (b) VPAC1 receptor antagonist PG97‐269 (3 × 10⁻⁵, 10⁻⁵, 10⁻⁶ M), (c) VPAC2 receptor antagonist PG99‐465 (10⁻⁶~10⁻⁸ M), (d) P2Y1 receptor antagonist MRS2500 (10⁻⁶~10⁻⁸ M), (e) NK1R antagonist CP96345 (10⁻⁶~10⁻⁸ M), (f) NSTR1 antagonist SR48962 (10⁻⁵~10⁻⁷ M). The data are presented as the means ±SE (n = 3 ~ 9 and Table 2), and the statistical analysis was performed using the nonparametric Mann–Whitney test. **p < 0.005 and *p < 0.05 compared with the control.

FIGURE 5

Effects of the small conductance Ca²⁺‐dependent K⁺ (SKCa) channel blocker apamin on the responses to Xenin25. (a) Treatment with 2.5 × 10⁻⁸ M apamin significantly shortened the inhibition time (n = 4, p < 0.05). (b) Treatment with 5 × 10⁻⁸ M and 10⁻⁷ M apamin significantly reduced the change in the frequency (n = 3, p < 0.05). (c) The change in the tension induced by 10⁻⁷ M Xenin25 was not affected by apamin. *p < 0.05 compared with the control. ▲Temporal analysis of the isometric tension signals on the inhibition time was impossible because the tension signals were unstable in the presence of 5 × 10⁻⁸ M (n = 3) and 10⁻⁷ M (n = 3) apamin. The data are presented as the mean ± SE, and the statistical analysis was performed using the Mann–Whitney test for nonparametric variables.

FIGURE 6

Distribution of the substance P (SP)‐, neurokinin receptor 1 (NK1R)‐, Calbindin‐, vasoactive intestinal polypeptide (VIP)‐, and NO synthase II (NOS)‐positive neurons in the myenteric plexus of the rat distal colon. (a) Double immunostaining for calbindin (green) and SP (red) in the myenteric plexus of the rat distal colon. Note that the immunoreactivity for Calbindin and SP colocalized in a subset of myenteric neurons (arrows). (b) Double immunostaining for VIP (green) and NK1R (red) in the myenteric plexus of the rat distal colon. Note that the immunoreactivity for VIP and NK1R colocalized in a subset of myenteric neurons (arrows). (c) Double immunostaining for VIP (green) and NOS (red) in the myenteric plexus of the rat distal colon. Note that the immunoreactivity for VIP and NOS colocalized in a subset of myenteric neurons (arrows).