Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice

Abstract

A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca(2+)] removal and L-type voltage-dependent Ca(2+) channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca(2+)]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders.

Figure 1. Serum spexin levels in FC patients.

(A) Characteristics of FC patients and control groups. The diagnosis of FC was based on the Rome III criteria. (B) Changes of serum spexin levels in FC patients. Control group n = 31, FC group n = 29. Data are expressed as means ± SEM, Statistical differences between individual groups were evaluated using Student’s t test. **P < 0.01 compared to control group.

Figure 2. Effects of spexin on the motor activity of gastrointestinal tract in mice in vivo.

(A) Expression level of spexin mRNA in intestine and colon of mice under the starvation condition. Mice were starved for 24 hours and then the total RNA of proximal colon, distal colon, jejunum and ileum were prepared for real-time PCR of spexin mRNA. (B) Effect of intraperitoneal (ip) spexin on propulsion of the carbon powder in the intestine. Saline or spexin (300 μg/kg and 1000 μg/kg) was injected ip and mice were placed in individual cages without water and food for 20 minutes. Then 0.2 ml 10% powdered carbon suspended in 5% gum arabic was intragastric administered. 15 minutes later, the mice were sacrificed and the distance of carbon-ink from the pylorus to the most distal point of the charcoal was recorded. (C) Effect of spexin (ip injection) on the efflux time of glass beads in the colon. A single 3-mm colored plastic bead was inserted into the distal colon (2.5 cm past the anus) with a lubricated plastic rod and then saline or spexin (300 μg/kg and 1000 μg/kg, respectively) were administrated by ip injection. The expulsion time of the bead for each mice was monitored. Statistical differences between individual groups were evaluated using One way ANOVA. *P < 0.05 and **P < 0.01 compared with paired saline-treated controls.

Figure 3. Effects of spexin on the contraction of mice jejunum and colon in the organ bath system in vitro.

The tissues were allowed to equilibrate for 1 hour and the mechanical activities of smooth muscles in the presence of PBS (A) or spexin 30 nM (B) 100 nM (C) 300 nM (D) and 100 nM (E) were recorded using the POWERLAB system and CHART5 software. KCl and ACH treatment were used as positive controls. (F) The active tensions in the colon and jejunum were calculated. Statistical differences between individual groups were evaluated using One way ANOVA. **P < 0.01 and ***P < 0.001 compared with paired PBS-treated controls.

Figure 4. Effects of GALR2/3 antagonists on spexin-induced intestinal and colonic motility.

The jejunum tissues were allowed to equilibrate for 1 hour and then treated with GALR2 antagonist M871 (0.05–50 nM, A &C) and GALR3 antagonist SNAP37889 (0.01–10 μM, B&D). The colon tissues were allowed to equilibrate for 1 hour and then treated with GALR2 antagonist M871 (50 nM, E) and GALR3 antagonist SNAP37889 (10 μM, F). 30 minutes later, the tissues were treated with spexin (1 μM) and the mechanical activities were recorded using the POWERLAB system and CHART5 software. The Emax% of 1 μM spexin in jejunum were calculated. Statistical differences between individual groups were evaluated using One way ANOVA. *P < 0.05 and **P < 0.01 compared with paired saline-treated controls.

Figure 5. Effects of Ca²⁺ influx and release on spexin-induced intestinal and colonic motility.

(A) The jejunum and colon tissues were allowed to equilibrate for 1 hour and then the nutrition buffers were replaced with Ca²⁺ free buffer supplemented with 1mM EGTA. 30 minutes later, the tissues were treated with spexin (1 μM) and the mechanical activities were recorded using the POWERLAB system and CHART5 software. The Emax% of 1 μM spexin in jejunum and colon were calculated. Statistical differences between individual groups were evaluated using Student’s t test. **P < 0.01compared with paired saline-treated controls. (B) Primary colonic smooth muscle cells were isolated, preloaded with the Ca²⁺-sensitive dye Fura-4 and challenged with 1 μM spexin. The fluorescence amplitude of Ca²⁺ signal was recorded. Further, The jejunum and colon tissues were allowed to equilibrate for 1 hour and then treated with L type-VSCC inhibitor nifedipine (1 μM, C) and IP3 receptor inhibitor 2-APB (100 μM, D). Thirty minutes later, the tissues were treated with spexin (1 μM) and the mechanical activities were recorded using the POWERLAB system and CHART5 software. The Emax% of 1 μM spexin in jejunum and colon were calculated. Statistical differences between individual groups were evaluated using Student’s t test. **P < 0.01compared with paired saline-treated controls.

Figure 6. Effects of TTX on spexin-induced intestinal and colonic motility.

The jejunum (A) and colon (B) tissues were allowed to equilibrate for 1 hour and TTX was then added into the nutrition buffer. 30 minutes later, the tissues were treated with spexin (1 μM) and the mechanical activities were recorded using the POWERLAB system and CHART5 software. The Emax% of 1 μM spexin in the colon and jejunum were calculated accordingly. Statistical differences between individual groups were evaluated using Student’s t test.