Calcitonin gene-related peptide facilitates serotonin release from guinea-pig colonic mucosa via myenteric neurons and tachykinin NK2/NK3 receptors

Abstract

The ability of calcitonin gene-related peptide (CGRP), to alter the outflow of 5-hydroxytryptamine (5-HT) from the guinea-pig proximal colon, was evaluated using three different isolated preparations: whole colon, mucosa-free muscle layer and submucosa/mucosa preparations. In the presence of the monoamine oxidase A inhibitor, clorgyline, CGRP elicited a concentration-dependent increase in 5-HT outflow from the whole colon, but not from mucosa-free muscle layer preparations. The CGRP-evoked 5-HT outflow was sensitive to tetrodotoxin (TTX) or hexamethonium, but was not detectable in submucosa/mucosa preparations. HCGRP8-37 (3 microM) inhibited the submaximal effect of CGRP on the 5-HT outflow. [Cys(ACM)2,7]hCGRP had a slight stimulant influence on the 5-HT outflow. The selective NK2 and NK3 receptor antagonists, SR48968 or SR142801, respectively, prevented the enhancing effect of CGRP. By contrast, a selective NK1 receptor antagonist L703606, failed to block the effect of CGRP. The enhancing effect of CGRP was mimicked by the NK2 receptor agonist [beta-Ala8]-neurokinin A (NKA)4-10 and the NK3 receptor agonist senktide. The effect of [beta-Ala8]-NKA4-10 on the 5-HT outflow was unaffected by TTX, while the effect of senktide was prevented by TTX, hexamethonium or SR48968. The present data also demonstrated a synergistic action of the NK2 and NK3 receptor agonists on the CGRP-evoked 5-HT outflow. We concluded that CGRP facilitates 5-HT release from the guinea-pig colonic mucosa through an action on myenteric neurons and that this effect is mediated by endogenously released tachykinins, acting via tachykinin NK2/NK3 receptors in cascade. British Journal of Pharmacology (2004) 141, 385-390. doi:10.1038/sj.bjp.0705624

Figure 1

(a) Effects of CGRP (100 nM) on the outflow of 5-HT from the guinea-pig isolated whole colon and mucosa-free muscle preparations. CGRP was present from 90 to 110 min of incubation, as indicated by the horizontal bar. Ordinate scale: outflow of 5-HT, expressed as pmolg tissue⁻¹ 10 min⁻¹. (b) Effects of increasing concentrations of CGRP on the outflow of 5-HT from the whole colon. Height of columns: peak outflow of 5-HT, expressed as % of the initial outflow (70–90 min of incubation) in the respective individual experiments. Mean values±s.e.m (vertical bars) of six to nine experiments are shown. Significance of differences from the control: ^*P<0.05.

Figure 2

Effects of CGRP (100 nM) in the absence or presence of tetrodotoxin (TTX) (1 μM) on the outflow of 5-HT from the isolated whole colon of guinea-pig. CGRP had no effect on the outflow of 5-HT from submucosa/mucosa preparations (SMP). CGRP was present from 90 to 110 min of incubation, as indicated by the horizontal bar. Ordinate scale: outflow of 5-HT, expressed as % of the initial outflow (70–90 min of incubation) in the respective individual experiments. Each point represents the mean±s.e.m (vertical bars) from six to nine experiments.

Figure 3

Effects of atropine (0.2 μM), hexamethonium (C6, 100 μM), L703606 (1 μM), SR48968 (1 μM) or SR142801 (1 μM) on the maximal outflow of 5-HT from the guinea-pig isolated whole colon evoked by CGRP (100 nM). Height of columns: maximal outflow of 5-HT, expressed as % of the initial outflow (70–90 min of incubation) in the respective individual experiments. Mean values±s.e.m (vertical bars) from six to nine experiments are shown. Significance of differences from the control: ^*P<0.001.

Figure 4

Effects of hCGRP_8–37 (1 and 3 μM) on the outflow of 5-HT from the guinea-pig isolated whole colon evoked by CGRP (10 nM). Height of columns: CGRP-evoked peak 5-HT outflow, expressed as % of the initial outflow (70–90 min of incubation) in the respective experiments. Mean values±s.e.m (vertical bars) from four to six experiments are shown. Significance of differences from the control: ^*P<0.05.

Figure 5

Effects of [β-Ala⁸]-NKA_4–10 (NK, 0.1 and 1 μM), in the absence or presence of tetrodotoxin (TTX, 1 μM) on the outflow of 5-HT from the guinea-pig isolated whole colon. [β-Ala⁸]-NKA_4–10 was present from 90 to 110 min of incubation, as indicated by the horizontal bar. Ordinate scale: outflow of 5-HT , expressed as % of the initial outflow (70–90 min of incubation) in the respective individual experiments. Each point represents the mean±s.e.m (vertical bars) from six to seven experiments.

Figure 6

Effects of tetrodotoxin (TTX, 1 μM), hexamethonium (C6, 100 μM) or SR48968 (1 μM) on the maximal outflow of 5-HT from the whole colon evoked by senktide (100 nM). Senktide had no effect on the outflow of 5-HT from submucosa/mucosa preparations (SMP). Height of columns: maximal outflow of 5-HT, expressed as % of the initial outflow (70–90 min of incubation) in the respective individual experiments. Mean values±s.e.m (vertical bars) from four to six experiments are shown. Significance of differences from the control: ^*P<0.001.

Figure 7

Effects of [β-Ala⁸]-NKA_4–10 (NK, 100 nM) and senktide (ST, 10 nM) on the outflow of 5-HT from the whole colon in the absence or presence of CGRP (10 nM). Height of columns: peak outflow of 5-HT, expressed as % of the initial outflow (70–90 min of incubation) in the respective individual experiments. Mean values±s.e.m (vertical bars) from four to six experiments are shown. Significance of differences from the CGRP alone: ^* P<0.001.