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. 2002 Mar;135(5):1144-51.
doi: 10.1038/sj.bjp.0704571.

5-HT(2B) Receptors Play a Key Role in Mediating the Excitatory Effects of 5-HT in Human Colon in Vitro

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Free PMC article

5-HT(2B) Receptors Play a Key Role in Mediating the Excitatory Effects of 5-HT in Human Colon in Vitro

R A Borman et al. Br J Pharmacol. .
Free PMC article

Abstract

1. 5-Hydroxytryptamine (5-HT) is known to produce a number of different effects in the gastrointestinal tract of various species, and has been proposed to play a key role in a number of intestinal disorders in man, including irritable bowel syndrome (IBS), although the receptors involved have yet to be established. The aim of the present study was to investigate the distribution and function of 5-HT(2B) receptors in human colon, and to establish their possible role in the aetiology of IBS. 2. The distribution of 5-HT(2B) receptor mRNA and protein were investigated by quantitative RT - PCR, Western analysis and immunocytochemistry. High levels of both mRNA and protein for 5-HT(2B) receptors were found throughout the human gastrointestinal tract, and in particular in colon, where 5-HT(2B) receptors were found predominantly in the longitudinal and circular smooth muscle layers within the muscularis externa, and in the myenteric nerve plexus lying between these two layers. 3. Electrical field stimulation of longitudinal muscle preparations of human colon mounted in organ baths resulted in neuronally-mediated contractile responses, that were significantly potentiated by application of 5-HT (up to 10(-7) M), with a pEC(50) of 8.2 +/- 0.1 (n=49 donors). The response to 5-HT was inhibited by a number of selective 5-HT(2B) receptor antagonists. 4. This study has shown for the first time that, in contrast to animal studies, the excitatory effects of 5-HT in human colon are mediated by 5-HT(2B) receptors. It is proposed that these receptors contribute to the putative 5-HT-induced colonic smooth muscle hypersensitivity associated with IBS.

Figures

Figure 1
Figure 1
Expression of mRNA for 5-HT2B receptors in human gastrointestinal tract. Data are expressed as mean log copy numbers (per 100 ng of total RNA) from three donors, with standard errors indicated by bars.
Figure 2
Figure 2
Dot-blotting of protein extracts from human ileum and colon with anti-5-HT2B antibody or control (mouse IgG1) antibody. Tissues are full thickness (whole), or sub-divided into mucosa and smooth muscle, and isolated taenia coli for colon. NA indicates not applicable to ileum. Data were obtained in tissues from a single donor.
Figure 3
Figure 3
5-HT2B receptor-like immunoreactivity in a paraffin-embedded section of human colon. In the muscularis externa, immunoreactivity was highest in the longitudinal muscle layer (L) and in the nerves of the myenteric nerve plexus (N), with lower expression in the circular muscle layer (C).
Figure 4
Figure 4
5-HT2B receptor-immunoreactivity (5-HT2BR-ir) and neurofilament-immunoreactivity (ir) in frozen sections of colon. 5-HT2BR-ir in muscle and nerve plexus (NP, upper panel), Neurofilament-ir in a neighbouring section showing localization of the nerve plexus (middle panel), and control IgG1 antibody (lower panel).
Figure 5
Figure 5
Typical contractile responses to 5-HT in electrically-stimulated human colon longitudinal smooth muscle. Figure shows the transient contractile response to EFS, and the potent, concentration-dependent potentiation of this neurally-mediated response by increasing concentrations of 5-HT (10−9 to 10−6.5M in half log increments). At concentrations in excess of 10−7M, 5-HT induces mild inhibition of electrically-stimulated contractions.
Figure 6
Figure 6
Effect of 5-HT on electrical field stimulation (EFS)-induced contractions of human colon longitudinal smooth muscle. Figure shows mean concentration-effect curve to 5-HT, with data expressed as increase in contractility (mN) over basal EFS-induced contractions. Data are given as mean±s.e.mean for n=49 donors, and have been fitted to the Hill equation according to a three parameter curve fit.
Figure 7
Figure 7
Antagonist effect of RS-127445 on the concentration-effect curves to 5-HT in human colon longitudinal smooth muscle. Figure shows mean concentration-effect curves to 5-HT in the absence and presence of RS-127445. Additional concentration-effect curves to 5-HT, in the presence of different concentrations of RS-127445 (see Figure 8), have been omitted for clarity. Data are expressed as percentage of the maximum response to 5-HT in the absence of antagonist, and are given as mean±s.e.mean for n>4 donors.
Figure 8
Figure 8
Schild plot of the antagonist effect of RS-127445 on responses to 5-HT in human colon longitudinal smooth muscle. The antagonism generated a pKB of 9.5±0.4 and slope of 0.9±0.2. Data are for n>4 donors.

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