An improved in vitro bioassay for the study of 5-HT4 receptors in the human isolated large intestinal circular muscle

Recently, it was demonstrated that 5-HT induces relaxation of human colon circular muscle through activation of 5-HT4 receptors and 5-HT7 receptors. The aim of the current study was to develop a new in vitro bioassay of human colon that would facilitate the pharmacological analysis of 5-HT responses mediated solely by 5-HT4 receptors. Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully-defined, agonist concentration-response curves by cumulative dosing. Under these conditions, 5-HT induced a concentration-dependent relaxation (pEC50 7.31, Hill slope 0.91). Neither methysergide (10 μM) nor granisetron (1 μM) affected the 5-HT-induced relaxation, suggesting that 5-HT1, 5-HT2, 5-HT3, 5-ht5, 5-HT6 or 5-HT7 receptors are not involved. The lack of effect of tetrodotoxin (0.3 μM) indicated a direct effect of 5-HT on the smooth muscle. The selective 5-HT4 receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5-HT-induced relaxation (pKB 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA2 10.34) and depression of the 5-HT curve. These affinity estimates are similar to those previously reported for 5-HT4 receptors. The selective 5-HT4 receptor agonists, prucalopride and R076186, induced relaxations (pEC50 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA2 estimates of 9.31 and 9.21, respectively. To summarise, in KCl (80 mM)-contracted muscle strips, 5-HT induces relaxation through activation of a homogeneous smooth muscle 5-HT4 receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5-HT4 receptors in vitro. British Journal of Pharmacology (2000) 129, 1601–1608; doi:10.1038/sj.bjp.0703254