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

摘要

class="enumerated" style="list-style-type:decimal">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.