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

1 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 hum an colon that would facilitate the pharmacological analysis of 5-HT respons es mediated solely by 5-HT4 receptors. 2 Contracting circular muscle strips with KCI (80 mM) yielded a stable cont ractile tension and, in contrast to muscarinic cholinoceptor agonists and h istamine, a profound reduction of spontaneous contractility. This allowed t he establishment of reproducible. fully-defined, agonist concentration-resp onse curves by cumulative dosing. Under these conditions, 5-HT induced a co ncentration-dependent relaxation (pEC(50) 7.31, Hill slope 0.91). 3 Neither methysergide (10 mu M) nor granisetron (1 mu M) affected the 5-HT -induced relaxation, suggesting that 5-HT1, 5-HT2, 5-HT3, 5-ht(5), 5-HT6 or 5-HT7 receptors are not involved. The lack of effect of tetrodotoxin (0.3 mu M) indicated a direct effect of 5-HT on the smooth muscle. 4 The selective 5-HT4 receptor antagonists GR 113808, GR 125487 and RS 3960 4 competitively antagonized the 5-HT-induced relaxation (pK(B) 9.43, 10.12 and 8.53, respectively). SE 204070 (1 nM) produced a rightward shift (pA(2) 10.34) and depression of the 5-HT curve. These affinity estimates are simi lar to those previously reported for 5-HT4 receptors. 5 The selective 5-HT4 receptor agonists, prucalopride and R076186, induced relaxations (pEC(50) 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA(2) estimates of 9.31 and 9.21, respectively. 6 To summarise, in KCl (80 mM)-contracted muscle strips, 5-HT induces relax ation through activation of a homogeneous smooth muscle 5-HT4 receptor popu lation. This new bioassay allows the focused, pharmacological characterizat ion of human colonic 5-HT4 receptors in vitro.