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We aimed to study 5‐HT4receptors in canine stomach contractility both in vivoand in vitro.In anaesthetized Beagle dogs, the selective 5‐HT4receptor agonist prucalopride (i.v.) induced dose‐dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5‐HT4receptor antagonist GR 125487 (10 μg kg−1, i.v.).Electrical field stimulation (EFS) of corpus longitudinal muscle strips resulted in atropine‐ and tetrodotoxin‐sensitive contractions (L‐NOARG (0.1 mM) present in all organ bath solutions). Prucalopride increased these contractions (maximal response after single‐dose addition (0.3 μM): 165% of initial value, or after cumulative addition: 188%). In the presence of methysergide (3 μM), 5‐HT also increased EFS‐contractions (after single‐dose addition (0.3 μM): increase to 192%, after cumulative addition: 148%).The selective 5‐HT4receptor antagonists GR 113808 (0.1 μM) or GR 125487 (10 nM) antagonized the prucalopride (0.3 μM)‐induced contraction increments. When EFS‐induced contractions were blocked by atropine or tetrodotoxin, prucalopride was ineffective. In the presence of methysergide (3 μM), the contraction increases to 5‐HT (0.3 μM) were prevented by GR 113808 (0.1 μM).The prucalopride curve (pEC507.9) was shifted in parallel to the right by GR 113808 3 nM(pA29.4). In the presence of methysergide (3 μM), the curve to 5‐HT (pEC508.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8±0.2 (pKBof 9.1 with unit Schild slope).In corpus circular muscle strips, the prucalopride (0.3 μM)‐induced augmentation of EFS‐contractions (258%) was also prevented by GR 113808 (0.1 μM) (124%).In conclusion, the effects of 5‐HT4receptor agonists on proximal stomach motor activity in vivocan be explained by an effect on 5‐HT4receptors on cholinergic nerves within the gastric muscle wall.

5‐HT4receptors mediate circular muscle relaxation in both human and canine large intestine, but this phenomenon alone can not explain the improvement in colonic motility induced by selective 5‐HT4receptor agonists in vivo. We set out to characterize 5‐HT4receptor‐mediated effects in longitudinal muscle strips of canine and human large intestine.Electrical field stimulation (EFS) was applied providing submaximal isotonic contractions. L‐NOARG (0.1 mM) was continuously present in the organ bath to preclude nitric oxide‐induced relaxation to EFS.The selective 5‐HT4receptor agonist prucalopride (0.3 μM) enhanced EFS‐evoked contractions, that were antagonized in both preparations by the selective 5‐HT4receptor antagonist GR 113808 (0.1 μM). The prucalopride‐induced increase was present in canine ascending and descending colon, but absent in rectum. Regional differences in response to prucalopride were not observed in human ascending and sigmoid colon and rectum. Incubation with atropine (1 μM) or tetrodotoxin (0.3 μM) inhibited EFS‐induced contractions, which were then unaffected by prucalopride (0.3 μM) in both tissues.In the presence of methysergide (3 μM; both tissues) and granisetron (0.3 μM; only human tissues), 5‐HT (0.3 μM) enhanced EFS‐induced contractions, an effect that was antagonized by GR 113808 (0.1 μM). In the presence of atropine or tetrodotoxin, EFS‐induced contractions were inhibited, leaving 5‐HT (0.3 μM) ineffective in both preparations.This study demonstrates for the first time that in human and canine large intestine, 5‐HT4receptors are located on cholinergic neurones, presumably mediating facilitating release of acetylcholine, resulting in enhanced longitudinal muscle contractility. This study and previous circular muscle strip studies suggest that 5‐HT4receptor agonism facilitates colonic propulsion viaa coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.

Recently, it was demonstrated that 5‐HT induces relaxation of human colon circular muscle through activation of 5‐HT4receptors and 5‐HT7receptors. The aim of the current study was to develop a new in vitrobioassay of human colon that would facilitate the pharmacological analysis of 5‐HT responses mediated solely by 5‐HT4receptors.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 (pEC507.31, 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‐HT6or 5‐HT7receptors 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‐HT4receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5‐HT‐induced relaxation (pKB9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA210.34) and depression of the 5‐HT curve. These affinity estimates are similar to those previously reported for 5‐HT4receptors.The selective 5‐HT4receptor agonists, prucalopride and R076186, induced relaxations (pEC507.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA2estimates 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‐HT4receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5‐HT4receptors in vitro.

This study aimed to characterize for the first time in vitro5‐HT4receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum.In the presence of methysergide (60 μM), 5‐HT induced relaxation of methacholine (1 μM)‐precontracted muscle strips, yielding a monophasic sigmoidal concentration‐relaxation curve (pEC507.2±0.07).Tetrodotoxin (0.3 μM) did not affect the curve to 5‐HT, suggesting the inhibitory 5‐HT receptor is located on the smooth muscle. Granisetron (0.3 μM) did also not affect the curve to 5‐HT, which excludes the 5‐HT3receptor mediating the relaxation to 5‐HT. The presence of methysergide rules out the involvement of 5‐HT1, 5‐HT2or 5‐HT7receptors.5‐HT, the selective 5‐HT4receptor agonists R076186, prucalopride (R093877) and SDZ HTF‐919 and the 5‐HT4receptor agonists cisapride and 5‐MeOT relaxed the muscle strips with a rank order of potency R076186=5‐HT>cisapride>prucaloprideSDZ HTF‐919>5‐MeOT.The selective 5‐HT4receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5‐HT, yielding pKBestimates of 9.7, 7.9 and 9.1, respectively. The selective 5‐HT4receptor antagonist SB 204070 shifted the curve to 5‐HT rightward and depressed the maximal response (apparent pA210.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5‐HT4receptor agonists R076186 (pA28.8).It is concluded that 5‐HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5‐HT4receptors. For the first time, a non‐human species was shown to exhibit relaxant 5‐HT4receptors in the large intestine.