J Schuurkes

Systematic review: Cardiovascular safety profile of 5-HT 4 agonists developed for gastrointestinal disorders

The nonselective 5‐HT4 receptor agonists, cisapride and tegaserod have been associated with cardiovascular adverse events (AEs).

5-HT4 receptors on cholinergic nerves involved in contractility of canine and human large intestine longitudinal muscle

5‐HT4 receptors 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‐HT4 receptor agonists in vivo. We set out to characterize 5‐HT4 receptor‐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‐HT4 receptor agonist prucalopride (0.3 μM) enhanced EFS‐evoked contractions, that were antagonized in both preparations by the selective 5‐HT4 receptor 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‐HT4 receptors 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‐HT4 receptor agonism facilitates colonic propulsion via a coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.

Effects of the enterokinetic prucalopride (R093877) on colonic motility in fasted dogs

The novel enterokinetic drug prucalopride was tested at various intravenous and oral doses in fasted dogs to assess: (i) the effects on colonic contractile motility patterns; and (ii) the mediation of these effects by 5‐hydroxytryptamine (5‐HT4) receptors. Colonic motility patterns were assessed in conscious dogs with four chronically implanted strain‐gauge force transducers that were sutured on the serosal side of the colon. Prucalopride altered colonic contractile motility patterns in a dose‐dependent fashion by stimulating high‐amplitude clustered contractions in the proximal colon and by inhibiting contractile activity in the distal colon. Prucalopride was equipotent after oral and intravenous administration, as reflected by the values for the effective dose that induced 50% of maximum effect (95% confidence limits): 0.04 mg kg–1 p.o. (0.01–0.1 mg kg–1) and 0.01 mg kg–1 i.v. (0.006–0.04 mg kg–1). Prucalopride also caused a dose‐dependent decrease in the time to the first giant migrating contraction (GMC); at higher doses of prucalopride, the first GMC generally occurred within the first half‐hour after treatment. Subcutaneous pretreatment with the 5‐HT4 receptor antagonist GR125487 (40 μg kg–1 bodyweight) completely prevented the effects of orally administered prucalopride (0.31 mg kg–1 bodyweight). Prucalopride, given orally or intravenously, alters colonic motility in the fasted conscious dog in a dose‐dependent fashion. It induces GMCs and causes proximal colon stimulation and distal colon inhibition of contractile motility patterns by stimulating 5‐HT4 receptors.

Pharmacological characterization of 5-HT4 receptors mediating relaxation of canine isolated rectum circular smooth muscle.

This study aimed to characterize for the first time in vitro 5‐HT4 receptors 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 (pEC50 7.2±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‐HT3 receptor mediating the relaxation to 5‐HT. The presence of methysergide rules out the involvement of 5‐HT1, 5‐HT2 or 5‐HT7 receptors. 5‐HT, the selective 5‐HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF‐919 and the 5‐HT4 receptor 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‐HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5‐HT, yielding pKB estimates of 9.7, 7.9 and 9.1, respectively. The selective 5‐HT4 receptor antagonist SB 204070 shifted the curve to 5‐HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5‐HT4 receptor agonists R076186 (pA2 8.8). It is concluded that 5‐HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5‐HT4 receptors. For the first time, a non‐human species was shown to exhibit relaxant 5‐HT4 receptors in the large intestine.

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.

Selective desensitization of the 5-HT4 receptor-mediated response in pig atrium but not in stomach.

Background and purpose:  The time dependency of the effect of 5‐HT4 receptor agonists depends on many specific regulatory mechanisms, which vary between tissues. This has important implications with regard to the effects of endogenous 5‐HT, as well as to the clinical use of 5‐HT4 receptor agonists, and might contribute to tissue selectivity of agonists.

Prejunctional muscarinic (M1)-receptor interactions on guinea-pig ileum: lack of effect of cisapride

1 Cisapride stimulates gastrointestinal motility, probably by enhancing the release of acetylcholine from myenteric nerve endings. Such an effect could be mediated via presynaptic muscarinic (M1)‐receptors. Our aim was to determine whether cisapride could antagonize the inhibitory effects of a M1‐agonist, McN‐A‐343 or mimic the effects of a M1‐antagonist, pirenzepine. 2 Longitudinal segments were suspended in Krebs solution (95% O2, 5% CO2, 37.5°C) for isometric tension recording (preload 1 g) during electrical transmural stimulation (0.1 Hz, 1 ms, sub‐ or supramaximal current). 3 McN‐A‐343 (2.0 times 10−6 m) reduced the contractile response to supramaximal stimulation (EC50 = 1.6 × 10−6 m), but had no effect on the contractions induced by exogenous acetylcholine. 4 The inhibitory effect of McN‐A‐343 on the contractile response to electrical stimulation could be reversed by pirenzepine (EC50 = 1.6 × 10−8m) but not by atropine. At these concentrations pirenzepine itself did not modify the contractile response to electrical stimulation. However, at 50 times higher concentrations pirenzepine inhibited the response to electrical stimulation as well as the response to exogenous acetylcholine (EC50 = 8.5 × 10−7m). 5 Cisapride enhanced the contractile response to submaximal electrical stimulation by 49 ± 10%. This stimulating effect of cisapride was not affected by the presence of pirenzepine but was reduced in the presence of McN‐A‐343 (22 ± 7%). 6 In conclusion: the effects of McN‐A‐343 and pirenzepine on the electrically stimulated guinea‐pig ileum are compatible with an interaction on presynaptic muscarinic‐(M1)‐receptors. Cisapride enhances the twitch amplitude via mechanisms independent of such M1‐receptor interactions.

Substance P-induced contractions of the guinea-pig proximal colon through stimulation of post-junctional tachykinin NK1 receptors

The effects of three tachykinin NK1 receptor antagonists and a tachykinin NK2 receptor antagonist against substance P-induced contractions of the guinea-pig proximal colon longitudinal muscle were investigated. Atropine, tetrodotoxin and phosphoramidon did not affect the concentration-response curve for substance P (pEC50 = 7.8). The tachykinin NK1 receptor antagonist, 2S,3S-cis-CP 96345, competitively inhibited the contractions due to substance P (pA2 = 8.5; constrained pA2 = 8.9), but at higher concentrations (> or = 3 x 10(-7) M), 2S,3S-cis-CP 96345 also depressed the concentration-response curve for methacholine. The species-selective tachykinin NK1 receptor antagonists, WIN 51708 and WIN 62577 (both 1 x 10(-6) M), and the tachykinin NK2 receptor antagonist, SR 48968 (3 x 10(-7) M), had no effect. It is concluded that substance P induces contractions through the stimulation of tachykinin NK1 receptors on the smooth muscle cells. In this preparation, tachykinin NK2 receptors do not seem to be involved in the contractile action of substance P.

Pharmacological characterization of 5‐hydroxytryptamine receptor types in the guinea‐pig proximal colon

It was investigated which 5‐hydroxytryptamine (5‐HT) receptors mediate the responses to 5‐HT in the longitudinal muscle layer of the guinea‐pig proximal colon, using selective 5‐HT receptor antagonists and the 5‐HT analogues α‐methyl‐5‐HT (2‐Me‐5‐HT), 2‐methyl‐5‐HT (2‐Me‐5‐HT), and 5‐methoxytryptamine (5‐MeOT). 5‐HT as well as its analogues induced concentration‐related contractions, at low concentrations preceded by relaxations. The 5‐HT concentration‐contractile response curve was biphasic whilst the curves to α‐Me‐5‐HT, 2‐Me‐5‐HT, and 5‐MeOT were monophasic. Tetrodotoxin (TTX) abolished the relaxations, and it inhibited the contractions to all agonists. In the presence of TTX, blockade of either 5‐HT2 (ketanserin) or 5‐HT3 receptors (ondansetron, tropisetron) reduced the contractions to 5‐HT, whereas blockade of both 5‐HT receptor types at the same time abolished them. In the absence of TTX, the contractions to 5‐HT were inhibited by antagonists at 5‐HT2 (ketanserin), 5‐HT3 (granisetron, nanomolar concentration of tropisetron) and also 5‐HT4 receptors (micromolar concentration of tropisetron). Contractions to α‐Me‐5‐HT did not seem to be sensitive to 5‐HT2 receptor blockage with ketanserin, but in the presence of TTX the contractions were abolished by the 5‐HT2 receptor antagonist. The 5‐HT3 receptor antagonist granisetron abolished contractions to 2‐Me‐5‐HT. In the presence of TTX, the 5‐HT2 receptor antagonist ketanserin abolished contractions to 5‐MeOT, and in the absence of TTX the contractions to 5‐MeOT were highly sensitive blockade of 5‐HT4 receptors with tropisetron. Blockage of either 5‐HT1 (methiothepin), 5‐HT2 (ketanserin), 5‐HT3 (ondansetron, granisetron, tropisetron) or 5‐HT4 (tropisetron) receptors did not abolish the relaxations to 5‐HT or 5‐MeOT. In conclusion, 5‐HT induces contractions of the longitudinal muscle of the guinea‐pig proximal colon, through the stimulation of 5‐HT2 receptors on the smooth muscle cells and 5‐HT3 receptors and putative neuronal 5‐HT4 receptors. 5‐HT evokes relaxations via an unknown neuronal receptor.