Jan A.J. Schuurkes

5-HT4 receptor agonists: similar but not the same

Abstract  5‐Hydroxytryptamine4 (5‐HT4) receptors are an interesting target for the management of patients in need of gastrointestinal (GI) promotility treatment. They have proven therapeutic potential to treat patients with GI motility disorders. Lack of selectivity for the 5‐HT4 receptor has limited the clinical success of the agonists used until now. For instance, next to their affinity for 5‐HT4 receptors, both cisapride and tegaserod have appreciable affinity for other receptors, channels or transporters [e.g. cisapride: human ether‐a‐go‐go‐related gene (hERG) is K+ channel and tegaserod: 5‐HT1 and 5‐HT2 receptors]. Adverse cardiovascular events observed with these compounds are not 5‐HT4 receptor‐related. Recent efforts have led to the discovery of a series of selective 5‐HT4 receptor ligands, with prucalopride being the most advanced in clinical development. The selectivity of these new compounds clearly differentiates them from the older generation compounds by minimizing the potential of target‐unrelated side effects. The availability of selective agonists enables the focus to shift to the exploration of 5‐HT4 receptor‐related differences between agonists. Based on drug‐ and tissue‐related properties (e.g. differences in receptor binding, receptor density, effectors, coupling efficiency), 5‐HT4 receptor agonists are able to express tissue selectivity, i.e. behave as a partial agonist in some and as a full agonist in other tissues. Furthermore, the concept of ligand‐directed signalling offers great opportunities for future drug development by enlarging the scientific basis for the generation of agonist‐specific effects in different cell types, tissues or organs. Selective 5‐HT4 receptor agonists might thus prove to be innovative drugs with an attractive safety profile for better treatment of patients suffering from hypomotility disorders.

Role of nitric oxide in the gastric accommodation reflex and in meal induced satiety in humans

Aims: In humans, impaired gastric accommodation is associated with early satiety and weight loss. In animals, accommodation involves activation of gastric nitrergic neurones. Our aim was to study involvement of nitric oxide in gastric accommodation and in meal induced satiety in humans. Methods: The effect of NG-monomethyl-l-arginine (l-NMMA) 4 mg/kg/h and 8 mg/kg/h on gastric compliance, on sensitivity to distension, and on gastric accommodation was studied with a barostat in double blind, randomised, placebo controlled studies. The effect of l-NMMA 8 mg/kg/h on meal induced satiety was studied using a drinking test. Results:l-NMMA had no significant effect on fasting compliance and sensitivity. Ingestion of a meal induced a relaxation of 274 (15) ml which was significantly smaller after l-NMMA 4 mg/kg/h (132 (45) ml; p=0.03) or l-NMMA 8 mg/kg/h (82 (72) ml; p=0.03). l-NMMA 8 mg/kg/h significantly decreased the amount of food ingested at maximum satiety from 1058 (67) to 892 (73) kcal (p<0.01). Conclusion: In humans, fasting gastric tone and sensitivity to distension are not influenced by nitric oxide synthase inhibition, but the gastric accommodation reflex involves activation of nitrergic neurones. Inhibition of nitric oxide synthase impairs accommodation and enhances meal induced satiety.

The in vitro pharmacological profile of prucalopride, a novel enterokinetic compound.

Prucalopride is a novel enterokinetic compound and is the first representative of the benzofuran class. We set out to establish its pharmacological profile in various receptor binding and organ bath experiments. Receptor binding data have demonstrated prucaloprides high affinity to both investigated 5-HT(4) receptor isoforms, with mean pK(i) estimates of 8.60 and 8.10 for the human 5-HT(4a) and 5-HT(4b) receptor, respectively. From the 50 other binding assays investigated in this study only the human D(4) receptor (pK(i) 5.63), the mouse 5-HT(3) receptor (pK(i) 5.41) and the human sigma(1) (pK(i) 5.43) have shown measurable affinity, resulting in at least 290-fold selectivity for the 5-HT(4) receptor. Classical organ bath experiments were done using isolated tissues from the rat, guinea-pig and dog gastrointestinal tract, using various protocols. Prucalopride was a 5-HT(4) receptor agonist in the guinea-pig colon, as it induced contractions (pEC(50)=7.48+/-0.06; insensitive to a 5-HT(2A) or 5-HT(3) receptor antagonist, but inhibited by a 5-HT(4) receptor antagonist) as well as the facilitation of electrical stimulation-induced noncholinergic contractions (blocked by a 5-HT(4) receptor antagonist). Furthermore, it caused relaxation of a rat oesophagus preparation (pEC(50)=7.81+/-0.17), in a 5-HT(4) receptor antagonist sensitive manner. Prucalopride did not cause relevant inhibition of 5-HT(2A), 5-HT(2B), or 5-HT(3), motilin or cholecystokinin (CCK(1)) receptor-mediated contractions, nor nicotinic or muscarinic acetylcholine receptor-mediated contractions, up to 10 microM. It is concluded that prucalopride is a potent, selective and specific 5-HT(4) receptor agonist. As it is intended for treatment of intestinal motility disorders, it is important to note that prucalopride is devoid of anti-cholinergic, anticholinesterase or nonspecific inhibitory activity and does not antagonise 5-HT(2A), 5-HT(2B) and 5-HT(3) receptors or motilin or CCK(1) receptors.

Focal activities and re-entrant propagations as mechanisms of gastric tachyarrhythmias.

BACKGROUND & AIMS Gastric arrhythmias occur in humans and experimental animals either spontaneously or induced by drugs or diseases. However, there is no information regarding the origin or the propagation patterns of the slow waves that underlie such arrhythmias. METHODS To elucidate this, simultaneous recordings were made on the antrum and the distal corpus during tachygastrias in open abdominal anesthetized dogs using a 240 extracellular electrode assembly. After the recordings, the signals were analyzed, and the origin and path of slow wave propagations were reconstructed. RESULTS Several types of arrhythmias could be distinguished, including (1) premature slow waves (25% of the arrhythmias), (2) single aberrant slow waves (4%), (3) bursts (18%), (4) regular tachygastria (11%), and (5) irregular tachygastria (10%). During regular tachygastria, rapid, regular slow waves emerged from the distal antrum or the greater curvature, whereas, during irregular tachygastria, numerous variations occurred in the direction of propagation, conduction blocks, focal activity, and re-entry. In 12 cases, the arrhythmia was initiated in the recorded area. In each case, after a normal propagating slow wave, a local premature slow wave occurred in the antrum. These premature slow waves propagated in various directions, often describing a single or a double loop that re-entered several times, thereby initiating additional slow waves. CONCLUSIONS Gastric arrhythmias resemble those in the heart and share many common features such as focal origin, re-entry, circular propagation, conduction blocks, and fibrillation-like behavior.

Origin and propagation of the slow wave in the canine stomach: the outlines of a gastric conduction system

Slow waves are known to originate orally in the stomach and to propagate toward the antrum, but the exact location of the pacemaker and the precise pattern of propagation have not yet been studied. Using assemblies of 240 extracellular electrodes, simultaneous recordings of electrical activity were made on the fundus, corpus, and antrum in open abdominal anesthetized dogs. The signals were analyzed off-line, pathways of slow wave propagation were reconstructed, and slow wave velocities and amplitudes were measured. The gastric pacemaker is located in the upper part of the fundus, along the greater curvature. Extracellularly recorded slow waves in the pacemaker area exhibited large amplitudes (1.8 +/- 1.0 mV) and rapid velocities (1.5 +/- 0.9 cm/s), whereas propagation in the remainder of the fundus and in the corpus was slow (0.5 +/- 0.2 cm/s) with low-amplitude waveforms (0.8 +/- 0.5 mV). In the antrum, slow wave propagation was fast (1.5 +/- 0.6 cm/s) with large amplitude deflections (2.0 +/- 1.3 mV). Two areas were identified where slow waves did not propagate, the first in the oral medial fundus and the second distal in the antrum. Finally, recordings from the entire ventral surface revealed the presence of three to five simultaneously propagating slow waves. High resolution mapping of the origin and propagation of the slow wave in the canine stomach revealed areas of high amplitude and rapid velocity, areas with fractionated low amplitude and low velocity, and areas with no propagation; all these components together constitute the elements of a gastric conduction system.

Evidence for 5-HT7 receptors mediating relaxation of human colonic circular smooth muscle

5‐HT4 receptors mediate relaxation of human colon circular muscle. However, after 5‐HT4 receptor blockade (SB 204070 10 nM), 5‐HT still induced a relaxation (pEC50 6.3). 5‐HT4 receptors were sufficiently blocked, as the curves to 5‐HT obtained in the presence of 10 and 100 nM SB 204070 were indistinguishable. This 5‐HT‐induced relaxation was tetrodotoxin‐insensitive, indicative of a smooth muscle relaxant 5‐HT receptor. This, and the rank order of potency (5‐CT=5‐MeOT=5‐HT) suggested involvement of 5‐HT1 or 5‐HT7 receptors. Mesulergine, a 5‐HT7 receptor antagonist at nanomolar concentrations, and a 5‐HT1 receptor antagonist at micromolar concentrations, competitively antagonized the 5‐HT‐induced relaxation (pKB 8.3) and antagonized the relaxation to 5‐CT. Methysergide antagonized the 5‐HT‐induced relaxation (pA2 7.6). It is concluded that the profile of the smooth muscle inhibitory 5‐HT receptor resembles that of the 5‐HT7 receptor. These data provide the first evidence for functional human 5‐HT7 receptors.

5-HT4 receptors located on cholinergic nerves in human colon circular muscle.

Abstract  5‐Hydroxytryptamine 4 (5‐HT4) receptor agonists promote colonic propulsion. The alteration of circular muscle (CM) motility underlying this involves inhibition of contractility via smooth muscle 5‐HT4 receptors and proximal colonic motility stimulation, the mechanism of the latter not having been characterized. Our aim was to identify and characterize a 5‐HT4 receptor‐mediated stimulation of human colon CM contractile activity. 5‐HT4 receptor ligands were tested on electrical field stimulation (EFS)‐induced contractions of human colonic muscle strips cut in the circular direction (called ‘whole tissue’ strips). Additionally, after incubation of tissues with [3H]‐choline these compounds were tested on EFS‐induced release of tritium in whole tissue strips and in ‘isolated’ CM strips, obtained by superficial cutting in the CM layer. Tetrodotoxin and atropine blocked EFS‐induced contractions of whole tissue CM strips. Prucalopride (0.3 μmol L−1) evoked a heterogenous response on EFS‐induced contraction, ranging from inhibition (most frequently observed) to enhancement. In the release experiments, EFS‐induced tritium efflux was blocked by tetrodotoxin. Prucalopride increased EFS‐induced tritium and [3H]‐acetylcholine efflux in whole tissue and in isolated CM strips. All effects of prucalopride were antagonized by the selective 5‐HT4 receptor antagonist GR113808. The results obtained indicate the presence of excitatory 5‐HT4 receptors on cholinergic nerves within the CM of human colon.

5-HT3 and 5-HT4 receptors and cholinergic and tachykininergic neurotransmission in the guinea-pig proximal colon

The pathways and possible transmitters involved in the contractile response to selective 5-HT3 and 5-HT4 receptor stimulation in the guinea-pig proximal colon were studied. In the presence of methysergide, 5-HT induced contractions, yielding a biphasic concentration-response curve that was changed into a monophasic curve in the presence of the 5-HT3 receptor antagonist, granisetron (1 microM) (low-affinity phase blocked), or the 5-HT4 receptor antagonist, SB 204070 ((1-butyl-4-piperidinyl methyl)-8-amino-7-chloro-1,4-benzodioxan-5-carboxylate) (10 nM) (high-affinity phase blocked) combination of the two antagonists abolished the contraction to 5-HT. The effectiveness and selectivity of both antagonists was confirmed by testing them against contractions in response to the 5-HT3 receptor-selective agonist, 2-methyl-5-HT, and the 5-HT4 receptor-selective agonist, 5-methoxytryptamine. Hexamethonium (100 microM) did not affect the 5-HT3 receptor-mediated contractions, whereas tetrodotoxin (0.3 microM) caused only slight inhibition. Both in the absence and presence of tetrodotoxin, atropine (0.3 microM) inhibited the 5-HT3 receptor-mediated contractions. Hence, the contractions to 5-HT are partly mediated by 5-HT3 receptors that are localized on the nerve endings of the motor neurons. Hexamethonium halved the 5-HT4 receptor-mediated contractions, whereas tetrodotoxin abolished them. The 5-HT4 receptor-mediated contractions were inhibited by atropine (0.3 microM). Thus, the 5-HT4 receptors seem to be localized in the soma of the motor neurons; they also occur on interneurons. The remaining contractions induced by 5-HT3 and 5-HT4 receptor stimulation in the presence of atropine were almost completely inhibited by the tachykinin NK1 receptor antagonist, CP 96345 ((2S,3S)-cis-2-(diphenyl methyl)-N-[(2-methoxy phenyl)-methyl]-1-azabicyclo-[2.2.2]-octan-3-amine) (0.1 microM). CP 96345 also abolished or strongly inhibited contractions in response to substance P (10 nM) and to neurokinin A (30 nM), but neither granisetron nor SB 204070 affected them. Hence, stimulation of either 5-HT3 or 5-HT4 receptors induced contractions that are partially mediated by acetylcholine, and partially by a tachykinin NK1 receptor-stimulating neurotransmitter, probably substance P and/or neurokinin A.

The role of nitric oxide (NO) in 5-HT-induced relaxations of the guinea-pig stomach

SummaryIn a previous study we showed that the relaxations induced after vagal stimulation of the guinea-pig stomach are mediated via nitric oxide (NO) or a NO-related substance. Intra-arterial injection (i.a.) of 5-hydroxytryptamine (5-HT) also induced relaxations in the guinea-pig stomach. Since it has been shown that in the guinea-pig colon 5-HT-induced relaxations are mediated via NO the aim of this study was to establish whether NO is involved in the 5-HT-induced relaxations in the guinea-pig stomach. Intra-arterial injection of 5-HT induced dose-dependent relaxations of the stomach. Since atropine and α- and β-adrenoceptor blocking agents did not influence the relaxation and since tetrodotoxin (TTX) blocked the relaxations, this effect is mediated via NANC-neurons. Administration of a NO-synthase-inhibitor NG-nitro-l-arginine (L-NNA) concentration-dependently reduced the 5-HT-induced relaxations. Haemoglobin (a NO-scavanger) did not affect the relaxations to 5-HT, while addition of methylene blue, an inhibitor of soluble guanylate cyclase, reduced the relaxations by 50%. Addition of an opioid receptor agonist (loperamide), a 5-HT1 antagonist (methiothepin or metergoline) or a 5-HT4 receptor agonist (cisapride) or-antagonist (tropisetron in micromolar concentrations) inhibited the 5-HT-induced relaxations. Neither the 5-HT4 receptor agonist renzapride, nor the novel 5-HT4 receptor antagonist SDZ 205-557, affected the relaxations to 5-HT. These data indicate that 5-HT-induced relaxations of the guinea-pig stomach are mediated via NANC-inhibitory nerves on which inhibitory opioid-receptors are present. The use of selective agonists and antagonists indicates that 5-HT does not act via 5-HT2, 5-HT3 or 5-HT4 receptors. 5-HT may act via 5-HT1 receptors but the subtype involved, if any, has not yet been identified. The inhibitory neurotransmitter which is involved is NO or a NO-related substance.

5‐HT4 receptors mediating enhancement of contractility in canine stomach; an in vitro and in vivo study

We aimed to study 5‐HT4 receptors in canine stomach contractility both in vivo and in vitro. In anaesthetized Beagle dogs, the selective 5‐HT4 receptor agonist prucalopride (i.v.) induced dose‐dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5‐HT4 receptor 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‐HT4 receptor 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 (pEC50 7.9) was shifted in parallel to the right by GR 113808 3 nM (pA2 9.4). In the presence of methysergide (3 μM), the curve to 5‐HT (pEC50 8.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8±0.2 (pKB of 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‐HT4 receptor agonists on proximal stomach motor activity in vivo can be explained by an effect on 5‐HT4 receptors on cholinergic nerves within the gastric muscle wall.