[3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion

Summary The binding characteristics of [³H]ICS 205-930, a 5-hydroxytryptamine 5-HT₃ receptor antagonist, were investigated in membranes prepared from cat and rabbit vagus nerve (VN) and superior cervical ganglion (SCG). The autoradiographic localisation of 5-HT₃ recognition sites was also assessed using [³H]ICS 205-930 in slices from cat medulla oblongata, nodose ganglion and vagus nerve.[³H]ICS 205-930 bound to a homogeneous population of high affinity recognition sites in cat VN: B_max = 201 ± 43 fmol/mg protein, pK_D = 9.26 ± 0.17 and SCG: B_max = 291 ± 40 fmol/mg, pK_D = 9.35 ± 0.80 (n = 3). Competition experiments performed in membranes from cat VN and SCG with agonists and antagonists suggested the presence of a homogeneous population of [³H]ICS 205-930 recognition sites. Competition curves were steep and monophasic and were best fitted by a 1 receptor site model. The following rank order of affinity for [³H]ICS 205-930 binding sites was observed with antagonists: SDZ 206-830 = ICS 205-930 > BRL 43694 > SDZ 206–792 > quipazine > MDL 72222 > metoclopramide > mCPP and agonists: 2-methyl-5-HT = 5-HT > phenylbiguanide. A similar profile was observed for a limited series of compounds in rabbit membranes. Drugs acting at 5-HT₁, 5-HT₂ and dopamine receptors (domperidone, spiperone and metergoline) showed very low affinities for [³H]ICS 205-930 recognition sites. The sites labelled with [³H]ICS 205-930 in vagus nerve and superior cervical ganglion of both species displayed the pharmacological profile of a 5-HT₃ receptor. There was a significant correlation between the rank order of affinity of the tested compounds for [³H]ICS 205-930 recognition sites in cat and rabbit membranes and their rank order of affinity for 5-HT₃ receptors from neuroblastoma-glioma NG 108-15 cells. Autoradiographic studies suggest that [³H]ICS 205-930 binding sites are present over and around the nodose ganglion cell somata, along certain fibers of the vagus nerve and in the terminal areas of this nerve in the medullar nucleus of the vagus.The present data demonstrate that [³H]ICS 205-930 identifies 5-HT₃ receptors in preparations of cat and rabbit vagus nerve and superior cervical ganglion.Send offprint requests to D. Hoyer at the above addressThe present results have been presented in part at the Winter Meeting of the British Pharmacological Society, London, December 20–22, 1988 (Hoyer et al. 1989)     

3H]zacopride: ligand for the identification of 5-HT3 recognition sites

[3H]Zacopride displayed saturable binding to homogenates of the rat entorhinal cortex as measured by the inclusion of the 5-HT3 receptor antagonist BRL43694 in the incubation media. Scatchard analysis indicated a single high affinity binding site (KD 0.76 +/- 0.08 nM, Bmax 77.5 +/- 6.5 fmol (mg protein)-1) with a Hill slope close to unity. Other 5-HT3 receptor antagonists (zacopride, ICS 205-930, GR38032F, GR65630, metoclopramide and cocaine) also competed for the binding site displacing 60% of the total [3H]zacopride binding. 5-HT and 2-methyl-5-HT also were competitive antagonists for [3H]zacopride binding whereas 5-HT1/5-HT2 agonists and antagonists, and agents acting on other neurotransmitter receptors had Ki values greater than 10(-5) M. It is concluded that [3H]zacopride may prove a useful ligand for the study of 5-HT3 recognition sites.     

Common pharmacological and physico-chemical properties of 5-HT3 binding sites in the rat cerebral cortex and NG 108-15 clonal cells

On account of the postulated existence of 5-HT3 receptor subtypes, the respective physico-chemical and pharmacological properties of specific binding sites for the potent 5-HT3 antagonist [3H]zacopride were compared using membranes from the rat posterior cortex or neuroblastoma-glioma NG 108-15 clonal cells. In both membrane preparations, [3H]zacopride bound to a single class of specific sites with a Kd close to 0.5 nM. However, the Bmax value in NG 108-15 cell membranes (970 +/- 194 fmol/mg protein) was approximately 50 times larger than that in cortical membranes (19 +/- 2 fmol/mg protein). The specific binding of [3H]zacopride was equally affected by temperature, pH and molarity of the assay medium, and equally insensitive to thiol- and disulfide-reagents (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dithiothreitol) and GTP in cortical as well as NG 108-15 cell membranes. Determination of the molecular size of [3H]zacopride specific binding sites by radiation inactivation yielded values close to 35 kDa for both membrane preparations. Finally, a highly significant positive correlation (r = 0.979) was found between the respective pKi values of 34 different drugs for their inhibition of [3H]zacopride specific binding to cortical or NG 108-15 cell membranes. Among them, the most potent was S(-)zacopride (pKi = 9.55), followed by BRL 43964, ICS 205-930, quipazine, R(+)zacopride, GR 38032F and MDL 72222. Atypical antidepressants (mianserin, amoxapine) and neuroleptics (clotiapine, loxapine and clozapine) were active in rather low concentrations (pKi less than 6.5), suggesting that recognition of 5-HT3 sites might be relevant to part of the in vivo effects of these drugs. Such identical physico-chemical and pharmacological properties of [3H]zacopride specific binding in cortical and NG 108-15 cell membranes strongly suggest that the same 5-HT3 receptor (subtype?) exists in these two preparations.     

三氯乙醇对5－HT和ICS_（205－930）与［~3H］BRL_（46470

用放射配体受体结合分析法，观察了三氯乙醇（ＴＣＥｔ）对５－ＨＴ３受体激动剂５－ＨＴ和５－ＨＴ３受体拮抗剂ＩＣＳ２０５－９３０与［３Ｈ］ＢＲＬ１６１７０竞争大鼠脑皮质５－ＨＴ３受体能力的影响。结果证实，ＴＣＥｔ（４ｍｍｏｌ·Ｌ－１）能使５－ＨＴ竞争受体的浓度－反应曲线左移，半数抑制结合浓度降低（Ｐ＜０．０１），斜率无显著改变；而对ＩＣＳ２０５－９３Ｏ未见明显影响。表明ＴＣＥｔ能增强５－ＨＴ与５－ＨＴ３受体结合的能力，提示它可能通过这一机制促进５－ＨＴ３配体闸门离子通道开放而发挥作用。     

Antagonism of [3H]zacopride binding to 5-HT3 recognition sites by its (R) and (S) enantiomers

[3H]Zacopride exhibits high affinity (Kd less than or equal to 1 nM) for 5-HT3 binding sites (inhibited by ICS 205-930) in rabbit intestinal muscularis and vagus nerve, human jejunum, rat intestinal muscularis and rat brain cortex. Its binding was inhibited by several 5-HT3 antagonists that displayed similar rank orders of potency in the tissues examined. Zacopride's (S) enantiomer was significantly more potent than its (R) enantiomer (21- to 42-fold in rabbit and human; 8- to 12-fold in rat) as an inhibitor of [3H]zacopride binding. These studies indicate that the utility of [3H]zacopride as a high affinity 5-HT3 ligand resides with the (S) enantiomer.     

Increased gut cholinergic activity and antagonism of 5-hydroxytryptamine M-receptors by BRL 24924: potential clinical importance of BRL 24924.

The mechanisms by which BRL 24924 ([(+/-)- (endo)])-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo-[3.3.1]-non- 4-yl) benzamide hydrochloride stimulates gut motility and the relationships between BRL 24924 and 5-hydroxytryptamine (5-HT) receptors have been studied. In guinea-pig isolated ileum, BRL 24924 (10(-14)-10(-6) M) increased electrically-evoked, cholinergically-mediated contractions, probably by increasing acetylcholine (ACh) release. This action of BRL 24924 was prevented by the presence of high concentrations of 5-HT, but not by hexamethonium, phentolamine and propranolol, methysergide or ICS 205-930. The mechanism by which BRL 24924 can increase gut ACh release is not certain, but most likely involves activation of an enteric 5-HT receptor which differs from those 5-HT M-receptors antagonized by ICS 205-930 or by higher concentrations of BRL 24924 in other test systems. BRL 24924 antagonized 5-HT-evoked, cholinergically-mediated contractions of guinea-pig isolated ileum (pA2 = 7.56 +/- 0.12). Similar and higher concentrations of BRL 24924 did not antagonize contractions evoked by nicotinic receptor stimulation. In rabbit isolated heart, BRL 24924 1-10 nM reduced the tachycardia evoked by 5-HT. In anaesthetized rats, BRL 24924 0.3-83 nmol kg-1 i.v. antagonized the Bezold-Jarisch reflex evoked by 5-HT; the ID50 for BRL 24924 was 10.2 +/- 3.0 nmol kg-1 (3.7 +/- 1.1 microgram kg-1). A direct action of BRL 24924 on nerve function was excluded. In rat cortex, BRL 24924 10(-6) M did not displace [3H]-5-HT or [3H]-ketanserin binding to 5-HT1 and 5-HT2 receptors. The actions of BRL 24924 are discussed in terms of its potential clinical use as a stimulant of gastric motility and as a 5-HT M-receptor antagonist.     

Identification of 5-HT3 recognition sites in the ferret area postrema

The 5-HT3 receptor antagonist [3H]zacopride was used to identify 5-HT3 recognition sites in the ferret area postrema. Specific binding was determined by the inclusion of the 5-HT3 receptor antagonist BRL 43694 in the incubation media, and was shown to be much higher in the area postrema than in other brain regions. The increased binding in the area postrema may reflect either a greater number of binding sites, a higher affinity for such sites, or both. The results indicate that 5-HT3 recognition sites are present within the area postrema and may afford an antiemetic site of action for zacopride and other 5-HT3 receptor antagonists.     

Association of [3H]zacopride with 5-HT3 binding sites

An assay was developed for [3H]zacopride binding to 5-HT3 specific sites in membranes from rabbit ileum muscularis. The binding was rapid, saturable, reversible, salt-insensitive, unaffected by pH between 6.5 and 9.5, and of high affinity (apparent KD = 0.65 +/- 0.15 nM). ICS 205-930, a potent 5-HT3 antagonist that inhibited competitively, was utilized to define 5-HT3 specific binding. Other 5-HT3 antagonists and agonists, although exhibiting marked differences in potency, were also effective inhibitors; whereas, antagonists of other classes of serotonin receptors, guanyl nucleotides and numerous receptor-specific ligands, including peptide hormones, were inactive. Vagus nerve exhibited the greatest amount of 5-HT3 specific binding amongst rabbit tissues and virtually all of the [3H]zacopride was bound to 5-HT3 binding sites. In rabbit, rat and ferret a fairly uniform distribution of 5-HT3 binding sites was observed along the muscularis of the small bowel. [3H]Zacopride is a high-affinity ligand for detecting 5-HT3 binding sites and rabbit small bowel muscularis membranes are a sensitive system for evaluating the potency of 5-HT3 antagonists or agonists.     

Peripherally administered serotonin 5-HT3 receptor antagonists reduce inflammatory pain in rats

Intraplantar administration of serotonin 5-HT3 receptor antagonists ICS 205-930 and MDL 72222 (1-100 micrograms; 50 microliters) produced dose-related analgesia against formalin-induced acute- and Freunds adjuvant-induced chronic-inflammatory pain in rats. 5-HT3 receptor antagonists had greater effect in the chronic pain test than in the acute paradigm. In both tests, ICS 205-930 was more potent than MDL 72222. These data further support the involvement of peripheral 5-HT3 sites in inflammatory pain, and suggest the utility of selective 5-HT3 receptor antagonists as peripheral analgesics.     

Behavioural effects in gerbils of the 5-HT3 receptor antagonists, BRL 43694 and ICS 205-930, under circumstances of high and low light intensity

The 5-HT3 receptor antagonists, BRL 43694 and ICS 205-930, were each given for 21 days in the drinking fluid at 1.3 mg/l (120 micrograms/kg daily), to Mongolian gerbils, while the controls received tap water to drink. Effects of the treatments in reducing aversion to a brightly lit environment were assessed on behaviour during social encounters with an unfamiliar untreated resident, under bright white light and in a two-compartment black and white test box, after 12-16 days of treatment. Effects on behaviour under dim red illumination, when encountering unfamiliar untreated residents, were examined after 17-19 days. Behaviour during social encounters was recorded by ethological procedures. During encounters under bright white light, the frequency and duration of the social element "attend" were increased by BRL 43694 and ICS 205-930 and the frequency of "nose" was increased by BRL 43694. In the light-dark box, BRL 43694, though not ICS 205-930, reduced the time spent in the dark compartment. Under dim red light, BRL 43694 and ICS 205-930 increased the occurrence of the social elements, "sniff", "follow" and "sniff chin", suggesting increased sensitivity to olfactory stimuli. Increases of social investigation were associated with compensatory changes to non-social behaviour. It is suggested that 5-HT3 receptor antagonists may, on the one hand, increase sensitivity to social stimuli under dim red illumination and, on the other hand, show an apparent anxiolytic potential, associated with increase of other elements of social investigation under the more aversive test conditions of bright white light.     

5-HT3 receptors are not involved in the modulation of the K(+)-evoked release of [3H]5-HT from spinal cord synaptosomes of rat.

The ability of 5-HT3 receptor agonists to modulate the resting efflux or K(+)-evoked release of [3H]5-HT from superfused synaptosomes from the spinal cord of the rat was investigated. Phenylbiguanide did not alter the resting efflux of [3H]5-HIAA or [3H]5-HT or modify the K(+)-evoked release of [3H]5-HT. 2-Methyl-5-HT (10 microM) caused an increase in resting efflux of [3H]5-HIAA, an effect that was blocked by the inhibitor of the uptake of 5-HT fluoxetine. No effect on K(+)-evoked release of tritium was observed. Bufotenine (100-1000 nM) increased the resting efflux of [3H]5-HT and [3H]5-HIAA. These effects were not antagonized by the 5-HT3 antagonist ICS 205-930 but were antagonized by fluoxetine. The drug ICS 205-930 (1 microM) did not alter resting efflux or block the ability of serotonin (30 and 100 nM) to decrease K(+)-evoked release of tritium. Quipazine, a potent antagonist of peripheral 5-HT3 receptors (subnanomolar concentrations), was also unable to alter resting or K(+)-evoked release of [3H]5-HT. It did, however, attenuate the inhibitory effect 5-HT on K(+)-evoked release. The concentrations required were in the micromolar range, consistent with the ability of the drug to antagonize the 5-HT1B autoreceptor. These results support the idea that 5-HT3 receptors do not act as nerve terminal autoreceptors in the spinal cord of the rat.     

The depolarizing action of 5-hydroxytryptamine on rabbit vagal afferent and sympathetic neurones in vitro and its selective blockade by ICS 205-930.

Depolarizing responses to 5-hydroxytryptamine (5-HT) were recorded from rabbit nodose (NG) and superior cervical (SCG) ganglia using the sucrose-gap technique. The antagonist potency and selectivity of ICS 205-930 ([3 alpha-tropanyl]-1H-indole-3-carboxylic acid ester) were investigated. In NG, 5-HT (5 to 80 nmol) evoked depolarizations of graded amplitude. The ED50 was 18.2 (10.9-30.5) nmol (geometric mean, 95% confidence limits). Responses were blocked surmountably by ICS 205-930, 10(-11) and 10(-10) M, the threshold for blockade being below 10(-11) M. Parallel, rightward shifts in dose-response curves were seen with these concentrations of antagonist, but at higher concentrations (10(-9) and 10(-8) M) there was a further rightward shift with reduction in slope and maximum of the curves. In SCG, where 5-HT (20 to 320 nmol) evoked depolarizations of graded amplitude and the ED50 was 55.8 (22.3-139.6) nmol (geometric mean, 95% confidence limits), ICS 205-930 had a similar inhibitory effect to that observed in NG. The apparent pA2 values for the surmountable blockade produced by ICS 205-930 at concentrations of 10(-11) and 10(-10) M were 10.2 +/- 0.2 for NG and 10.4 +/- 0.1 for SCG (means +/- s.e. mean). ICS 205-930 was selective in its action since it had no effect on dimethylphenylpiperazinium (DMPP) responses in either ganglion or on GABA responses in NG. This study provides quantitative evidence on the blocking action of ICS 205-930 at neuronal 5-HT receptors using a technique that allows the depolarizing responses evoked by the amine to be directly recorded.     

Effects of p-chloroamphetamine on release of [3H]gamma-aminobutyric acid from slices of rat caudate-putamen.

The effect of endogenous serotonin on the release of [3H]gamma-aminobutyric acid ([3H]GABA) from slices of rat caudate-putamen was studied. p-Chloroamphetamine was used to release endogenous serotonin. p-Chloroamphetamine (100 nM) enhanced the release of [3H]GABA induced by 20 mM K+, while 1000 nM p-chloroamphetamine decreased it. The 5-HT3 receptor antagonists ICS 205-930 (50 nM) and MDL 72222 (100 nM) prevented this facilitation caused by 100 nM p-chloroamphetamine. ICS 205-903 (50 nM), when used alone, reduced the release of [3H]GABA caused by 23 mM K+. This finding confirmed the hypothesis that endogenous serotonin can enhance the release of [3H]GABA via 5-HT3 receptors. In contrast, an effect of 5-HT1 and 5-HT2 receptors could not be clearly established. It is likely that the release of endogenous GABA from striatonigral GABA neurons may also be affected by serotonin via 5-HT3 receptors.     

Interaction of the atypical neuroleptic clozapine with 5-HT3 receptors in the cerebral cortex and superior cervical ganglion of the rat

Clozapine, an atypical neuroleptic drug devoid of extrapyramidal side effects, was a moderately potent, competitive inhibitor of the binding of [3H]quaternised ICS 205-930 to 5-HT3 receptor sites in rat cortical membranes, possessing a pKi value of 7.0. In contrast, several other antipsychotic agents, including fluphenazine, alpha-flupenthixol, haloperidol, spiperone and (-)-sulpiride were essentially inactive. Clozapine also antagonised the 2-methyl 5-HT-induced depolarisation of the rat isolated superior cervical ganglion, a response known to be mediated via 5-HT3 receptors. Clozapine (0.1-1 microM) induced parallel displacements to the right of the dose-response curve to 2-methyl 5-HT in this tissue, possessing a pKb value of 7.3. These data suggest that the atypical antipsychotic profile of clozapine may be related, at least, in part to its ability to interact with central 5-HT3 receptor sites.     

Solubilization of a 5-HT3 binding site from rabbit small bowel muscularis membranes

A 5-HT3 binding site, with high affinity for (S-)[3H]zacopride, was solubilized from rabbit small bowel muscularis membranes utilizing 0.5% sodium cholate and 400 mM (NH4)2SO4. Approximately 72% of the (S-)[3H]zacopride binding activity was recovered in a form that retained the high affinity (Kd = 0.7 nM) and specificity for this radioligand that is characteristic of the membrane-bound receptor. ICS 205-930 and other 5-HT3 compounds were effective inhibitors and exhibited the same rank order of potency in the solubilized and membrane-bound preparations. The receptor-detergent complex did not sediment after centrifugation for 1 h at 150,000 x g and eluted between thyroglobulin (MW = 669,000) and apoferritin (MW = 443,000) when fractionated by high-performance liquid chromatography gel filtration. This is the first report of the solubilization of a 5-HT3 binding site.     

Gastrointestinal motility stimulating drugs and 5-HT receptors on myenteric neurons

5-HT3 receptor antagonists may have both antiemetic and gastric and intestinal motility stimulating properties, but they differ in their relative potencies and efficacies for these two activities. Since the 5-HT3 receptor is present on enteric neurons, intracellular recordings of myenteric neuronal transmembrane potential were used to assess the actions of four proposed motility stimulating drugs, metoclopramide, BRL 24924, ICS 205-930 and cisapride. BRL 24924 (10(-6) M), ICS 205-930 (10(-7) M) and cisapride (5 x 10(-6) M) each antagonized the 5-HT3-mediated fast depolarization of myenteric neurons. Metoclopramide (10(-5) M) was less consistent in its ability to antagonize this response, and the response often returned in the continued presence of metoclopramide. In the present study, BRL 24924 (10(-6) M) and, as previously shown, cisapride (5 x 10(-6) M) antagonized the slow depolarization of myenteric neurons induced by 5-HT. Metoclopramide (10(-5) M), BRL 24924 (10(-6) M) and cisapride (5 x 10(-6) M), but not ICS 205-930 (10(-7) M) depolarized myenteric neurons within the first 2 min of contact with myenteric neurons. These data support the view that there are separate receptors that may be responsible for the prokinetic actions of these drugs and a series of 5-HT3-mediated actions which include antiemesis.     

Dual effects of 5-hydroxytryptamine on the release of gamma-aminobutyric acid from myenteric neurones of the guinea-pig ileum.

The effects of 5-hydroxytryptamine (5-HT) on the release of gamma-aminobutyric acid (GABA) were examined in the longitudinal muscle-myenteric plexus (LM-MP) preparation of guinea-pig ileum. 5-HT increased the spontaneous release and inhibited the electrically-evoked release of [3H]-GABA. The 5-HT-evoked release was Ca2+-dependent and tetrodotoxin-sensitive, and was antagonized by (3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930), but not by methysergide and ketanserin. The inhibitory effect of 5-HT was antagonized by methysergide, but not by ketanserin and ICS 205-930. 8-Hydroxy-2-(di-n-propylamino)tetralin mimicked the inhibitory effect of 5-HT. Thus, 5-HT may exert an excitatory effect on the enteric GABAergic neurone via the 5-HT3 receptor and an inhibitory effect via the 5-HT1A receptor.     

Activation of 5-HT3 receptor subtypes causes rapid excitation of rabbit parasympathetic neurones.

Intracellular recordings were made from parasympathetic neurones of the rabbit vesical pelvic ganglia maintained in vitro. 5-Hydroxytryptamine (5-HT) caused a membrane depolarization which was antagonized by ICS 205-930 ([3 alpha-tropanyl]-1H-indole-3-carboxylic acid ester) but not by methysergide. ICS 205-930 caused a parallel shift to the right of the dose-response curve for 5-HT. These results suggest that the 5-HT3 receptor is involved in the membrane depolarization.     

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prokinetic benzamides stimulate peristaltic activity in the isolated guinea pig ileum by activation of 5-HT4 receptors.

Substituted benzamides such as metoclopramide, cisapride, zacopride, renzapride or BRL 20627, stimulate intestinal motility in various species. As they are antagonists at 5-HT3 and agonists at 5-HT4 receptors and as both mechanisms could potentially contribute to their gastrointestinal prokinetic effect, the underlying mechanism is unclear. To clarify this, the effect of some substituted benzamides on gut motility was investigated in the isolated guinea pig ileum using the Trendelenburg technique, in which pressure-induced peristaltic contractions are measured. All benzamides stimulated the peristaltic reflex with the rank order of potency: renzapride greater than cisapride greater than BRL 20627 greater than (+/-)-zacopride greater than metoclopramide. ICS 205-930, granisetron and 2-methyl-5-HT did not change the peristaltic response. 5-HT and 5-methoxytryptamine potently mimicked the effect of the benzamides. The effect of 5-HT was not blocked by ICS 205-930 (10(-7) M). These results indicate that the Trendelenburg preparation is suitable for the investigation of intestinal prokinetic effects of the substituted benzamides. Furthermore, the results suggest that the intestinal effect of benzamides results from activation of 5-HT4 receptors rather than from blockade of 5-HT3 receptors.