The interaction of dexamethasone with ondansetron on drug-induced emesis in the ferret.

The potential of dexamethasone (0.5-20 mg/kg i.p.) to antagonize the emesis induced by cisplatin (10 mg/kg i.v.), apomorphine (0.25 mg/kg s.c.), morphine (0.5 mg/kg s.c.) and copper sulphate (100 mg/kg intragastric) was investigated alone and in combination with ondansetron in the ferret. There was a trend for dexamethasone 0.1-5 mg/kg to reduce cisplatin-induced emesis and for 0.05-2.5 mg/kg to delay the onset of apomorphine-induced emesis but doses of dexamethasone up to 20 mg/kg were without effect to modify morphine- or copper sulphate-induced emesis. The combination of dexamethasone 2.5 mg/kg with ondansetron 0.1 mg/kg did not have additive effects to reduce cisplatin-induced emesis but did reduce significantly the total numbers of episodes recorded. Ondansetron 1 mg/kg was without effect to modify apomorphine-, morphine- or copper sulphate-induced emesis but the combination pretreatment of ondansetron 1 mg/kg with dexamethasone 2.5 and 5 mg/kg reduced significantly apomorphine-induced retching. Data are discussed in terms of the antiemetic effectiveness of dexamethasone to antagonize chemotherapy-induced emesis in man.

The actions of fentanyl to inhibit drug-induced emesis.

The ability of fentanyl to inhibit drug-induced emesis was investigated in the ferret. Initial studies established that morphine, in small doses (0.025-0.5 mg/kg s.c.), induced emesis in the ferret that decreased at the larger doses of 1 and 2 mg/kg (s.c.). Fentanyl (10-80 micrograms/kg s.c.) failed to induce emesis but in this dose range prevented the emesis induced by morphine (0.5 mg/kg s.c.), apomorphine (0.25 mg/kg s.c.), copper sulphate (100 mg/kg intragastric) and cisplatin (10 mg/kg i.v.). The antiemetic effects could be obtained in the absence of sedation or motor impairment. The antagonism by fentanyl of apomorphine-, copper sulphate- and cisplatin-induced emesis was inhibited by naloxone (0.1 or 0.5 mg/kg s.c.). It is concluded that fentanyl exerts a broad spectrum of actions to inhibit drug-induced emesis. An autoradiographic study of the binding of [3H]DAGO to the brainstem of the ferret indicated high densities of mu recognition sites in the area postrema, nucleus tractus solitarius, dorsal motor nucleus of the vagus, reticular medulla and other sites. The results are discussed in terms of balanced facilitatory and inhibitory opioid systems, regulating emesis and that the antiemetic actions of fentanyl reflect an important, although not necessarily an exclusive, action at mu opioid receptors.

The effects of the stable thromboxane A2-mimetic, U46619, on gastric mucosal damage and gastric non-parietal secretion in the rat.

The effects of the thromboxane A2-mimetic, U46619, and the thromboxane receptor antagonist, AH23848, on ethanol-induced gastric mucosal damage and gastric non-parietal secretion have been examined in the rat. Oral dosing with U46619 or AH23848 produced a dose-related inhibition of ethanol-induced gastric mucosal damage in the conscious rat, and these effects were partially blocked by indomethacin treatment. Intragastric application of U46619 or AH23848 to the stomach of the anaesthetized rat stimulated the gastric secretion of a juice which consisted principally of Na+ and Cl- ions. These secretagogue effects of both compounds were blocked by indomethacin treatment. These results show that U46619 and AH23848 induce secretory and protective effects in the stomach of the rat, although these responses probably do not involve thromboxane receptors and are mediated, at least in part, by endogenous prostaglandins. The results are discussed in relation to the role of endogenous thromboxane A2 in gastric mucosal protection, and of the possible protective function of non-parietal secretion in the stomach.

The effect of chlorpromazine on the function of colonic and ileal mucosa in the anaesthetized rat.

1 The effect of chlorpromazine (Cpz) on the net fluxes of water and sodium in the ileum and colon has been studied in the anaesthetized rat. 2 Under control conditions both the ileum and colon absorbed water and sodium. Cpz (100 mumol/kg, s.c.) had no significant effect on these basal rates of absorption. 3 Intestinal secretion was stimulated by a combination of intra-arterial prostaglandin E1 (PGE1; 10 nmol kg-1 min-1) and intraluminal theophylline (25 mM). A marked potentiation occurred between PGE1 and theophylline in the stimulation of colonic and ileal secretion. 4 In the ileum, a dose-related inhibition of the PGE 1/theophylline-induced changes in the net fluxes of water and sodium was produced by Cpz (0.1 to 100 mumol/kg, s.c.). Under these conditions the inhibition of the secretagogue-induced changes was significant using Cpz at 1 mumol/kg (s.c.) and a dose of Cpz of 100 mumol/kg (s.c.) returned the net fluxes of both water and sodium to basal levels. 5 In the colon, Cpz, at doses of 0.1 and 1 mumol/kg subcutaneously did not inhibit the PGE1/theophylline-induced changes in the net fluxes of water and sodium, and, in contrast with the ileum, significant changes were only obtained with Cpz at 10 mumol/kg. Increasing the dose of Cpz to 100 mumol/kg further inhibited the net flux of sodium, but not that of water in the colon, and in each case the secretagogue-induced changes were not returned to basal levels.

Prostanoid stimulation of anion secretion in guinea-pig gastric and ileal mucosa is mediated by different receptors.

1. The receptors that mediate stimulation of anion secretion by prostanoids in isolated preparations of guinea-pig gastric and ileal mucosa have been compared by use of selective prostanoid agonists and antagonists. 2. In gastric mucosa, the relative potency of agonists suggested that the control of anion secretion in this tissue was complex and may be mediated by EP2, EP3, and TP receptors. A role for TP receptors was confirmed with the TP-selective antagonist AH23848 which inhibited short circuit current responses to the TP receptor agonist U-46619 with a pA2 value of 8.44 but was without effect on responses to prostaglandin E2 (PGE2) or the EP selective agonist, sulprostone. 3. In ileal mucosa, the relative potency of agonists differed from that observed in gastric mucosa and was consistent with the view that anion secretion in this region of intestine was controlled by DP and EP2 receptors. 4. These studies suggest that anion secretion in gastric and ileal mucosa is controlled by different prostanoid receptor subtypes and so provide important information for the design of prostanoids which may protect gastric mucosa and that are free from side effects such as diarrhoea.

The effect of cimetidine on basal and stimulated pepsin secretion in the isolated whole stomach of the rat.

1 The isolated stomach preparation of the immature rat has been used to study the stimulation and inhibition of pepsin secretion. 2 The isolated stomach secretes a basal level of pepsin. High concentrations (10(-3)M) of the H2-receptor antagonist, cimetidine, and the muscarinic receptor blocking drug, atropine, did not affect this secretion in a manner which was consistently of statistical significance. 3 Concentrations of histamine of 10(-5)M, 10(-4)M and 10(-3) M stimulated maximum levels of pepsin output of 126%, 155% and 299% respectively of control. There was no evidence that this secretion was secondary to the stimulation of acid secretion. 4 Cimetidine (10(-4)M and 10(-3)M) produced a dose-related inhibition of the pepsin output to 10(-3)M histamine, suggesting that histamine H2-receptors mediate this response. 5 Atropine (10(-3)M) had no effect on the pepsin response to 10(-3)M histamine, suggesting that muscarinic mechanisms play no part, even modulatory, in this secretion.

Characterization of the 5-hydroxytryptamine receptor type involved in inhibition of spontaneous activity of human isolated colonic circular muscle.

1. Experiments were carried out to characterize pharmacologically the 5-hydroxytryptamine (5-HT) receptor types which mediate inhibition of spontaneous contractions of the intertaenial circular muscle in human isolated colon. 2. 5-HT caused a reproducible concentration-dependent inhibition of spontaneous contractions of the circular muscle of human colon in vitro with a mean EC50 value of 0.2 microM and 95% confidence limits of 0.1-0.5 microM. No evidence for a contractile action of 5-HT was found. Tetrodotoxin (TTX, 1.5 microM) caused a rightward shift of the concentration-response curve of 5-HT with a concentration-ratio of 2.9. 3. The inhibitory response to 5-HT was mimicked by several indoles with the rank order of potency 5-HT > 5-methoxytryptamine = alpha-methyl-5-HT > 5-carboxamidotryptamine >> 2-methyl-5-HT. 5-Hydroxyindalpine was inactive. 4. The substituted benzamides were agonists with the following rank order of potency, 5-HT > renzapride > zacopride > metoclopramide > cisapride. 5. The inhibitory responses to 5-HT were not inhibited by methysergide (10 microM) or methiothepin (1 microM), which are antagonists selective for 5-HT1-like and 5-HT2 receptors, nor by ondansetron (10 microM) which is an antagonist at 5-HT3 receptors. 6. The inhibitory responses induced by 5-HT and 5-methoxytryptamine were competitively antagonized by a weak 5-HT4 receptor antagonist, tropisetron, with pKB values of approximately 6. Tropisetron had no significant effect on the inhibitory response curve produced by isoprenaline (0.01-100 microM). 7. The pharmacological profile of the 5-HT-evoked relaxations of human colon circular muscle are consistent with activation of a 5-HT4-like receptor.

Investigation of the role of extracellular calcium in the control of acid secretion in the isolated whole stomach of the rat.

1 An isolated stomach preparation from immature rats has been used to study the role of extracellular calcium in the control of gastric acid secretion. Calcium was removed from both the serosal and mucosal solutions either in the absence of a chelating agent or in the presence of EGTA.2 Removal of calcium in the absence of EGTA had no significant effect on basal acid secretion or the acid responses to gastrin and dibutyryl cyclic adenosine 3',5'-monophosphate (db cyclic AMP). Under the same conditions there was a marked potentiation of the acid response to histamine, and a reduction of the acid response to acetylcholine which was readily reversed on restoring calcium to the bathing solutions.3 Removal of calcium in the presence of EGTA caused an inhibition of basal acid secretion and of the acid responses to histamine and db cyclic AMP. In each case this reduction in acid output was readily reversed on bathing the stomachs in normal calcium-containing (2.5 mM Ca(2+)) EGTA-free solutions.4 The inhibition of the acid response to histamine produced by Ca(2+)-free solutions which contained EGTA was not reversed on bathing the stomachs in solutions that contained both EGTA (0.1 mM) and an excess of calcium (2.5 mM).5 The removal of extracellular calcium in the absence of EGTA provided evidence that the secretion of H(+) ions is dependent under some conditions on calcium ions. The possibility cannot be excluded that EGTA itself exerts an inhibitory influence on the process of acid secretion.

The effect of atropine on acid secretion stimulated by acetylcholine, histamine and gastrin in the isolated whole stomach of the rat.

1 An isolated stomach preparation from immature rats has been used to study the effect of atropine on gastric acid secretion. 2 The acid secretory response to acetylcholine was not inhibited by atropine at a concentration of 0.3 micrometer. Concentrations of atropine of 1 to 3 micrometer produced a measurable inhibition of acid secretion, and a concentration of atropine of 10 micrometer caused a complete block of acid secretion which could not be surmounted by high concentrations of acetylcholine. 3 The acid secretory response to histamine was not inhibited by concentrations of atropine of up to 1 mM. 4 Concentrations of atropine of 1 micrometer and 10 micrometer did not inhibit gastrin-stimulated acid secretion, although a significant inhibition of acid output was observed with atropine at concentrations of 0.1 mM and 1 mM. 5 These findings are discussed in relation to the role of cholinergic mechanisms in the control of gastric acid secretion.

Investigation of the 5-hydroxytryptamine receptor mechanism mediating the short-circuit current response in rat colon.

1. 5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (SCC) in rat isolated colonic mucosa with an EC50 value of approximately 4 microM. The purpose of the present study was to investigate the 5-HT receptor mechanism(s) involved in this response. 2. The relatively selective 5-HT receptor agonists 5-carboxamidotryptamine (5-CT) and alpha-methyl-5-HT stimulated SCC and were 6 to 8 times less potent than 5-HT. 2-Methyl-5-HT was inactive both as an agonist and an antagonist. 3. The following compounds produced no significant inhibition of the SCC response to 5-HT: ketanserin (1 microM), methysergide (1 microM), methiothepin (0.3 microM), GR38032F (0.3 microM), tetrodotoxin (0.3 microM) and sulpiride (1 microM). 4. Both metoclopramide (3 and 10 microM) and cisapride (0.1 and 1 microM) inhibited the SCC responses to 5-HT in a concentration-related manner, and the higher doses similarly inhibited the responses to 5-CT. With both agonists the inhibitory effects of metoclopramide and cisapride were insurmountable. However, these inhibitory actions appeared to be selective since neither metoclopramide nor cisapride affected the basal SCC or the SCC response to prostaglandin E2. 5. The SCC responses to 5-HT and 5-methoxytryptamine were selectively inhibited by ICS205-930 at 3 microM, and respective pKB values of 6.0 and 6.6 were calculated. 6. It is concluded that 5-HT stimulates an SCC response in rat colon via a receptor mechanism that cannot be clearly identified as 5-HT1-like, 5-HT2 or 5-HT3. This receptor is selectively antagonized by ICS 205-930 and by the benzamides, metoclopramide and cisapride. The 5-HT receptor in rat colon therefore exhibits some of the properties associated with the so-called 5-HT4 receptor.

Investigation into the 5-hydroxytryptamine receptor mediating smooth muscle relaxation in the rat oesophagus.

1. An investigation has been made into the 5-hydroxytryptamine (5-HT) receptor mediating relaxation of rat oesophagus in preparations precontracted with carbachol. 2. In tissues treated with pargyline (100 microM) and in the presence of corticosterone (30 microM) and cocaine (30 microM) the potency of 5-HT and 5-methoxytyramine (5-MeOT) was not changed but the maximum response to these agonists was reduced. Thus there was no evidence of metabolism and/or uptake through an amine depleting mechanism. 3. The relaxant concentration-effect curves to 5-HT were shifted to the left in a concentration-related manner by isobutylmethylxanthine (1 and 10 microM), suggesting the involvement of adenosine 3':5'-cyclic monophosphate in these responses. 4. 5-HT produced concentration-related relaxations of rat oesophagus with an EC50 value of 0.24 microM. Several indole agonists were tested and the following rank order of potency of key agonists obtained: 5-HT greater than alpha-methyl-5-hydroxytryptamine = 5-carboxamidotryptamine (5-CT) greater than 5-MeOT. In contrast, 2-methyl-5-hydroxytryptamine, sumatriptan and 8-hydroxy-2-(di-n-propylamino) tetralin were weak or inactive. 5. The substituted benzamides, metoclopramide, cisapride, renzapride and R,S-zacopride acted as partial agonists, producing 60-70% of the 5-HT maximum. 6. The relaxation responses to 5-HT were neither inhibited by antagonists selective for 5-HT1 or 5-HT2 receptors nor by the 5-HT3 receptor antagonists, ondansetron, granisetron or MDL 72222. 7. The relaxation responses induced by 5-HT, 5-CT, 5-MeOT and renzapride were selectively inhibited by high concentrations of ICS 205-930 with pKB values of approximately 6. 8. The 5-HT receptor mediating relaxation in rat oesophagus cannot be designated 5-HT1, 5-HT2 or 5-HT3 under the current 5-HT classification, but the observed effects are consistent with stimulation of the putative 5-HT4 receptor.

Investigation of the 5-hydroxytryptamine receptor mediating the 'maintained' short-circuit current response in guinea-pig ileal mucosa.

1. 5-Hydroxytryptamine (5-HT) stimulated a biphasic increase in short-circuit current (SCC) in guinea-pig isolated ileal mucosa. The initial 'spike' response to 5-HT was inhibited by tetrodotoxin (0.3 microM). We have investigated the 5-HT receptor mechanism(s) controlling the second 'maintained' component of the response which remained after treatment with tetrodotoxin. 2. 5-HT stimulated concentration-related increases in SCC with an EC50 value of 5.4 microM. Isobutyl-methylxanthine (IBMX, 10 microM) produced a six fold leftward shift of this concentration-response curve, suggesting the involvement of a cyclic nucleotide(s) in these responses. 3. In the presence of IBMX, 5-HT stimulated reproducible increases in SCC with an EC50 value of 0.9 microM. The rank order of potency of indole agonists in these tests was 5-HT greater than or equal to 5-methoxytryptamine greater than 5-carboxamidotryptamine = alpha-methyl-5-HT much greater than 2-methyl-5-HT. 4. The substituted benzamides were partial agonists. Metoclopramide and cisapride produced approximately 20% of the 5-HT maximum, and renzapride and R,S-zacopride produced approximately 50% of the 5-HT maximum. Metoclopramide and cisapride inhibited the SCC responses to 5-HT with apparent pKB values of 4.8 and 7.0 respectively. 5. The SCC responses to 5-HT were not inhibited by antagonists selective for 5-HT1 (methysergide, methiothepin), 5-HT2 (ketanserin) or 5-HT3 (ondansetron, ICS205-930) receptors. 6. The SCC responses to 5-methoxytryptamine, 5-carboxamidotryptamine, alpha-methyl-5-HT and R,S-zacopride, but not 5-HT, were selectively inhibited by high concentrations of ICS205-930 with apparent pKB values of approximately 6.7. A possible interpretation of these results is that the 'maintained' SCC response to 5-HT is mediated by a heterogeneous population of 5-HT receptors. One of these receptors exhibits the characteristics of the putative 5-HT4 receptor.

Differences in response to 5-HT4 receptor agonists and antagonists of the 5-HT4-like receptor in human colon circular smooth muscle.

1. In isolated circular smooth muscle strips of human colon 5-hydroxytryptamine (5-HT) produced a concentration-related inhibition of spontaneous motility. 2. The azabicycloalkyl benzimidazolones, BIMU 8 and BIMU 1, which have 5-HT4 receptor stimulant properties, inhibited motility with EC50 values of 0.76 microM and 3.19 microM respectively and their Emax values were not significantly different from 5-HT (EC50, 0.13 microM). 3. The 5-HT4 receptor antagonist, DAU 6285 (1-10 microM), displaced the 5-HT concentration-response curve to the right in a parallel concentration-dependent manner without depressing the maximum. The Schild plot was linear and the slope did not differ significantly from unity giving a pA2 value of 6.32. 4. The high affinity selective 5-HT4 receptor antagonist, GR 113808, at a concentration of 3 nM displaced the 5-HT concentration-response curve in a parallel manner giving an apparent pKB estimate of 8.9 +/- 0.24. However, higher concentrations of 10-100 nM GR 113808 did not result in a further significant displacement of the 5-HT concentration-response curve and there was no suppression of Emax. 5. GR 113808 (10 nM) also caused a parallel displacement of the concentration-response curve to the 5-HT4 receptor agonist, 5-methoxytryptamine (5-MeOT) giving apparent pKB values ranging from 8.3-9.3. 6. GR 113808 (3-100 nM) failed to displace 5-HT or 5-MeOT concentration-response curves in tissue strips from 3 patients out of a total of 10 patients studied in whom the response to 5-HT and 5-MeOT was normal. 7. The 5-HT4 receptor antagonist, SDZ 205-557 (0.3-10 microM), had no significant effect on 5-HT-induced inhibition of spontaneous motility.8. The present results are discussed in the light of variability of response to GR 113808 and SDZ205-557 in other tissues.9. Overall, our data indicate that human colon circular smooth muscle can be regarded as a site in which 5-HT4-like receptors are present but it is as yet unclear whether these results are also an indication of receptor variation.

Development of a radioligand binding assay for 5-HT4 receptors in guinea-pig and rat brain.

1. The 5-HT4 receptor antagonist, GR113808, has been radiolabelled to a high specific activity with tritium. 2. Characterization of specific [3H]-GR113808 binding in homogenates of guinea-pig striatum and hippocampus revealed a single site of high affinity (Kd values 0.20 and 0.13 nM respectively). 3. [3H]-GR113808 binding was reversible and displayed rapid kinetics such that association and dissociation were complete within 3 min. 4. Specific [3H]-GR113808 binding was potently and stereoselectively inhibited by agonists and antagonists acting at the 5-HT4 receptor but not by compounds selective for other 5-HT receptors or other neurotransmitter receptors. 5. Autoradiographic analysis revealed a discrete localization in both guinea-pig and rat brain with high concentrations of binding in brain areas such as the striatum, substantia nigra and olfactory tubercle. 6. [3H]-GR113808 binding to homogenates of guinea-pig striatum meets the criteria for labelling of the 5-HT4 receptor and, as such, represents the first characterization of this receptor in a radioligand binding assay.

Relationship between lesion formation and permeability of rat gastric mucosa to H+ and other cations.

The relationship between lesion formation and ionic permeability has been investigated in rat gastric mucosa in vivo. Changes in these parameters were measured in the mucosa treated topically with prostaglandins E2 and A2 and/or aspirin. Particular attention was paid to the net flux of H+ ions across the gastric mucosa. The effect of aspirin concentrations of 5 mM, 20 mM and '40 mM' (the latter, a suspension in a saturated solution) was investigated. Aspirin concentrations of 20 mM and '40 mM' produced a marked increase in lesion formation and increased the net mucosal to serosal flux of H+ ions. Aspirin 5 mM produced a significant increase in lesion formation but did not cause a significant change in net H+ ion flux. This result suggests that aspirin can have a direct irritant effect on the gastric mucosa and that the back diffusion of H+ ions is not a pre-requisite for the development of overt mucosal ulceration. The effect of topically applied prostaglandin E2 (PGE2) on aspirin-induced gastric mucosal damage was investigated. Concentrations of PGE2 of 10(-5) M and 10(-4) M ameliorated aspirin-induced damage, but these changes were not necessarily accompanied by a significant reduction in net H+ ion flux. Again, this result is not consistent with a direct relationship between lesion formation and mucosal permeability to H+ ions. Since PGA2 did not ameliorate aspirin-induced mucosal damage, the protective effect of PGE2 could not be attributed to its conversion to PGA2 in the acidic environment of the gastric lumen. 5 Changes in gastric mucosal potential difference (p.d.) and net fluxes of Na+ and K+ ions may occur without a concomitant change in the permeability of the gastric mucosa to acid back-diffusion. Thus, the assumption cannot be made that a change in the permeability of the gastric mucosa to one particular ion reflects a general increase in ionic permeability.

The effect of metiamide on acid secretion stimulated by gastrin, acetylcholine and dibutyryl cyclic adenosine 3',5'-monophosphate in the isolated whole stomach of the rat.

1 An isolated stomach preparation from immature rats is described. The lumen of the stomach was perfused and the hydrogen ion activity of the perfusate recorded continuously. 2 The preparation gave dose-dependent responses to gastrin, acetylcholine and dibutyryl cyclic adenosine 3',5'-monophosphate and these responses were readily reversed on washing out the agonist. 3 The acid secretory response to gastrin was inhibited by metiamide at concentrations of 10(-5) M and 3 X 10(-5)M. 4 The acid secretory responses to acetylcholine and dibutyryl cyclic adenosine 3',5'-monophosphate were not inhibited by concentrations of metiamide up to 10(-3) M. 5 These findings are discussed in relation to the role of histamine in the control of gastric acid secretion.

5-HT3 receptor antagonists injected into the area postrema inhibit cisplatin-induced emesis in the ferret.

1. The purpose of the present study was to identify and investigate the role of 5-hydroxytryptamine3 (5-HT3) receptors in the area postrema in the control of cisplatin-induced emesis in the ferret. 2. Homogenate binding and autoradiography experiments using the high affinity 5-HT3 receptor ligand, [3H]-GR65630, identified the presence of a high concentration of 5-HT3 receptors in the area postrema of the ferret. 3. Intraperitoneal injection of the 5-HT3 receptor antagonists, GR38032F, GR65630A and MDL72222, at doses of 1, 0.1 and 1 mg kg-1 respectively, inhibited emesis induced by cisplatin, 9 mg kg-1 i.p. 4. Discrete injection of low doses of the 5-HT3 receptor antagonists directly into the area postrema region also inhibited cisplatin-induced (9 mg kg-1 i.p.) emesis. The dose ranges used were: GR38032F, 0.01-1 microgram; GR65630A, 0.001-0.1 microgram; MDL72222, 0.1-10 micrograms. 5. Cisplatin-induced emesis was not inhibited by discrete injection of ketanserin (30 micrograms) or methiothepin (30 micrograms) into the area postrema. Injection of the 5-HT3 receptor agonist, 2-methyl-5-HT, directly into the area postrema produced an incomplete emetic response. 6. These results confirm a role of 5-HT, and in particular 5-HT3 receptors, in the control of cisplatin-induced emesis, and show that at least one functional site for these receptors in modulating the emetic response is the area postrema, the locus of the chemoreceptor trigger zone.

GR113808: a novel, selective antagonist with high affinity at the 5-HT4 receptor.

1. The 5-HT4 receptor has only recently been identified but has yet to be cloned. This paper describes the pharmacology of a potent and selective 5-HT4 receptor antagonist, GR113808, which will be useful in the further characterization of this receptor. 2. On the guinea-pig ascending colon, GR113808 (1 nM-0.1 microM) behaved as an antagonist of 5-hydroxytryptamine (5-HT)-induced contraction, producing rightward displacements of the concentration-effect curve to 5-HT and a concentration-related depression of the maximum effect. However, the compound had no effect on cholecystokinin (CCK-8)-induced contraction in concentrations up to 1 microM. 3. In the guinea-pig colon preparation, onset and offset of the antagonism by GR113808 of 5-HT-induced contraction was examined. Incubation of the tissues for either 15 min, 30 min or 60 min produced similar rightward displacements of the concentration-effect curves to 5-HT, with no increase in the degree of depression of the maxima with increasing time of incubation. Experiments examining offset of antagonism (0.01 microM) demonstrated that washout for 30 min was required to reverse fully the effects of the antagonist. 4. Potency estimates in the colon for GR113808 were made by determining approximate pA2 values (30 min) using the Gaddum equation. The values obtained were 9.2, 9.7 and 9.2 when tested against the agonists 5-HT, 5-methoxytryptamine and R,S-zacopride respectively. 5. On the carbachol-contracted tunica muscularis mucosae preparation of the rat thoracic oesophagus, GR113808 behaved as an antagonist of 5-HT-induced relaxation, producing no reduction in maximum response. Analysis of these data yielded a pA2 of 9.3. GR1 13808 also antagonised the relaxant effects of 5-methoxytryptamine (pA2 = 9.0) and R,S-zacopride (pA2 = 9.4). The compound had no effect on isoprenaline-induced relaxation of the carbachol-contracted oesophagus at a concentration of 1 MicroM.6. In tests of selectivity, GR113808 had only low affinity for 5-HT3 receptors (pKi = 6.0) and had no functional activity at either 5-HT2 or 5-HT1-like receptors on vascular smooth muscle preparations. In a range of binding assays, GRi 13808 was shown to have no appreciable affinity for any other receptor type investigated.7. In the anaesthetized piglet, GRI13808 was a potent antagonist of 5-methoxytryptamine-induced tachycardia (mean DRo = 97.2 microg kg-1 h-1). The compound was ineffective against isoprenaline-induced tachycardia.8. The present results are discussed in comparison with those for existing antagonists at the 5-HT4receptor. The results of this study indicate that GRI13808 will be a valuable antagonist for studying 5-HT4 receptor mechanisms in vitro and in vivo and validate its use as a radioligand for determining 5-HT4 receptor distribution.

The pharmacological characterization of 5-HT3 receptors in three isolated preparations derived from guinea-pig tissues.

1. The pharmacological characterization of the 5-HT3 receptors in guinea-pig isolated tissues is described. The tissues used were ileum (longitudinal muscle-myenteric plexus), colon and vagus nerve. The guinea-pig isolated colon is a novel preparation. 2. In the guinea-pig isolated ileum, 5-hydroxytryptamine (5-HT, 1 x 10(-8)-3 x 10(-5) M) and the selective 5-HT3 receptor agonist 2-methyl-5-HT (3 x 10(-7)-1 x 10(-4) M) caused concentration-related contractions. The 5-HT concentration-response curve was biphasic whilst the 2-methyl-5-HT curve was monophasic. The EC50 value for the low potency portion of the 5-HT curve was 4.1 x 10(-6) M. The EC50 for 2-methyl-5-HT was 1.23 x 10(-5) M. Selective 5-HT3 receptor antagonists caused rightward shifts of the 2-methyl-5-HT curve and the lower potency portion of the 5-HT curve. Neither ketanserin (1 x 10(-6) M) nor methysergide (1 x 10(-5) M) antagonized the responses to 5-HT or 2-methyl-5-HT. 3. In the guinea-pig isolated colon, 5-HT (3 x 10(-7)-3 x 10(-5) M; EC50 2.4 x 10(-6) M) caused contractions which were mimicked by 2-methyl-5-HT (1 x 10(-6)-1 x 10(-4) M; EC50 7.2 x 10(-6) M). Selective 5-HT3 receptor antagonists caused rightward displacements of the 5-HT concentration-response curves. Neither ketanserin (1 x 10-6 M) nor methysergide (1 x 10- 5M) had any effect on responses to 5-HT or 2-methyl- 5-HT. 4. In the guinea-pig isolated vagus nerve, 5-HT (1 x 10-6-3 x 1O-4M) and 2-methyl-5-HT (1 x i0-S- 1 X 10-3m; EC50 7.6 x 10- M) caused depolarizations; at higher concentrations there were afterhyperpolarizations. The maximum response to 2-methyl-5-HT was less than half that to 5-HT. Selective 5-HT3 receptor antagonists caused rightward displacements of the 5-HT concentration-response curves. Antagonists at other 5-HT receptors (ketanserin, 1 x 10- M and methysergide, 1 x 10-6 M) had no effect. 5. The estimated affinity values of 5-HT3 receptor antagonists correlated well between the three models. Phenylbiguanide was inactive as an agonist or antagonist (up to 1 x 1O-4M) in each preparation. 6. Comparisons with antagonist affinity values obtained in the rat isolated vagus nerve revealed marked differences. Antagonists were generally more potent on the rat isolated vagus nerve, although the differences varied considerably between antagonists. 7. The results are discussed in terms of species-related receptor differences.

Identification of putative 5-HT4 receptors in guinea-pig ascending colon.

Experiments were carried out to characterise pharmacologically a neuronal, non-5-HT3, 5-hydroxytryptamine (5-HT) receptor in guinea-pig isolated ascending colon. In preparations pretreated with methysergide (1 microM) and ondansetron (10 microM), 5-HT (0.003-1 microM) produced repeatable concentration-related contractions of guinea-pig ascending colon with an EC50 value of 29 (20-41) nM. The responses to 5-HT could be antagonised substantially by tetrodotoxin (0.3 microM) and atropine (1 microM) indicating a neuronal cholinergically mediated effect. The 5-HT-induced response was mimicked by 5-methoxytryptamine and alpha-methyl-5-HT with equipotent concentration ratios of 26 and 28, respectively. In contrast, 2-methyl-5-HT, 8-OH-DPAT, sumatriptan, phenylbiguanide and 5-hydroxyindalpine had no agonist activity up to 10 microM. The benzamides, metoclopramide, cisapride, R,S-zacopride and renzapride, mimicked the contractile action of 5-HT, acting like partial agonists. ICS205-930 (3 microM) acted as a competitive antagonist against 5-HT and 5-methoxytryptamine with estimated pKB values of 6.4 and 6.7 respectively. ICS205-930 (1 microM) also antagonised responses to R,S-zacopride and renzapride. Ketanserin (1 microM), phenylbiguanide (10 microM) and sulpiride (1 microM) had no effect on responses to 5-HT. We conclude that the pharmacological characteristics of the receptor, which mediates contraction of guinea-pig ascending colon by activation of cholinergic nerves, are consistent with it being of the putative 5-HT4 receptor type.