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The objective of this study was to explore the role of 5-HT3 receptors in modulating potassium (K+)-evoked release of [3H]-acetylcholine ([3H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [3H]-choline. Rat entorhinal cortices were cross-chopped into 300 microns slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/l Ca2+ and stimulated with two consecutive exposures of 20 mmol/l K+ for 4 min (S1 and S2, respectively). Compounds were added 20 min before S2 stimulation and remained in the superfusion buffer for the duration of the experiment. The S2/S1 ratio was then calculated. Stimulated release of [3H]-ACh was dependent on extracellular Ca2+ and K+ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10(-9)-10(-6) mol/l), in the presence of 1 mumol/l ritanserin or 1 mumol/l ondansetron, had no influence on K(+)-evoked release of [3H]-ACh. In slices prepared from Hooded Lister rats, 2 mumol/l 5-HT but not 2-Me-5-HT significantly (P0.05) inhibited K(+)-evoked [3H]-ACh release only 17% in the presence of 1 mumol/l ritanserin. However, 2 mumol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K(+)-evoked ACh release was observed with either 5-HT3 receptor agonists or antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT4 receptors in isolated distal colon myenteric plexus of guinea-pig, mediating contraction of longitudinal smooth muscle, have been further characterized by selective agonists and antagonists. The indole agonists, 5-HT and 5-methoxytryptamine (5-MeOT), were full agonists (relative to 5-HT) with potency values (pEC50) of 8.0 +/- 0.1 (n = 50) and 7.8 +/- 0.1 (n = 12), respectively. 5-HT4 receptor agonists of other structural classes, including benzimidazolones (BIMU 1 and BIMU 8), and benzamides ((S)-zacopride, (R)-zacopride, renzapride, SC 49518) were partial agonists with intrinsic activities less than that of 5-HT. In general, the potencies for these compounds at 5-HT4 receptors in guinea-pig colon were similar to the potencies seen in the rat isolated oesophagus, where 5-HT4 receptors mediate relaxation. GR 113808 ¿[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl] methyl1-methyl-1H-indole-3-carboxylate¿, RS 39604 ¿1-[4-amino-5-chloro-2-(3, 5-dimethoxybenzyloxy)phenyl]-3[1-[2-[(methylsulfonyl)amino] ethyl]-4-piperidinyl]-1-propanone hydrochloride and SB 204070 ¿(1-n-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1, 4-benzodioxane-5-carboxylate¿ antagonized 5-HT responses with pA2 values of 9.1 +/- 0.1, 9.0 +/- 0.2 and 11.0 +/- 0.1, respectively. These affinity values were similar to those obtained at 5-HT4 receptors in isolated rat oesophagus (9.0+/- 0.4, 9.3 +/- 0.1 and 10.6 +/- 0.1 respectively). Despite these operational similarities between 5-HT4 receptors in guinea-pig colon and rat oesophagus, several novel compounds have revealed important differences between 5-HT4 receptors in the two tissues. For example, the substituted benzoate, RS 23597 ¿3-(piperidine-1-yl) propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride, acted as a partial agonist (intrinsic activity 0.5) in guinea-pig colon with a potency of 7.6 +/-0.1 (n = 16). In isolated rat oesophagus, however, this compound was a surmountable antagonist (pA2 = 7.8 +/- 0.1) with no intrinsic activity. In contrast, the substituted naphthalimide (S)RS 56532 ¿(S)-6-amino-5-chloro-2-(1-azabicyclo[2, 2, 2]octan-3-yl) 2,3-dihydro-1H-benz[de] isoquinoline-1,3-dione hydrochloride¿, was a potent (pEC50 = 7.9 +/- 0.1), efficacious partial agonist (intrinsic activity = 0.8) in the rat oesophagus. However, in guinea-pig colon, it was a surmountable antagonist with an affinity (pKB) of 9.4 +/- 0.1. Furthermore, several novel, selective, 5-HT4 compounds also showed opposing patterns of intrinsic activities similar to those described for RS 23597 and (S)RS 56532. It is concluded that these differences are inconsistent with differences in 5-HT4 receptor reserves, and may suggest that 5-HT4 receptors in the guinea-pig colon and the rat oesophagus can be operationally distinguished.

Previous reports on a series of benzoylthiophenes, including PD 81,723 [2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl) thiophene], have shown specific enhancement of agonist binding at the adenosine A1 receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 (thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester) and RS-74513-000 [2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene] on response elicited by adenosine A1 receptors in isolated guinea pig left atrium and ileum. In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA [N6-cyclopentyladenosine] with a pKB value of 6.2 +/- 0.2 (n = 4). At a low concentration which had no antagonistic effect (0.1 microM), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4-11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 microM) did not enhance or antagonize effects of CPA. At concentrations above 1 microM, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A1 antagonist CPX [8-cyclopentyl-1,3-dipropyl-xanthine] (1 microM). Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 microM) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

It is well documented that certain substituted benzamides, such as cisapride, and benzimidazolones, such as BIMU 8, enhance gastric emptying in rats. As these compounds possess 5-HT3 antagonistic and 5-HT4 agonistic properties, the precise mechanisms (5-HT3 or 5-HT4) underlying their gastroprokinesic effects is still unclear. In the present study, we used SC 49518 (a benzamide and selective 5-HT4 receptor agonist) and two selective 5-HT4 receptor antagonists (RS 23597-190 and SB 204070) to elucidate the role of 5-HT4 receptors in gastroprokinesis. SC 49518 (1–316 μg/kg; ip) produced significant and dose-dependent stimulation of gastric emptying in rats (ED50 = 2.3 μg/kg; ip). SC 49518 also produced dose-dependent inhibition of bradycardia induced by 2-methyl 5-HT (von Bezold-Jarisch reflex) but with a 156 fold lower potency (ID50 = 0.36 mg/kg; ip). The gastroprokinetic effects of SC 49518 (3–316 μg/kg; ip) were significantly antagonized by the selective 5-HT4 receptor antagonist RS 23597-190 (0.1 mg/kg/min; iv). SB 204070 (0.003-1 mg/kg; ip), another selective 5-HT4 receptor antagonist, produced dose-dependent inhibition of the gastroprokinesic effects of SC 49518 (10 μg/kg; ip), the inhibition attaining statistical significance at the dose of 0.1 mg/kg; ip. RS 23597-190 had no effects on gastric emptying per se whereas SB 204070 significantly increased gastric emptying by itself at 1 mg/kg; ip but not at 0.1 mg/kg; ip. These findings show, for the first time, that SC 49518, a selective 5-HT4 receptor agonist, produces potent stimulation of gastric emptying in rats via a mechanism involving activation of 5-HT4 receptors. It is suggested that a similar mechanism may account for the gastroprokinetic effects of other non-selective benzamides and benzimidazolones.

The action of the cervane alkaloid, imperialine, has been assessed at M1, M2 and M3 receptors in functional assays and at M1, M2, M3 and putative M4 sites in binding studies. In functional studies, imperialine acted as a selective surmountable antagonist at M2 receptors in guinea-pig isolated atria and uterus (-log KB = 7.7 and 7.4, respectively), in comparison to M1 receptors in canine isolated saphenous vein (-log KB = 6.9) or M3 receptors in a range of guinea-pig isolated smooth muscles including ileum, trachea, fundus, seminal vesicle or oesophagus (-log KB = 6.6-6.8). In rat aorta, the -log KB value at the M3 receptor (5.9) was slightly, but significantly, lower. In competition radioligand binding studies, imperialine was also selective toward to M2 sites in rat myocardium (-log Ki = 7.2) with respect to M1 and M3 sites (rat cerebral cortex, rat submaxillary gland; -log Ki = 6.1 and 5.7, respectively). However, it did not significantly discriminate between rat cardiac M2 sites and putative M4 sites in rabbit lung (-log Ki = 6.9). Imperialine resembles the alkaloid himbacine in terms of its pharmacological profile at muscarinic receptor subtypes in that it acts as an M2 selective antagonist with respect to M1 or M3 sites. It may also provide a second, commercially available, antagonist with which to discriminate between M1 and M4 receptors.

1. The muscarinic pharmacology of a novel oxadiazole muscarinic agonist, (+/-) L-660,863, [+/-3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine] has been studied using pharmacological, radioligand binding and biochemical techniques, in vitro. 2. In isolated tissue experiments, (+/-)L-660,863 was a more potent agonist than carbachol in all preparations studied, being most potent at muscarinic receptors mediating negative chronotropy in guinea-pig right, spontaneously beating atria and least potent at receptors mediating contractions in canine saphenous vein and endothelial denuded rabbit aorta (-log EC50 values were 8.8, 6.6 and 6.3, respectively. The apparent affinities (-log KA) of (+/-)L-660,863) estimated by receptor inactivation, showed some selectivity toward the atrial M2 muscarinic receptor (-log KA = 7.6) in comparison to the M1 or M3 muscarinic receptors (-log KA = 5.4 and 6.2) respectively. This degree of selectivity was also observed in competition radioligand binding studies. 3. At M3 muscarinic receptors mediating inositol phosphates (IPs) accumulation in longitudinal muscle of guinea-pig ileum, the potency of (+/-)L-660,863 (-log EC50 value = 6.2) was similar to the apparent affinity calculated at M3 muscarinic receptors in the functional studies (see above). In contrast, at muscarinic receptors mediating IPs accumulation in guinea-pig atria and ventricles, the potency for (+/-)L-660,863 (-log EC50 = 6.2 and 6.4, respectively) was lower than the apparent affinity calculated at M2 muscarinic receptors from inotropic and binding studies in cardiac tissue (see above).(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptors mediating the release of epithelial derived relaxant factor (EpDRF) have been studied by using both contractions of the guinea-pig tracheal strip (with epithelium intact or denuded) or a coaxial bioassay assembly (rat anococcygeus-recipient; guinea-pig trachea-donor tissue). Indomethacin (1 microM/1) and physostigmine (0.1 microM/1) were both present throughout the study. In the tracheal strip studies, the potencies and maximal effects of all agonists studied (acetylcholine, arecoline, bethanechol, carbachol, (+)cis-dioxolane, ethoxyethyltrimethylammonium, L-660,863, (+/-)methacholine and OXA-22) were not affected or were only slightly (but significantly) reduced by removal of the epithelium. The -log KB for the muscarinic antagonists, atropine, pirenzepine, methoctramine and 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) were also not affected and the -log KB values were consistent with M3 muscarinic receptor function. However, the -log KB value of para-fluoro-hexahydro-siladifendol (p-F-HHSiD) was significantly (P less than 0.05) increased upon epithelial denudation (epithelium intact, 7.1; epithelium removed, 7.6). The coaxial bioassay assembly provided more convincing evidence for release of EpDRF in that all muscarinic agonists studied caused relaxations of a precontracted anococcygeus tissue. These relaxations were observed only in the presence of a tracheal tube possessing an intact epithelium. The rank order of potencies for agonists at receptors mediating EpDRF dependent relaxation were similar to those estimated at receptors causing contraction. These data suggested that a substantial receptor reserve was associated with the receptors mediating both EpDRF release and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

1. para-Fluoro-hexahydrosila-difenidol (p-FHHSiD) has been proposed as an M3 selective antagonist. However, the M3 selectivity is variable in that it exhibits a high pA2 value for M3 muscarinic receptors in guinea-pig ileum but a low value at muscarinic receptors in guinea-pig trachea. 2. The pA2 value in the trachea was found to be agonist independent since similar pA2 values were found when acetylcholine, carbachol, (+)-cis-dioxolane or OXA-22 were used (7.13, 7.03, 6.85 and 6.97, respectively). The pA2 value was not meaningfully increased when the equilibrium period was increased from 60 to 180 min. The pA2 value was unaffected by blockade of M1 or M2 receptors, using 0.1 μM pirenzepine or methoctramine (7.03 and 7.14, respectively). p-F-HHSiD and atropine appeared to act at the same site, as adjudged by combination concentration-ratio studies. 3. The pA2 values for p-F-HHSiD vary by 10 fold between ileal (8.0) and tracheal M3 receptors (7.0). The precise reason for this is unknown, but appears to be unrelated to conditions of disequilibrium that could be detected. The antagonist should therefore only be employed to distinguish M3 or M1 from M2 receptors. In this respect, although the M1/M3 VS M2 discrimination is relatively large (68 fold), p-F-HHSiD exhibits similar properties to other putative M3 selective antagonists such as 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) or the parent compound, hexahydrosiladifenidol (HHSiD).

Agonist-induced desensitization has been utilized to discriminate and independently “isolate” the neuronal excitatory receptors to 5-hydroxytryptamine (5-HT) in the guinea pig ileum (5-HT3 and putative 5-HT4 receptors). Electrically stimulated longitudinal muscle myenteric plexus preparations, and non-stimulated segments of whole ileum were used. Exposure to 5-methoxytryptamine (10 μmol/l) inhibited completely responses to 5-HT at the putative 5-HT4 receptor without affecting 5-HT3-mediated responses. Conversely, exposure to 2-methyl-5-HT (10 μmol/l) inhibited completely responses to 5-HT at the 5-HT3 receptor without affecting putative 5-HT4-mediated responses. The inhibition with 5-methoxytryptamine and 2-methyl-5-HT, either alone or in combination, appeared selective as responses to KCI, DMPP, carbachol, histamine, and substance P were unaffected or only very slightly modified. Furthermore, the pA2 values for ICS 205–930 at the putative 5-HT4 (pA2 = 6.2 to 6.5) and 5-HT3 (pA2 = 7.6 to 8.1) receptors (estimated in the presence of 2-methyl-5HT and 5-methoxytryptamine, respectively) were consistent with those estimated in the absence of desensitization. 5-Methoxytryptamine, but not 2-methyl-5-HT, suppressed completely but reversibly the concentration-effect curve to renzapride, suggesting that responses to this agent are mediated exclusively via agonism at the putative 5-HT4 receptor. It is concluded that 5-methoxytryptamine and 2-methyl-5-HT can be utilized as selective probes to discriminate the putative 5-HT4 receptor from the 5-HT3 receptor in guinea pig ileum. This finding is of importance as no selective antagonist exists for the putative 5-HT4 receptor. Furthermore, the presently described method of agonist-induced desensitization and 5-HT receptor discrimination may be useful for the identification and characterization of the putative 5-HT4 receptor in other tissues and species.