We investigated the eect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical ®eld stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or coincubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentrationdependently inhibited twitch responses (IC 50 73 nM), but had no additive eect with atropine or TTX. The cannabinoid CB 1 receptor antagonist SR 141716 (pA 2 8.2), but not the CB 2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction.These results provide functional evidence of the existence of prejunctional cannabinoid CB 1 -receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical ®eld stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB 1 receptors naturally expressed in the human gut.

A number of benzoates derived from 4-amino-5-chloro-2-methoxybenzoic acid and substituted 1-piperidineethanol were synthesized and found to be potent 5-HT4 receptor agonists in the electrically-stimulated myenteric plexus and longitudinal muscle of the guinea pig ileum and the rat esophagus muscle. Monosubstitution of the piperidine ring with Me, OH, NH-Ac, or CONH2 groups gave compounds equipotent to 7a (ML 10302), a 5-HT4 receptor agonist previously reported to have nanomolar affinity. 7a,k were as potent as serotonin (5-HT) but had maximal responses which were only 60-80% of that of 5-HT, suggesting a partial agonist profile for these compounds. Binding assays were performed with [3H]GR 113808 in the rat striatum, and several of these compounds were found to have nanomolar affinity for 5-HT4 receptors (7a, Ki = 1.07 +/- 0.5 nM; 7k, Ki = 1.0 +/- 0.3 nM). The introduction of two methyl groups on the piperidine ring brought about a dramatic change in the pharmacological profile of 2-[(cis- and trans-3,5-dimethylpiperidinyl)ethyl]-4-amino-5-chloro-2- methoxybenzoate, 7g,h. 7g (Ki = 0.26 +/- 0.06 nM) inhibited the relaxant action of 5-HT in the rat esophagus muscle with a pA2 value of 8.6. The advantage of the ester function was demonstrated by comparing the activity of several such compounds at 5-HT4 receptors with those of the corresponding amidic derivatives. This difference was less marked when the basic moiety was sterically constrained as in the quinuclidine and tropane moieties. Structural analyses of 7a,g were performed by determining their X-ray crystal structures and by molecular modeling (SYBYL). A relatively limited number of minimum energy conformers was found for both compounds. They were characterized by the cis folded conformation of the ethyl chain and by the orientation of the lone pair of the nitrogen atom pointing out of the molecule as seen in conformationally-constrained benzamides such as zacopride and renzapride. A hypothetical model for the 5-HT4 receptor with two sites for the binding of agonist and antagonist molecules was proposed.

1 Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose-dependently contracted by neurokinin A (EC 50 , 4.9 nM), the tachykinin NK 2 -receptor selective agonist (SR 144743) and N,N-demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [b-Ala 8 ]NKA (4 ± 10), with similar potency (Schild plot pA 2 values 9.4, 9.4 and 9.3, slope=1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept*9.3) and a low (X intercept 8.4, slope 1.6) a nity site. Co-incubation of SR 48968 (10, 100 nM) and SR 144782 (10 nM) produced additive e ects; in this experimental condition, SR 48968 apparent a nity (pK B ) was 8.2. In addition, SR 144782 (0.1 mM) antagonized responses to neurokinin A, substance P and neurokinin B, with pK B consistent with its a nity for tachykinin NK 2 -receptors. The potent and selective NK 1 and NK 3 -receptor antagonists, SR 140333 and SR 142801 (both 0.1 mM), failed to inhibit contractions induced by SP or NKB. 4 In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non-neuronal tachykinin NK 2 -receptors, but not tachykinin NK 1 -or NK 3 -receptors. Our ®ndings with SR 48968 suggest the existence of two tachykinin NK 2 -receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric e ects at the tachykinin NK 2 -receptor might well account for the complexity of the observed interaction.

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Background and aims-The subtype and species related heterogeneity of adrenoceptors prompted a functional reappraisal of these molecular targets of motility inhibition in the human colon. -6 mol/l) had no eVect on taenia relaxation by SR 58611A and rendered isoprenaline and terbutaline virtually inactive on circular strips, although not on taenia, which was relaxed at higher than control concentrations and maximally by isoprenaline. Propranolol, a 1 , 2 non-selective antagonist, at high concentrations (10 -5 mol/l) prevented taenia relaxation by CGP 12177 and SR 58611A; its quantitative antagonism of isoprenaline (in common with that of CGP 12177 used as an antagonist) was competitive in circular strips but not on taenia. Conclusions-1 , 2 , and 3 adrenoceptors are functionally detectable in the human colon; agonist stimulation of any one type relaxed taenia but only isoprenaline was fully eVective at the 3 subtype. (Gut 2000;47:337-342) Methods-Relaxation