1 The interaction of melatonin (N-acetyl-5-methoxytryptamine) with 5-hydroxytryptamine 4 (5-HT 4 ) receptors and/or with melatonin receptors (ML 1 , ML 2 sites) has been assessed in isolated strips of the guinea-pig proximal colon. In the same preparation, the pharmacological pro®le of a series of melatonin agonists (2-iodomelatonin, 6-chloromelatonin, N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT), 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT)) was investigated. 2 In the presence of 5-HT 1/2/3 receptor blockade with methysergide (1 mM) and ondansetron (10 mM), melatonin (0.1 nM ± 10 mM), 5-HT (1 nM ± 1 mM) and the 5-HT 4 receptor agonist, 5-methoxytryptamine (5-MeOT: 1 nM ± 1 mM) caused concentration-dependent contractile responses. 5-HT and 5-MeOT acted as full agonists with a potency (7log EC 50 ) of 7.8 and 8.0, respectively. The potency value for melatonin was 8.7, but its maximum e ect was only 58% of that elicited by 5-HT. 3 Melatonin responses were resistant to atropine (0.1 mM), tetrodotoxin (0.3 mM), and to blockade of 5-HT 4 receptors by SDZ 205,557 (0.3 mM) and GR 125487 (3, 30 and 300 nM). The latter antagonist (3 nM) inhibited 5-HT-induced contractions with an apparent pA 2 value of 9.6. GR 125487 antagonism was associated with 30% reduction of the 5-HT response maximum. Contractions elicited by 5-HT were not modi®ed when melatonin (1 and 10 nM) was used as an antagonist. 4 Like melatonin, the four melatonin analogues concentration-dependently contracted colonic strips. The rank order of agonist potency was: 2-iodomelatonin (10.8) 46-chloromelatonin (9.9) 5 N-acetyl-5-HT (9.8) 55-MCA-NAT (9.6) 4melatonin (8.7), an order typical for ML 2 sites. In comparison with the other agonists, 5-MCA-NAT had the highest intrinsic activity. 5 The melatonin ML 1B receptor antagonist luzindole (0.3, 1 and 3 mM) had no e ect on the concentration-response curve to melatonin. Prazosin, an a-adrenoceptor antagonist possessing moderate/ high a nity for melatonin ML 2 sites did not a ect melatonin-induced contractions at 0.1 mM. Higher prazosin concentrations (0.3 and 1 mM) caused a non-concentration-dependent depression of the maximal response to melatonin without changing its potency. Prazosin (0.1 and 1 mM) showed a similar depressant behaviour towards the contractile responses to 5-MCA-NAT. 6 In the guinea-pig proximal colon, melatonin despite some structural similarity with the 5-HT 4 receptor agonist 5-MeOT, does not interact with 5-HT 4 receptors (or with 5-HT 1/2/3 receptors). As indicated by the rank order of agonist potencies and by the ine cacy of luzindole, the most likely sites of action of melatonin are postjunctional ML 2 receptors. However, this assumption could not be corroborated with the use of prazosin as this`ML 2 receptor antagonist' showed only a nonconcentration-dependent depression of the maximal contractile response to both melatonin and 5-MCA-NAT. Further investigation with the use of truly selective antagonists at melatonin ML 2 receptors is required to clarify this issue.

In this study the functional interaction of the antidepressant drugs amitriptyline, mianserin, maprotiline, imipramine, fluoxetine and the putative antidepressant drug flibanserin has been studied on 5-HT7-mediated responses to 5-carboxamidotryptamine (5-CT) in the guinea-pig ileum. 5-CT induced a concentration-dependent inhibition of the contractile response to substance P (100 nM). Except for fluoxetine and flibanserin, all the antidepressants antagonized by different degrees the 5-CT inhibitory response with the following rank affinity order: mianserin > maprotiline > imipramine > amitriptyline. Mianserin was the only antidepressant to show a profile of competitive antagonism at 5-HT7 receptors in a tenfold range of concentrations (0.1-1 microM), with an affinity (pA2) value of 8.1 +/- 0.6. The antagonism of the other antidepressants was not concentration-dependent (amitriptyline) or was associated with slight or moderate reduction of the maximal 5-CT response (imipramine or maprotiline). The apparent affinity (pKB) values were: amitriptyline, 7.0 +/- 0.2; maprotiline, 7.3 +/- 0.6; imipramine, 7.2 +/- 0.4. Our results show that various antidepressant drugs belonging to different chemical classes behave as antagonists at enteric 5-HT7 receptors through competitive or allosteric mechanisms. This evidence extends our previous findings demonstrating the interaction of antidepressants with other 5-HT receptors, namely 5-HT3 and 5-HT4 receptors.

1 A combined study of receptor binding in central neuronal cell membranes and functional responses in isolated segments of guinea-pig small intestine allowed characterization of the interaction of four antidepressant drugs with central and peripheral 5-HT3 and 5-HT4 receptors. 3 In whole ileal segments, concentration-response curves to 5-HT were biphasic, with the high-and low-potency phases involving 5-HT4 and 5-HT3 receptors, respectively. Curves to 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: a 5-HT3 receptor agonist) and 5-methoxytryptamine (5-MeOT: a 5-HT4 receptor agonist) were monophasic. All antidepressants were used at concentrations lacking anticholinoceptor properties, as demonstrated in both electrically stimulated longitudinal musclemyenteric plexus preparations (LMMPs) and in unstimulated LMMPs following addition of acetylcholine (100 nM). 4 Fluoxetine (0.1-1 JM) and litoxetine (0.3-3JM) antagonized both the high-and low-potency phases of the 5-HT curve. Schild analysis for the low-potency phase yielded pA2 estimates of 6.6 ± 0.3 (Schild slope of 1.1) and of 6.6 ± 0.1 (Schild slope of 1.1), respectively. At higher concentrations (3 JM), fluoxetine markedly inhibited the 5-HT response maximum. Clomipramine (10-300 nM) inhibited, by a mechanism independent of concentration, both phases of the 5-HT curve with a reduction of the maximum response. Paroxetine (1 JM) was ineffective on the high-potency phase, but caused a rightward shift of the low-potency phase (pKB: 6.1 ± 0.01).5 Responses to 2-methyl-5-HT were inhibited by 1 JAM fluoxetine (pKB: 5.4 ± 0.02). Like clomipramine (30 and 100 nM), litoxetine (1 and 3 JAM) produced rightward displacements of 2-methyl-5-HT-induced contractions, which were virtually independent of antidepressant concentration (pKB values: 6.0 ± 0.02 and 5.5 + 0.01, respectively). At higher concentrations, fluoxetine (3 JM) and clomipramine (300 nM) markedly reduced the 2-methyl-5-HT response maximum. Paroxetine (1 JAM) was ineffective.6 Responses to 5-MeOT were shifted to the right by fluoxetine (0.1-1 JAM) and litoxetine (1 and 3fJM) in a concentration-dependent manner. At higher concentrations, fluoxetine (3 JlM) markedly reduced the 5-MeOT response maximum, an effect also observed with 100 and 300 nM clomipramine. Paroxetine (1 JM) was ineffective.7 In unstimulated LMMPs, the excitatory effects evoked by 5-HT, 2-methyl-5-HT and 5-MeOT and the antagonism produced by 300 nM clomipramine were comparable to those obtained in whole ileal segments. This suggests that 5-HT contained in the mucosa of whole preparations does not interfere with agonist-induced contractile responses and with the inhibitory effect of antidepressant drugs.

1 Experiments were carried out to characterize the receptors mediating the indirect excitatory response to 5-hydroxytryptamine (5-HT) in the guinea-pig isolated trachea. 2 5-HT caused concentration-dependent contractions of tracheal strips, and the resulting concentration-response curve was biphasic in nature. The first phase was obtained with agonist concentrations in the range of 0.01-3 nM and achieved a maximum which was 30% of the total 5-HT response, while the second phase was in the range 1O nM-L M. 3 Atropine (0.1I M) and tetrodotoxin (TTX: 0.3 LM) significantly reduced both phases of the 5-HT curve. Morphine (10 gIM), which can act to inhibit neuronal acetylcholine release, abolished the first phase and reduced the second phase. This suggests that the first phase is mainly neurogenic (cholinergic) in nature, while the second phase is in part neurogenic and in part due to direct activation of the effector 6 Our results suggest that neural 5-HT2A and, to a lesser extent, 5-HT3 receptor subtypes mediate the first phase of the 5-HT curve in the guinea-pig trachea. The second phase is mediated by 5-HT2A receptors, which are probably located at both the neural and muscular level. No evidence for the participation of 5-HT, receptors in the 5-HT response has been obtained.