1 Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2 In detrusor strips preincubated with [ 3 H]-choline, electrical ®eld stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium out¯ow and a contractile response. In the presence of 10 mM paraoxon (to prevent ACh degradation) the tritium out¯ow was characterized by HPLC analysis as [ 3 H]-ACh (76%) and [ 3 H]-choline (24%).3 Electrically-evoked [ 3 H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and una ected by hexamethonium (10 mM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM ± 1 mM), and enhanced by atropine (0.1 ± 100 nM). These ®ndings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. 4 The e ects of various subtype-preferring muscarinic receptor antagonists were evaluated on [ 3 H]-ACh release and muscle contraction. The rank potency (7log EC 50 ) orders at pre-and postjunctional level were: atropine 54-diphenyl-acetoxy-N-piperidine (4-DAMP)4mamba toxin 3 (MT-3)4tripitramine4para-¯uorohexahydrosiladiphenidol (pF-HHSiD)5methoctramine5pirenze-pine4tripinamide, and atropine54-DAMP4pF-HHSiD> >pirenzepine=tripitramine4tripinami-de4methoctramine> >MT-3, respectively. 5 The comparison of pre-and post-junctional potencies and the relationship analysis with the a nity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M 4 receptor, while the receptor involved in muscular contraction belongs to the M 3 subtype.

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.

Clinical depression is viewed as a physical and psychic disease process having a neuropathological basis, although a clear understanding of its ethiopathology is still missing. The observation that depressive symptoms are influenced by pharmacological manipulation of monoamines led to the hypothesis that depression results from reduced availability or functional deficiency of monoaminergic transmitters in some cerebral regions. However, there are limitations to current monoamine theories related to mood disorders. Recently, a growing body of experimental data has showed that other classes of endogenous compounds, such as neuropeptides and amino acids, may play a significant role in the pathophysiology of affective disorders. With the development of neuroscience, neuronal networks and intracellular pathways have been identified and characterized, describing the existence of the interaction between monoamines and receptors in turn able to modulate the expression of intracellular proteins and neurotrophic factors, suggesting that depression/antidepressants may be intermingled with neurogenesis/neurodegenerative processes.

1 In isolated detrusor strips from the guinea-pig urinary bladder, contractile responses to electrical field stimulation were mostly mediated by neurally released acetylcholine (ACh) and adenosine 5'-triphosphate (ATP). 2 5-Hydroxytryptamine (5-HT) produced a concentration-dependent increase in the amplitude of stimulated detrusor strip contractions. The 5-HT concentration-response curve showed a biphasic profile: the high potency phase was obtained at sub-micromolar concentrations (10-300 nM), while the low potency phase in the range 1-30 gM. The maximum response of the first phase was 30% of the total 5-HT response. 3 Like 5-HT, the 5-HT3 receptor agonist, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: 0.3-100 Mm), the 5-HT2 receptor agonist, (±)-l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI: 30 nM-3 gM) and the 5-HT4 receptor agonist, 5-methoxytryptamine (5-MeOT: 0.1-30 FM) potentiated, though with lower potency, detrusor contractions. The resulting concentration-response curves were monophasic in nature. 2-Methyl-5-HT had a maximum effect comparable to that of 5-HT. By contrast, the maximal effects of DOI and 5-MeOT were only 20% and 30% of that elicited by 30 gM 5-HT, respectively. 4 The 5-HT3 receptor antagonist, granisetron (0.3 gM) had no effect on the high potency phase, but caused a rightward parallel shift of the low potency phase of the 5-HT curve (pKB = 7.3). Granisetron 6 In a separate set of experiments, the interactions of 5-HT with either the purinergic or cholinergic components of excitatory neuromuscular transmission were investigated. In the presence of hyoscine (1 gM), 5-HT was mostly effective at sub-micromolar concentrations, while in the presence of the P2-purinoceptor antagonist, suramin (300 gM), 5-HT-induced potentiation was mainly obtained with micromolar concentrations. 7 Thus, in electrically stimulated detrusor strips from guinea-pig, 5-HT potentiated excitatory neuromuscular transmission by activating at least three separate neural 5-HT receptors. These include the 5-HT2A and 5-HT4 receptors, which mediate the 5-HT high potency phase mainly by activation of purinergic transmission. On the other hand, the potentiating effect caused by micromolar concentrations of 5-HT mostly involves cholinergic transmission and is mediated by the 5-HT3 receptors.

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.