Agomelatine (S20098) displayed pK i values of 6.4 and 6.2 at native (porcine) and cloned, human (h)5-hydroxytryptamine (5-HT) 2C receptors, respectively. It also interacted with h5-HT 2B receptors (6.6), whereas it showed low affinity at native (rat)/cloned, human 5-HT 2A (Ͻ5.0/5.3) and 5-HT 1A (Ͻ5.0/5.2) receptors, and negligible (Ͻ5.0) affinity for other 5-HT receptors. In antibody capture/ scintillation proximity assays, agomelatine concentration dependently and competitively abolished h5-HT 2C receptor-mediated activation of Gq/11 and Gi 3 (pA 2 values of 6.0 and 6.1). As measured by [ 3 H]phosphatidylinositol depletion, agomelatine abolished activation of phospholipase C by h5-HT 2C (pK B value of 6.1) and h5-HT 2B (pK B value of 6.6) receptors. In vivo, it dose dependently blocked induction of penile erections by the 5-HT 2C agonists (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (Ro60,0175) and 1-methyl-2-(5,8,8-trimethyl-8H-3-aza-cyclopenta[a]inden-3-yl) ethylamine (Ro60,0332). Furthermore, agomelatine dose dependently enhanced dialysis levels of dopamine in frontal cortex of freely moving rats, whereas they were unaffected in nucleus accumbens and striatum. Although the electrical activity of ventrotegmental dopaminergic neurons was unaffected by agomelatine, it abolished their inhibition by Ro60,0175. Extracellular levels of noradrenaline in frontal cortex were also dose dependently enhanced by agomelatine in parallel with an acceleration in the firing rate of adrenergic cell bodies in the locus coeruleus. These increases in noradrenaline and dopamine levels were unaffected by the selective melatonin antagonist N- [2-(5-ethyl-benzo[b]thien-3-yl)ethyl] acetamide (S22153) and likely reflect blockade of 5-HT 2C receptors inhibitory to frontocortical dopaminergic and adrenergic pathways. Correspondingly, in distinction to agomelatine, melatonin showed negligible activity at 5-HT 2C receptors and failed to modify the activity of adrenergic and dopaminergic pathways. In conclusion, in contrast to melatonin, agomelatine behaves as an antagonist at 5-HT 2B and 5-HT 2C receptors: blockade of the latter reinforces frontocortical adrenergic and dopaminergic transmission.

The mitogen-activated protein kinase (MAPK) cascade is stimulated by both receptor tyrosine kinases and G protein-coupled receptors. We show that recombinant human dopamine D 3 receptors expressed in Chinese hamster ovary cells transiently activate MAPK via pertussis toxin-sensitive Gi and/or Go proteins. The involvement of D 3 receptors was confirmed by use of the D 3 agonists PD 128,907 and (ϩ)-7-hydroxy-2-dipropylaminotetralin, which mimicked the response to dopamine (DA). Furthermore, haloperidol and the selective D 3 receptor antagonists S 14297 and GR 218,231 attenuated DA-induced MAPK activation; however, when tested alone, S 14297 weakly stimulated MAPK activity, suggesting partial agonist activity. The transduction mechanisms by which hD 3 receptors activate MAPK were explored with specific kinase inhibitors. Genistein and lavendustin A, inhibitors of tyrosine kinase activity, did not reduce DA-induced MAPK activation. In contrast, PD 98059, an inhibitor of MAPK kinase, and Ro 31-8220 and Gö 6983, inhibitors of protein kinase C (PKC), blocked DA-induced MAPK activation. However, MAPK activation was insensitive to PKC down-regulation by phorbol esters, indicating the involvement of an "atypical" PKC. Furthermore, MAPK activation involved phosphatidylinositol 3-kinase inasmuch as its inhibition by LY 294002 and wortmannin reduced DA-induced MAPK activation. In conclusion, this study demonstrates that stimulation of hD 3 receptors activates MAPK. This action is mediated via an atypical isoform of PKC, possibly involving cross-talk with products of phosphatidylinositol 3-kinase activation.Extracellular signal-regulated kinases ERK1 and ERK2, also known as mitogen-activated protein kinases (MAPK), are involved in the control of cell growth and differentiation by growth factors (Schlessinger and Ullrich, 1992). Recently, various G protein-coupled receptors (GPCRs) have also been shown to stimulate MAPK, although activation cascades differ according to receptor subtype and G protein family (Lopez-Ilasaca, 1998).Intracellular mechanisms leading to MAPK activation by growth factor receptors are now well defined, and mainly involve Ras GTP-binding protein activation via SH2 and SH3 domain adaptor proteins and subsequent Raf/MAPK kinase activation (Schlessinger and Ullrich, 1992;Pawson, 1995). In contrast, the nature of G protein coupling to MAPK activation is less well characterized, although both ␣ and ␤␥ subunits of G proteins are implicated. It has, thus, been shown that G␤␥ subunits stimulate Ras protein via Src-like proteins, possibly involving other intermediates, such as phosphatidylinositol 3-kinase (PI 3-kinase), and subsequent tyrosine phosphorylation of Shc and recruitment of the Grb2-Sos complex (Faure et al., 1994Hawes et al., 1995;Igishi and Gutkind, 1998). As concerns ␣o and ␣q subunits, their potential involvement in MAPK activation remains unclear, but may involve protein kinase C (PKC) and Pyk2 activation, respectively Dikic and al., 1996;Igishi et al., 1998;Berts et al., 1999).The dopam...

As determined by a guanosine 5Ј-O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding assay, which does not distinguish G protein subtypes, 5-hydroxytryptamine (5-HT) and 2(S)-1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) behaved as full agonists at human 5-HT 2C (h5-HT 2C ) receptors (VSV isoform) stably expressed in Chinese hamster ovary (CHO) cells, whereas 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), d-lysergic acid diethylamide (LSD), and lisuride exhibited partial agonist properties. After treatment with pertussis toxin to uncouple 5-HT 2C receptors from Gi/Go but not Gq/11, DOI and LSD were as efficacious as 5-HT and Ro600175 in stimulating [35 S]GTP␥S binding, whereas lisuride still exhibited low efficacy (40%). Correspondingly, in a scintillation proximity assay employing specific antibodies against Gq/11, 5-HT, Ro600175, DOI, and LSD behaved as high-efficacy agonists, whereas lisuride showed efficacy of 36%. In contrast, when employing a specific antibody recognizing Gi 3 , DOI and LSD were less efficacious (80 and 30%, respectively) than 5-HT and Ro600175, and lisuride was inactive. Agonist actions were specifically mediated by h5-HT 2C receptors inasmuch as the selective 5-HT 2C antagonist SB242,084 blocked [35 S]GTP␥S binding at both Gq/11 and Gi 3 . Agonist potency for stimulation of Gi 3 was ϳ6-to 8-fold less than for Gq/11, indicating that the latter was preferentially engaged by h5-HT 2C receptors. Inactivation of h5-HT 2C receptors with the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline did not modify the efficacy of 5-HT, Ro600175, and DOI at Gq/11, whereas their efficacies were substantially reduced at Gi 3 , indicating a greater receptor reserve for the former. Finally, the preferential activation of Gq/11 versus Gi 3 by DOI, LSD, and lisuride was diminished in the presence of lower receptor number. In conclusion, h5-HT 2C receptors couple to both Gq/11 and Gi 3 in CHO cells, and efficacy for G protein subtype activation is both ligand-and receptor reserve-dependent.