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The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.

The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at the h5-HT1B receptor and only L694247 at the h5-HT1D receptor. GR127935 (10 microM) exerted little effect on [35S]GTP gamma S binding via h5-HT1B receptors (10% stimulation), but potently (pA2: 9.11) antagonized h5-HT1B receptor-stimulated [35S]GTP gamma S binding. Ketanserin and methiothepin inhibited [35S]GTP gamma S binding (by 13-28%) in the absence of an agonist, but were potent and competitive antagonists in the presence of an agonist via h5-HT1B (methiothepin) and h5-HT1D (methiothepin and ketanserin) receptors. The results document the utility of using [35S]GTP gamma S binding studies to assess agonist efficacy, and to characterize 5-HT1B/D receptor ligands as apparently neutral antagonists and inverse agonists at the G-protein level.

The functional activity of various 5-HT receptor agonists, including 5-CT, sumatriptan, CP 93, 129 and 1-naphtylpiperazine, and of drugs known to bind with high affinity to 5-HT1B (pindolol, propranolol, cyanopindolol, SDZ 21,009 and isamoltane) or 5-HT1D binding sites (yohimbine and rauwolscine) was measured at 5-HT receptors that are negatively coupled to adenylate cyclase in cultures of the renal epithelial cell line OK. 5-HT receptor-mediated inhibition of adenylate cyclase was studied by measuring inhibition of cAMP formation, induced by 100 microM forskolin. Besides 5-HT, various other compounds with affinity for 5-HT receptors behaved as agonists with the following rank order of potency: RU 24,969 > 5-CT > dihydroergotamine = 5-HT > CP 93,129 > d-LSD > 1-naphtylpiperazine > sumatriptan > TFMPP = mCPP > CGS 12066B = metergoline > methysergide. The beta-adrenergic receptor blockers cyanopindolol, SDZ 21,009, (-)-pindolol and (-)-propranolol, and the alpha 2-adrenergic blockers yohimbine and rauwolscine yielded agonist activity at nanomolar and micromolar concentrations, respectively. Isamoltane acted as a partial agonist. Methiothepin was the only compound that antagonised the OK cell 5-HT receptor-mediated inhibition of forskolin-induced cAMP formation. We conclude that the OK cell 5-HT receptor has properties consistent with a 5-HT1B receptor, although differences are apparent with regard to potencies of some compounds. Methiothepin is probably the only effective antagonist at 5-HT1B receptor sites, whereas the described putative 5-HT1B receptor antagonists have to be considered as partial agonists, yielding agonist or antagonist activity depending on the system that is studied.

Most antidepressants produce changes in monoamine receptors in brain after chronic administration in animals. The most commonly described alterations are a decreased density and function of beta-adrenergic receptors and have been postulated to be the mechanisms by which antidepressants exert their therapeutic effect. Milnacipran (previous name midalcipran) is a new, clinically-effective antidepressant, which inhibits the uptake of both serotonin and noradrenaline but has no affinity for any pre- or postsynaptic receptor studied. When given either orally at 7.5 mg/kg twice daily for 3 days, at 30 mg/kg once daily for 3 weeks, by osmotic mini-pump at 30 mg/kg/day for 27 days, or in drinking water at approximately 15 mg/kg/day for 6 weeks and after a washout period of 24 hr, milnacipran produced no down-regulation of beta-adrenoceptors. In addition, there were no alterations of alpha 1- or alpha 2-adrenoceptors, 5-HT1, 5-HT2 receptors or benzodiazepine binding sites. Moreover, uptake and accumulation of serotonin and noradrenaline were unmodified. In addition, the potency for milnacipran to inhibit monoamine uptake in vitro in the cortex was not altered in treated rats, compared to control animals. Thus, in spite of its action on both the uptake of serotonin and noradrenaline, milnacipran appears to be without long-term action on beta-adrenoceptors or the other receptors studied, suggesting that, at least for this antidepressant, these modifications are not essential for clinical activity.