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The role of antidepressants in the treatment of visceral pain has not been extensively examined. Milnacipran, a serotonin/noradrenalin reuptake inhibitor, has recently been approved in the USA for fibromyalgia, a chronic pathology characterized by diffused/chronic musculoskeletal pain, and a high prevalence of irritable bowel syndrome. Here, we determined its antinociceptive efficacy in two visceral pain tests in rodents: the acetic acid-induced writhing model in mice and the butyrate/colonic distension assay in rats, a model of irritable bowel syndrome. Acute milnacipran (5-40 mg/kgi.p.) significantly and dose-dependently reduced writhing (72.2 ± 3.2 versus 17.0 ± 4.1 writhes at 40 mg/kg). Following repeated administration (40 m/kgi.p. for 5 days), milnacipran preserved its ability to significantly reduce writhing (76 ± 8.3 versus 21.1 ± 6.7 writhes). Similarly, in the butyrate model, acute milnacipran (17.5 and 35 mg/kg, i.p.) significantly and dose-dependently increased cramps induction thresholds (from 45.7 ± 5.7 to 66.3 ± 4.8 and 75.6 ± 2.9 mm Hg, for 17.5 and 35 mg/kg, respectively) and reduced the number of cramps (from 3.0 ± 0.8 to 1.2 ± 0.8 and 0.3 ± 0.3 following inflation of an intra-rectal balloon. To summarise, milnacipran was efficacious in the writhing test, after acute and semi-chronic administration. This effect was confirmed after acute administration in a more specific model of colonic hypersensitivity induced by butyrate. This suggests that milnacipran has potential clinical application in the treatment of visceral pain, such as in irritable bowel syndrome, highly co-morbid with fibromyalgia.

Small rodents (mice, rats) are the species of choice for evaluating the pharmacology of centrally acting compounds, such as antipsychotics, whereas toxicology data are routinely obtained from other species (rabbits, dogs, monkeys). Whilst there is a substantial number of "therapeutically relevant" pharmacological models for "antipsychotic-like" activity in small rodents, based on hyperdopaminergic or hypoglutamatergic/NMDA approaches, there is a remarkable paucity of such models in other species. Here, we compared the efficacy and potency of reference and new generation dopamine D2/5-HT(1A) putative antipsychotics, administered orally, against apomorphine-induced emesis in dogs, a model of central D2 receptor activation that can be implemented with relative ease. Risperidone potently and fully (10 microg/kg) prevented emesis/retching induced by 0.1 mg/kg s.c. apomorphine. SLV313 and F15063 (D2 receptor antagonists/5-HT(1A) receptor agonists) also abolished emesis/retching, albeit less potently than risperidone (minimal effective dose, MEDs: 10 and 40 microg/kg, respectively). The D2 receptor partial agonists/5-HT(1A) receptor agonists aripiprazole and bifeprunox, (up to 80 microg/kg) only partially attenuated emesis, as did the peripheral D2 receptor antagonist domperidone. Under the present experimental conditions, haloperidol was only efficacious at the highest dose tested (320 microg/kg). To summarize, dogs are very sensitive to the dopaminergic blocking effects of antipsychotics in this model of D2 receptor activation. This model can thus be advantageously used to investigate the pharmacological activity of novel D2 receptor antagonists/partial agonists in dogs.

The serotonin 5-HT 1A receptor antagonist WAY-100635 was recently reported to have potent agonist properties at dopamine D 4 receptors (Chemel et al., 2006, Psychopharmacology 188, 244–251.). Herein WAY-100635 (p K i at human (h) serotonin 5-HT 1A receptors = 9.51; p K i at dopamine hD 4.4 receptors = 7.42) stimulated [ 35 S]GTPγS incorporation in membranes of Chinese Hamster Ovary cells expressing dopamine hD 4.4 receptors with only moderate potency and modest efficacy (pEC 50 = 6.63; E max = 19% of dopamine). Moreover, in antagonism experiments, WAY-100635 had a much lower potency at dopamine hD 4.4 receptors (p K B = 7.09), than at serotonin h5-HT 1A receptors (p K B = 9.47). These data demonstrate that WAY-100635 has high selectivity for serotonin h5-HT 1A versus dopamine hD 4.4 receptors.

The effects of antipsychotics targeting dopamine D2 and serotonin 5-HT1A receptors were compared with conventional antipsychotics on phosphorylation of Extracellular signal-Regulated Kinase 1/2 (ERK 1/2) in CHO cell lines stably expressing either the human serotonin 5-HT1A or human dopamine D2S receptor. All antipsychotics except haloperidol and olanzapine exhibited agonist properties at serotonin 5-HT1A receptors. Emax values (% effect of 10 microM 5-HT) were: bifeprunox (74), SSR181507 (73), SLV313 (72), aripiprazole (60), ziprasidone (56), clozapine (33). At dopamine D2S receptors, partial agonist activity (% effect of 10 microM dopamine) was observed for bifeprunox (76), SSR181507 (66) and aripiprazole (59). Other antipsychotics attenuated dopamine-induced ERK phosphorylation, with pK(B) values of : SLV313 (8.5), haloperidol (8.1), olanzapine (7.8), ziprasidone (7.7), and clozapine (6.4). Amongst the dopamine D2/serotonin 5-HT1A receptor compounds, aripiprazole acts as a partial dopamine D2S and serotonin 5-HT1A receptor agonist. SSR181507 and bifeprunox possess a profile of action similar to each other, efficaciously stimulating both serotonin 5-HT1A and dopamine D2S receptors. In contrast, SLV313, also an efficacious serotonin 5-HT1A receptor agonist, acted as a high potency dopamine D2 receptor antagonist. Thus, antipsychotics display varying efficacies at serotonin 5-HT1A and dopamine D2S receptors which may play a major role in their differential functional profiles in blocking the diverse symptoms of schizophrenia.

5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.]. The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

In analogy to the dopamine D3 receptor agonist, (+)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin) (0.01–0.63 mg/kg s.c.), clozapine dose-dependently (0.63–40.0 mg/kg s.c.) elicited hypothermia in rats. Haloperidol and raclopride, mixed dopamine D 2 D 3 receptor antagonists, failed, in contrast, to modify core temperature. Further, they dose-dependently inhibited the action of clozapine with inhibitory dose50 values (ID50) of 0.3 mg/kg s.c., in each case. The preferential dopamine D3 versus D2 receptor antagonist, (+)-AJ 76 (cis-(+)-5-methoxy-1-methyl-2-(n-propylamino)tetralin) (ID50 = 2.8), and the selective dopamine D3 versus D2 receptor antagonist, (±)-S 11566 ((±)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho(2,3b) dihydro,2,3-furane]) (ID50 = 1.6) likewise blocked the action of clozapine without reducing core temperature alone. The action of (±)-S 11566 was stereospecific in that its active eutomer, (+)-S 14297 (ID50 = 1.0), also inhibited the action of clozapine whereas its inactive distomer, (−)-S 17777 (ID50 > 10.0), was not effective. Antagonist potency for blockade of clozapine-induced hypothermia correlated powerfully both with potency for blockade of (+)-7-OH-DPAT-induced hypothermia (r = 0.98) and with affinity at cloned human dopamine D3 receptors transfected into Chinese hamster ovary (CHO) cells (r = 0.92). In conclusion, these data suggest that dopamine D3 receptors may be involved in the induction of hypothermia by clozapine in the rat.

Milnacipran, a serotonin/norepinephrine reuptake inhibitor (SNRI), has shown efficacy against several chronic pain conditions, including fibromyalgia. Here, we evaluated, in rats, its anti-allodynic effects following acute or sub-chronic treatment in a model of neuropathic pain (chronic constriction injury, CCI, of the sciatic nerve). Amitriptyline, a tricyclic antidepressant active pre-clinically and clinically against neuropathic pains, was added as a comparison compound. Upon acute i.p. administration, milnacipran was potently efficacious in the CCI model. It significantly reduced thermal allodynia in the cold (4 °C) plate test (MED = 2.5 mg/kg), and attenuated mechanical allodynia in the von Frey filaments test (MED = 10 mg/kg). Given sub-chronically (7 day, b.i.d.), milnacipran was effective at 10 mg/kg i.p. in both tests. Acute amitriptyline (10 mg/kg i.p.) was efficacious against mechanical, but less so against cold allodynia; under sub-chronic conditions, it was only active against mechanical allodynia. These data show that milnacipran is as efficacious as the reference compound amitriptyline in a pre-clinical model of injury-induced neuropathy, and demonstrate for the first time that it is active acutely and sub-chronically against cold allodynia. They also suggest that milnacipran has the potential to alleviate allodynia associated with nerve compression-induced neuropathic pain in the clinic (for example following discal hernia, avulsion or cancer-induced tissue damage).

Brain region-specific modulation of immediate-early gene (IEG) may constitute a marker of antipsychotic drug-like activity. We investigated the effects of the putative antipsychotic drug N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine (F15063), a compound that targets both dopamine D(2) and serotonin 5-HT(1A) receptors, in comparison with haloperidol and clozapine on rat mRNA expression of IEG i.e. the zinc-fingered transcription factors c-fos, fosB, zif268, c-jun and junB, two transcription factors of the nuclear receptor family nur77 and nor1, and the effector IEG arc. F15063 (10 mg/kg) and clozapine (10 mg/kg), but not haloperidol (0.63 mg/kg), induced c-fos and fosB mRNA expression in prefrontal cortex, a region associated with control of cognition and negative symptoms of schizophrenia. In striatum, only c-fos, fosB, junB and nur77 were induced by clozapine whereas all IEG mRNAs were increased by haloperidol and F15063 (from 2.5 mg/kg) with similar high efficacy despite a total absence of F15063-induced catalepsy. However, at 0.63 mg/kg, F15063 induced a lower degree of striatal IEG mRNA expression than haloperidol and pretreatment with the serotonin 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride (WAY100635) (0.63 mg/kg) increased the level of IEG mRNA induction by F15063. Furthermore, (+)-8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] at 0.16 mg/kg decreased haloperidol-induced striatal IEG mRNA expression although it exerted no effects on its own. These results are consistent with an activation of serotonin 5-HT(1A) receptors by F15063, thus reducing D(2) blockade-induced striatal IEG mRNA. Furthermore, the substantial F15063-induced expression of IEGs such as c-fos in striatum is not related to cataleptogenic activity and may act more as a marker of efficacious dopamine D(2) receptor blockade.

F15063 (N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)-ethyl]-(3-cyclopenten-1-yl-benzyl)-amine) is a potential antipsychotic with dopamine D2/D3 receptor antagonist, 5-HT1A receptor agonist and dopamine D4 receptor partial agonist properties. Herein, we compared its effects on rat ventral tegmental area dopamine and dorsal raphe serotonin electrical activity with those of the dopamine D2 receptor partial agonist/5-HT1A receptor agonist, SSR181507. Further, we investigated the modulation of extracellular dopamine and noradrenaline in the medial prefrontal cortex and serotonin in the hippocampus of freely moving rats by F15063 using in vivo microdialysis. In the ventral tegmental area, F15063 (200-700 microg/kg, i.v.) did not alter the electrical activity of dopamine neurons whereas SSR181507 (250-1000 microg/kg, i.v.) partially inhibited it, consistent with dopamine D2 receptor partial agonism. Both compounds reduced the inhibition of firing rate induced by the full agonist apomorphine. In the dorsal raphe, both ligands suppressed firing activity, consistent with agonism at 5-HT1A autoreceptors, although SSR181507 (25-75 microg/kg, i.v.) was more potent than F15063 (100-300 microg/kg, i.v.). F15063 (0.63-40 mg/kg, i.p.) dose-dependently increased dopamine levels in the prefrontal cortex and decreased hippocampal 5-HT. These effects were reversed by the selective 5-HT1A receptor antagonist WAY100635 (0.16 mg/kg, s.c.), indicating that they were mediated by 5-HT1A receptors (at post- and pre-synaptic levels, respectively). In the medial prefrontal cortex, noradrenaline levels were moderately but significantly increased by F15063 at 2.5 mg/kg. In conclusion, whereas SSR181507 exhibits (partial) agonism at dopamine D2 and 5-HT1A receptors, F15063 blocks dopamine D2-like receptors whilst activating 5-HT1A receptors. Such a profile distinguishes F15063 from SSR181507 and currently available antipsychotic drugs.

Several novel antipsychotics activate serotonin 5-HT1A receptors as well as antagonising dopamine D2/3 receptors. Such a pharmacological profile is associated with a lowered liability to produce extrapyramidal side effects and enhanced efficacy in treating negative and cognitive symptoms of schizophrenia. However, 5-HT1A receptor agonists increase plasma corticosterone and many antipsychotics disturb the regulation of glucose. Here, we compared the influence on plasma glucose and corticosterone of acute treatments with 'new generation' antipsychotics which target dopamine D2/3 receptors and 5-HT1A receptors, with that of atypical antipsychotics, and with haloperidol. Olanzapine and clozapine, antipsychotics that are known to produce weight gain and diabetes in humans, both at 10 mg/kg p.o., substantially increased plasma glucose (from 0.8 to 1.7 g/l) at 1 h after administration, an effect that returned to control levels after 4 h. In comparison, F15063 (40 mg/kg p.o.) was without effect at any time point. Olanzapine and clozapine dose-dependently increased plasma glucose concentrations as did SLV313 and SSR181507. Haloperidol and risperidone had modest effects whereas aripiprazole, ziprasidone and bifeprunox, antipsychotics that are not associated with metabolic dysfunction in humans, and F15063 had little or no influence on plasma glucose. The same general pattern of response was found for plasma corticosterone levels. The present data provide the first comparative study of conventional, atypical and 'new generation' antipsychotics on glucose and corticosterone levels in rats. A variety of mechanisms likely underlie the hyperglycemia and corticosterone release observed with clozapine and olanzapine, whilst the balance of dopamine D2/3/5-HT1A interaction may contribute to the less favourable impact of SLV313 and SSR181507 compared with that of bifeprunox and F15063.

Several examples of agonist-directed trafficking of receptor signalling at 5-HT2A and 5-HT2C receptors have been reported that involve independent downstream transduction pathways. We now report the functional selectivity of a series of chemically diverse agonists at human (h)5-HT2A, h5-HT2B and h5-HT2C-VSV by examining two related responses, the upstream activation of Gq/11 proteins in comparison with its associated cascade of calcium mobilisation. At the h5-HT2A receptor, d-lysergic acid diethylamide (LSD) and the antiparkinsonian agents lisuride, bromocriptine and pergolide exhibit a higher potency for Gq/11 activation than calcium release in contrast with all the other tested ligands such as 5-HT, mCPP and BW723C86, that show an opposite preference of signalling pathway. Comparable observations are made at h5-HT2B and h5-HT2C-VSV receptors, suggesting a similar mechanism of functional selectivity for the three serotonin receptors. Interestingly, the non-hallucinogenic compound lisuride behaves as a partial agonist for both Gq/11 activation and calcium release at the three 5-HT2 receptors, in contrast with DOI, LSD, pergolide and bromocriptine, which are known to provoke hallucinations, and behave as more efficacious agonists. Hence, a functional selectivity for Gq/11 activation together with a threshold of efficacy at h5-HT2A (and possibly h5-HT2B and/or h5-HT2C-VSV) may contribute to hallucinogenic liability. Thus, our results extend the notion of agonist-directed trafficking of receptor signalling to all the 5-HT2-receptor family and indicate that measures of Gq/11 activation versus calcium release may be useful to identify more effective therapeutic drugs with limited side effects.

Dopamine D2 receptor antagonists induce hyperprolactinemia depending on the extent of D2 receptor blockade. We compared the effects of the new antipsychotic agents SSR181507 ((3-exo)-8-benzoyl-N-[[(2 s)7-chloro-2,3-dihydro-1,4-benzodioxin-1-yl]methyl]-8-azabicyclo[3.2.1]octane-3-methanamine monohydrochloride), bifeprunox (DU127090: 1-(2-Oxo-benzoxazolin-7-yl)-4-(3-biphenyl)methylpiperazinemesylate) and SLV313 (1-(2,3-dihydro-benzo[1,4]dioxin-5-yl)-4-[5-(4-fluorophenyl)-pyridin-3-ylmethyl]-piperazine) with those of aripiprazole (7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]-butyloxy)-3,4-dihydro-2(1 H)-quinolinone), clozapine and haloperidol, on functional measures of dopamine D2 receptor activity in vitro and in vivo: [35S]-GTPgammaS binding to membranes from Sf9 insect cells expressing human dopamine D2 Long (hD2 L) receptors, and serum prolactin levels in the rat. All compounds antagonized apomorphine-induced G protein activation at dopamine hD2 L receptors. Antagonist potencies of aripiprazole, bifeprunox and SLV313 were similar to haloperidol (pK(b) = 9.12), whereas SSR181507 (8.16) and clozapine (7.35) were less potent. Haloperidol, SLV313 and clozapine were silent antagonists but SSR181507, bifeprunox and aripiprazole stimulated [35S]-GTPgammaS binding by 17.5%, 26.3% and 25.6%, respectively, relative to 100 microM apomorphine (Emax = 100%). pEC50s were: SSR181507, 8.08; bifeprunox, 8.97; aripiprazole, 8.56. These effects were antagonized by raclopride. Following oral administration in vivo, the drugs increased prolactin release to different extents. SLV313 and haloperidol potently (ED50 0.12 and 0.22 mg/kg p.o., respectively) stimulated prolactin release up to 86 and 83 ng/ml. Aripiprazole potently (ED50 0.66 mg/kg p.o.) but partially (32 ng/ml) induced prolactin release. SSR181507 (ED50 4.9 mg/kg p.o.) also partially (23 ng/ml) enhanced prolactin release. Bifeprunox only weakly increased prolactin at high doses (13 ng/ml at 40 mg/kg) and clozapine only affected prolactin at the highest dose tested (41 ng/ml at 40 mg/kg). Prolactin levels of the corresponding vehicle-treated animals were4.3 ng/ml. These data show that (1) SSR181507, aripiprazole and bifeprunox, but not SLV313, are partial agonists at dopamine hD2 L receptors in vitro; (2) SSR181507, bifeprunox and aripiprazole exhibit reduced prolactin release in vivo compared with drugs that are neutral antagonists at dopamine D2 receptors.

At h5-HT1A receptors, stably transfected into Chinese Hamster Ovary Cells (CHO-h5-HT1A), the selective 5-HT1A receptor agonist, (+)8-hydroxy-dipropyl-amino-tetralin, ((+)8-OH-DPAT), transiently activated mitogen-activated protein kinase (MAPK) with a pEC50 of 8.5. The arylalkylamine, (-)-pindolol, also behaved as an agonist with a maximal effect of 57% relative to (+)8-OH-DPAT (100%), and with a pEC50 of 7.2. The selective 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-hexane carboxamide (WAY100,635), blocked (+)8-OH-DPAT- and (-)-pindolol-induced MAPK activation with pK(B)s of 9.7 and 9.9, respectively, whereas the selective 5-HT(1B) receptor antagonist, 1'-Methyl-5-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5H-spiro[furo[2,3-f]indole-3,4'-piperidine] (SB224,289) was inactive. Pertussis toxin blocked the actions of (+)8-OH-DPAT and (-)-pindolol demonstrating implication of G(i)/G(o) proteins. Thus, stimulation of MAPK provides an intracellular marker and signal for expression of the agonist actions of (-)-pindolol at h5-HT1A receptors.

The novel benzopyranopyrrole, S33084 ((3aR,9bS)-N[4-(8-cyano-1,3a,4, 9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl] (4-phenyl)benzamide)), and the aminotetralin derivative, GR218,231 (2(R,S)-(di-n-propylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3 , 4-tetrahydro naphthalene), displayed high affinity at cloned, rat dopamine D(3) receptors (pK(i)s of 8.72 and 8.67, respectively), as well as dopamine D(3) receptors in rat olfactory tubercle (8.62 and 8.94, respectively). In contrast, they showed low affinities at striatal dopamine D(2) receptors (6.82 and 6.64, respectively). Unlike S33084 and GR218,231, the arylpiperazine, L741,626 (4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol), showed lower affinity for cloned (6.46) and native (6.92) dopamine D(3) receptors than for striatal dopamine D(2) receptors (7.52). S33084, GR218,231 and L741,626 should prove useful tools for exploration of the functional roles of dopamine D(3) vs. dopamine D(2) receptors.

Native brain 5-HT1B/1D) receptors were studied using the novel antagonist, [3H]GR 125,743 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyri dyl)benzamide). In guinea-pig striatal membranes, [3H]GR 125,743 displayed rapid association (t1/2 = 4.5 min), high (90%) specific binding and high affinity (K(d) = 0.29 nM), although B(max) values (fmol/mg protein) varied according to brain region-striatum: 199; frontal cortex: 89; hippocampus: 79; cerebellum: 26. In frontal cortex, the B(max) determined with [3H]5-CT ([3H]carboxamidotryptamine) was significantly higher (178; P0.05), suggesting that it also labels other binding sites. In striatal membranes, guanylylimidodiphosphate (GppNHp) inhibited [3H]5-CT but not [3H]GR 125,743 binding, suggesting that the latter has antagonist properties. Nevertheless, in competition binding experiments, the pK(i) values obtained with [3H]GR 125,743 and [3H]5-CT for 20 serotonergic ligands, including L 694,247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl ]-1H-indole-3-yl]ethylamine), GR46,611 (3-[3-(2-dimethylamino-ethyl)-1H-indol-6-yl]-N-(4-methoxybenzyl)acrylami de), sumatriptan and alniditan, were highly correlated (r = 0.99). Ketanserin and ritanserin showed low affinity for [3H]GR 125,743 binding to guinea-pig striatal sites (K(i) = 12600 and 369 nM), suggesting that 5-HT1B (rather than 5-HT1D) receptors are predominantly labelled in this tissue. The present data indicate that [3H]GR 125,743 is a useful tool for studying native 5-HT1B/1D receptors.

The activity of monoamine neurotransmitters was examined at dopamine D4 receptors. In competition binding with [3H]spiperone, noradrenaline and adrenaline exhibited a high affinity binding component (KH = 12.1 nM and 5.0 nM, respectively), similar to that of dopamine (KH = 2.6 nM), whereas serotonin (5-hydroxytryptamine, 5-HT) and histamine had low affinity (Ki > 1000 nM). Noradrenaline and adrenaline acted as agonists at dopamine D4 receptors, stimulating receptor-mediated [35S]guanylyl-gamma-thiotriphosphate ([35S]GTP gamma S) binding (EC50 = 7.8 and 5.8 microM, respectively, versus 0.1 microM for dopamine). The dopamine D4 receptor-selective ligand, 3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2,3b]-pyridi ne (L 745,870) and the dopaminergic antagonists, spiperone, haloperidol and clozapine, inhibited noradrenaline-stimulated [35S]GTP gamma S binding whereas alpha 1-, alpha 2- and beta-adrenoceptor antagonists did not. These results indicate that dopamine D4 receptors are activated by noradrenaline and adrenaline, although at 50-100-fold higher concentrations than dopamine.

In Chinese hamster ovary (CHO) cells expressing cloned human 5-HT1A receptors, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) exhibited high affinity (Ki = 0.79 nM), similar to that of 5-HT (0.61 nM), (+/-)-8-hydroxy-3-(di-n-propylamino)tetralin ((+/-)-8-OH-DPAT; 0.58 nM) and N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N- (2-pyridinyl)cyclo-hexanecarboxamide (WAY 100.635; 0.56 nM). In these cells, 5-HT stimulated [35S]GTP gamma S binding 3-fold (EC50 = 15 nM) whereas (+/-)-8-OH-DPAT exhibited 73% efficacy relative to 5-HT (EC50 = 6.0 nM). WAY 100.635 completely blocked 5-HT- and (+/-)-8-OH-DPAT-stimulated [35S]GTP gamma S binding. Likewise, S 15535 antagonised 5-HT-stimulated [35S]GTP gamma S binding, reducing it to 30.1% of control values. S 15535 (100 nM) also shifted the 5-HT and (+/-)-8-OH-DPAT stimulation curves to the right, to EC50 values of 870 and 313 nM, respectively. However, unlike WAY 100.635, which by itself did not stimulate [35S]GTP gamma S binding, S 15535 alone increased it by 34.7% relative to 5-HT (EC50 = 5.8 nM). In conclusion, S 15535 antagonises the stimulation of 5-HT1A receptors by 5-HT, whilst itself exerting weak partial agonist activity at these sites.