Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPγS binding study

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Abstract

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]GTPγS 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]GTPγS 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]GTPγS 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]GTPγS 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]GTPγS 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.

Introduction

The 5-HT1A receptor is a member of the superfamily of monomeric transmembrane G-protein-coupled receptors and has been heterologously expressed in COS7 (african green monkey kidney), HeLa, CHO (Chinese hamster ovary), and other cell lines, enabling the study of its coupling to G-proteins and second messenger systems (Raymond et al., 1992; Newman-Tancredi et al., 1992; Boess and Martin, 1994; Kenakin, 1996). 5-HT1A receptors are localised as somatodendritic autoreceptors in raphe nuclei and postsynaptically in corticolimbic structures such as hippocampus and cortex, reflecting a key role in the modulation of affective disorders, including anxiety disorders and depression (De Vry, 1995; Maes and Meltzer, 1995; Millan et al., 1997b).

5-HT1A receptors have also attracted interest as potential targets for novel antipsychotic agents. First, clinical studies have reported that the 5-HT1A receptor partial agonist, buspirone, markedly ameliorates the lowered mood and social withdrawal of schizophrenics, through its antianxiety effects (Sovner and Parnell-Sovner, 1989; Goff et al., 1991; Harvey and Balon, 1995). Second, post-mortem studies have shown that frontal cortex 5-HT1A receptor density is increased in schizophrenic patients (Hashimoto et al., 1991; Burnet et al., 1997). Third, 5-HT1A receptor agonists, such as 8-OH-DPAT (8-hydroxy-dipropylamino-tetralin), block the catalepsy induced in rats by neuroleptics such as haloperidol or raclopride (McMillen et al., 1988; Andersen and Kilpatrick, 1995), suggesting that an antipsychotic agent which displays 5-HT1A receptor agonist activity may show a lower incidence of extrapyramidal symptoms in humans. Fourth, schizophrenic patients suffer from a functional deficiency in the frontal cortex, and in rat dialysis studies, 5-HT1A receptor agonists selectively reinforce dopamine and noradrenaline release in this region, suggesting that 5-HT1A receptor agonism may alleviate `hypofrontality' (Millan et al., 1997b). Fifth, the `atypical' antipsychotic, clozapine, which is clinically effective against both positive and negative symptoms in the absence of extrapyramidal symptoms, displays marked affinity at human brain 5-HT1A receptors (Mason and Reynolds, 1992) and partial agonism at recombinant human 5-HT1A receptors (Newman-Tancredi et al., 1996a). Indeed, Corbett et al. (1993)suggested that the capacity of antipsychotic agents to treat psychotic symptoms in the absence of extrapyramidal symptoms is due to agonist actions at 5-HT1A receptors. However, no direct evidence was provided to support this hypothesis and the present study therefore undertook a systematic analysis of this issue by investigating the intrinsic activity of antipsychotic agents for modulation of 5-HT1A receptor-mediated signal transduction.

5-HT1A receptors modulate the activity of diverse second messenger pathways, including potassium and calcium channels, inositol phosphate metabolism, arachidonic acid production and adenylyl cyclase activity (Schoeffter and Hoyer, 1988; Pauwels et al., 1993; Boess and Martin, 1994). Whilst these approaches yield valuable information about 5-HT1A receptor activation, they measure responses which are several steps `downstream' of the receptor per se and depend on the cell types investigated (Liu and Albert, 1991; Raymond et al., 1993). In addition, 5-HT1A receptors can modulate different second messenger systems with different potencies (Fargin et al., 1989; Gudermann et al., 1996) or the same second messenger systems in a different manner depending on brain region (Clarke et al., 1996; Johnson et al., 1997). These differing responses complicate the interpretation of second messenger effects. In contrast, both adenylyl cyclase inhibition and phosphoinositide hydrolysis are mediated by the same G-protein, at least in HeLa cells (Fargin et al., 1991), suggesting that investigation of agonist activity at the G-protein level may overcome some of these difficulties. Odagaki and Fuxe (1995)measured G-protein activation by determining GTPase activity in rat hippocampal membranes, whilst guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding methodology can be applied to 5-HT1A receptors in recombinant systems (Newman-Tancredi et al., 1996b, Newman-Tancredi et al., 1997a; Stanton and Beer, 1997).

The present study had several aims. First, to characterise the optimal experimental conditions for determination of agonist stimulation of 5-HT1A-receptor-mediated [35S]GTPγS binding in a CHO cell line stably expressing recombinant human 5-HT1A receptors. Second, to evaluate the agonist/antagonist activity (by stimulation of [35S]GTPγS binding) and binding affinity (by competition with [3H]8-OH-DPAT) of serotonergic reference compounds. These include serotonergic agonists, antagonists, and agents proposed for the treatment of affective disorders, such as buspirone and (+)flesinoxan. Third, to investigate the 5-HT1A receptor activity of a range of antipsychotic drugs. These included older antipsychotic agents, such as chlorpromazine, haloperidol, pimozide and raclopride, which are known to provoke marked extrapyramidal symptoms (Meltzer, 1996; Baldessarini, 1996). Several recent drugs, intended to display clozapine-like clinical efficacy without extrapyramidal symptoms induction, were also evaluated, including risperidone, ziprasidone, quetiapine, olanzapine and sertindole (Schwartz and Brotman, 1992; Fleischhacker and Hummer, 1997).

Section snippets

Determination of affinity (Ki) at CHO-h5-HT1A receptors

Membranes were prepared from CHO-h5-HT1A cells stably expressing the human 5-hydroxytryptamine 5-HT1A receptor (Newman-Tancredi et al., 1992). Cells grown in suspension culture were harvested by centrifugation and homogenised in buffer A (HEPES 20 mM, pH 7.5 and MgSO4 5 mM) using a Kinematica Polytron. The homogenate was centrifuged at 50,000×g for 30 min and the membrane pellet resuspended in buffer A. For competition binding experiments, membranes (10–20 μg protein) were incubated with [3H

Definition of [35S]GTPγS binding conditions

5-HT (10 μM) stimulated [35S]GTPγS binding to 5-HT1A membranes in a linear manner over the first 20 min (3) of time course experiments, and a standard incubation time of 20 min was therefore used. In contrast, no stimulation of [35S]GTPγS binding was observed in membranes of untransfected CHO cells (results not shown). Basal (non-agonist-stimulated) binding of [35S]GTPγS to CHO-h5-HT1A membranes was dependent on the concentration of GDP present in the buffer (Fig. 1B) and was reduced from about

Affinity/efficacy of 5-HT agonists at 5-HT1A receptors

A range of serotonergic agonists and partial agonists were tested for their capacity to stimulate 5-HT1A receptor mediated [35S]GTPγS binding in CHO-h5-HT1A membranes. The methoxynaphtylpiperazine ligand, S 14671, was the most potent agonist tested, with virtually full agonist activity, relative to 5-HT (Table 1; Fig. 2C) consistent with its exceptionally potent and efficacious actions in in vivo functional paradigms (Millan et al., 1992; Schreiber et al., 1994). Its analogue, S 14506 was also

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