Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a []GTPγS binding study
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-[]thio)-triphosphate ([]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 []GTPγS binding in a CHO cell line stably expressing recombinant human 5-HT1A receptors. Second, to evaluate the agonist/antagonist activity (by stimulation of []GTPγS binding) and binding affinity (by competition with []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 [
Definition of [35S]GTPγS binding conditions
5-HT (10 μM) stimulated []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 []GTPγS binding was observed in membranes of untransfected CHO cells (results not shown). Basal (non-agonist-stimulated) binding of []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 []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
References (81)
- et al.
Constitutively active 5-hydroxytryptamine2C receptors reveal novel inverse agonist activity of receptor ligands
J. Biol. Chem.
(1994) - et al.
5-HT1A receptors are not involved in clozapine's lack of cataleptogenic potential
Neuropharmacology
(1996) - et al.
Molecular biology of 5-HT receptors
Neuropharmacology
(1994) Serotonin receptors in cognitive behaviors
Current Opinion in Neurobiology
(1997)- et al.
[]WAY 100,635 for 5-HT1A receptor autoradiography in human brain: a comparison with []8-OH-DPAT and demonstration of increased binding in the frontal cortex in schizophrenia
Neurochem. Int.
(1997) - et al.
Radioreceptor binding profile of the atypical antipsychotic olanzapine
Neuropsychopharmacology
(1996) - et al.
Effects of atypical antipsychotic agents on social behavior in rodents
Pharmacol. Biochem. Behav.
(1993) - et al.
Effector coupling mechanisms of the cloned 5-HT1A receptor
J. Biol. Chem.
(1989) - et al.
Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein
Cell. Signal.
(1991) - et al.
NAN-190: an arylpiperazine analog that antagonizes the stimulus effects of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)
Eur. J. Pharmacol.
(1988)
The selective 5-HT1A antagonist radioligand []WAY 100635 labels both G-protein-coupled and free 5-HT1A receptors in rat brain membranes
Eur. J. Pharmacol.
The putative 5-HT1A receptor antagonists NAN-190 and BMY 7378 are partial agonists in the rat dorsal raphe nucleus in vitro
Eur. J. Pharmacol.
Increase in serotonin 5-HT1A receptors in prefrontal cortex and temporal cortices of brains from patients with chronic schizophrenia
Life Sci.
[]8-OH-DPAT labels a 5-HT site coupled to inhibition of phosphoinositide hydrolysis in the dorsal raphe
Eur. J. Pharmacol.
Clozapine inhibits serotonergic transmission by an action at α1-adrenoceptors not at 5-HT1A receptors
Eur. J. Pharmacol.
Clozapine has sub-micromolar affinity for 5-HT1A receptors in human brain tissue
Eur. J. Pharmacol.
S 15535: a highly selective benzodioxopiperazine 5-HT1A receptor ligand which acts as an agonist and an antagonist at presynaptic and postsynaptic sites respectively
Eur. J. Pharmacol.
The dopamine D3 receptor antagonist, (+)S 14297, blocks the cataleptic properties of haloperidol in rats
Eur. J. Pharmacol.
Further evidence for differential affinity states of the serotonin1A receptor in rat hippocampus
Brain Res.
Clozapine is a partial agonist at cloned, human serotonin 5-HT1A receptors
Neuropharmacology
S 15535 and WAY 100,635 antagonise 5-HT-stimulated []GTPγS binding at cloned human 5-HT1A receptors
Eur. J. Pharmacol.
Activity of serotonin (5-HT) receptor agonists, partial agonists and antagonists at cloned human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells
Biochem. Pharmacol.
Clozapine increases dopamine release in prefrontal cortex by 5-HT1A receptor activation
Eur. J. Pharmacol.
The potent activity of the 5-HT1A receptor agonists S 14506 and S 14671, in the rat forced swim test is blocked by novel 5-HT1A receptor antagonists
Eur. J. Pharmacol.
Atypical neuroleptics have low affinity for dopamine D2 receptors or are selective for D4 receptors
Neuropsychopharmacology
Characterisation of a cloned human 5-HT1A receptor cell line using []GTPγS binding
Eur. J. Pharmacol.
Characterisation of recombinant human serotonin 5-HT1A receptors expressed in Chinese hamster ovary cells: []spiperone discriminates between the G-protein-coupled and uncoupled forms
Biochem. Pharmacol.
Prevention by (±)-8-hydroxy-2-(di-n-propylamino)tetralin of both catalepsy and the rises in rat striatal dopamine metabolism caused by haloperidol
Br. J. Pharmacol.
The 5-HT1A receptor antagonist (S)UH 301 blocks (R)-8-OH-DPAT-induced inhibition of serotonergic dorsal raphe cell firing in the rat
J. Neural Transm. Gen. Sect.
Comparative profiles of interaction of atypical antipsychotics at cloned human dopamine hD2, hD3 and hD4 receptors: S 16924 shows a marked preference for hD4 sites
Eur. Neuropsychopharmacol.
Pharmacology of the novel 5-hydroxytryptamine1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)-tetralin: inhibition of (R)-8-hydroxy-2-(dipropylamino)-tetralin-induced effects
J. Pharmacol. Exp. Ther.
Potency of 5-hydroxytryptamine1A agonists to inhibit adenylyl cyclase activity is a function of affinity for the `low-affinity' state of []8-hydroxy-N,N-dipropylaminotetralin ([]8-OH-DPAT) binding
J. Pharmacol. Exp. Ther.
Evaluation of butaclamol in chronic schizophrenic patients
J. Clin. Pharmacol.
Lack of 5-hydroxytryptamine1A-mediated inhibition of adenylyl cyclase in dorsal raphe of male and female rats
J. Pharmacol. Exp. Ther.
Intrinsic activity of enantiomers of 8-hydroxy-2-(di-n-propylamino)tetralin and its analogs at 5-hydroxytryptamine1A receptors that are negatively coupled to adenylate cyclase
Mol. Pharmacol.
Electrophysiological evidence for a large receptor reserve for inhibition of dorsal raphe neuronal firing by 5-HT1A agonists
Synapse
Dopamine receptor of the porcine anterior pituitary gland: evidence for two affinity states discriminated by both agonists and antagonists
Mol. Pharmacol.
Characterization of the 5-hydroxytryptamine1A receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes
J. Pharmacol. Exp. Ther.
5-HT1A receptor agonists: recent developments and controversial issues
Psychopharmacology
Cited by (238)
Prevention of MK-801-induced amnestic effect with combined activation of 5-HT<inf>1A</inf> and muscarinic receptors in mice
2024, Pharmacology Biochemistry and BehaviorNew dual 5-HT1A and 5-HT7 receptor ligands derived from SYA16263
2021, European Journal of Medicinal ChemistryConstitutive activity of 5-HT receptors: Factual analysis
2020, NeuropharmacologyFunctional characterization of 5-HT<inf>1A</inf> and 5-HT<inf>1B</inf> serotonin receptor signaling through G-protein-activated inwardly rectifying K<sup>+</sup> channels in a fluorescence-based membrane potential assay
2020, Biochemical PharmacologyCitation Excerpt :Conversely, GR 127935 potently inhibited the response induced by 5-HT EC80 (3–7 nM 5-HT, determined on the day of the experiment) in 5-HT1B/GIRK2-HEK293 cells [IC50 (pIC50 ± S.E.M.): 9.8 nM (8.01 ± 0.09), n = 3], while not significantly inhibiting the 5-HT EC80-induced response in 5-HT1A/GIRK2-HEK293 cells at concentrations up to 3 µM (Fig. 2B). Thus, the inhibitory potencies and subtype-selectivity profiles exhibited by the two antagonists at the two cell lines in this assay were in good agreement with the literature [37–40]. Next, the effect of selective inhibition of the endogenous Gαi/o protein population in the 5-HT1A/GIRK2- and 5-HT1B/GIRK2-HEK293 cell lines on the 5-HT-induced responses was investigated using the well-established Gαi/o-protein inhibitor PTX.
Serotonin and schizophrenia
2020, Handbook of Behavioral Neuroscience