Characterization of 5-HT1A receptor functional coupling in cells expressing the human 5-HT1A receptor as assessed with the cytosensor microphysiometer

doi:10.1016/S1056-8719(98)00035-5

Journal of Pharmacological and Toxicological Methods

Volume 40, Issue 1, July 1998, Pages 47-55

https://doi.org/10.1016/S1056-8719(98)00035-5 Get rights and content

Abstract

The functional activity of a series of 5-HT_1A receptor ligands has been evaluated in a cell line expressing the human 5-HT_1A receptor (h5-HT_1A · CHO) using the agonist-stimulated increase in extracellular acidification rate, measured with the microphysiometer, as a functional assay. Both 5-CT and 8-OH-DPAT were potent agonists in stimulating an increase in extracellular acidification rate in h5-HT_1A · CHO cells with estimated EC₅₀ values of 1.2 and 7.8 nM, respectively. Additionally, these two 5-HT_1A receptor agonists elicited a similar maximum response. Concentration-dependent agonist activity was also observed in the presence of buspirone, ipsapirone, BMY7378, NAN-190 and WAY100135, and each of these compounds behaved as partial 5-HT_1A receptor agonists. The selective 5-HT_1A receptor antagonist WAY100635 produced a potent (IC₅₀, 2.3 nM) and complete block of the 8-OH-DPAT-stimulated response. An evaluation of the inhibitory activity of a series of 5-HT_1A receptor antagonists produced the following rank order of potency; WAY100635 > LY206130 (IC₅₀, 7.1 nM) > WAY100135 (30.8 nM) > pindolol (76.2 nM) > (−)UH-301 (92.8 nM). Parallel studies on the inhibition of forskolin-stimulated adenylyl cyclase activity in h5-HT_1A · CHO cells revealed that agonist potencies were generally similar between the two functional assays and were in good agreement with the estimated 5-HT_1A receptor binding affinities. However, the relative efficacies determined for the partial agonists in the cAMP assay were substantially greater than those observed with the microphysiometer. Finally, antagonists were considerably weaker in the cAMP assay compared with the microphysiometer. The evaluation of 5-HT_1A ligands using the microphysiometer, which represents a very distinct indice of 5-HT_1A receptor function compared with the cAMP assay, results in a different profile of functional activity.

Introduction

Of the 15 different cloned 5-HT receptor subtypes identified to date (reviewed in Saxena, 1995), the 5-HT_1A receptor subtype has been studied most extensively. In the CNS, this receptor subtype is located both presynaptically where it functions as an inhibitory autoreceptor suppressing the firing of dorsal raphe 5-HT neurons and postsynaptically in 5-HT projection regions within the frontal cortex and hippocampus. The therapeutic potential of 5-HT_1A receptor ligands has largely driven the interest in this receptor subtype, and in this regard 5-HT_1A partial agonists such as buspirone (Goa and Ward, 1986) and ipsapirone (Glaser, 1988) are clinically effective anxiolytics. In addition to an established role in anxiety, 5-HT_1A receptors have been implicated as a therapeutic target in a number of other psychiatric disorders including obsessive compulsive disorder and depression (Deakin, 1993). Recently, it has been proposed that the combined action of a 5-HT_1A receptor antagonist with a selective 5-HT reuptake inhibitor may result in a faster-acting antidepressant that may be efficacious in treatment-resistant patients (reviewed in Artigas et al., 1996).

One of the major challenges in the development of 5-HT_1A receptor selective ligands has been the identification of selective antagonists which lack intrinsic activity in models of presynaptic 5-HT_1A receptor function. For example, the earliest described 5-HT_1A receptor antagonists BMY7378 and NAN-190 have subsequently been shown to exhibit partial agonist activity at presynaptic 5-HT_1A receptors. Specifically, BMY7378 and NAN-190 act as partial 5-HT_1A receptor agonists to suppress firing of rat dorsal raphe neurons in vitro (Greuel and Glaser, 1992). In addition, BMY7378 (Sharp et al., 1990) and NAN-190 (Hjorth and Sharp, 1990) decrease hippocampal 5-HT release in vivo via an agonist action at presynaptic 5-HT_1A autoreceptors. However, major advances in the development of 5-HT_1A receptor antagonists have been made with the development of WAY100135 (Fletcher et al., 1993) and WAY100635 (Forster et al., 1995) as selective 5-HT_1A receptor antagonists. WAY100635 appears to act truly as a selective 5-HT_1A receptor antagonist devoid of intrinsic agonist activity, while in vivo microdialysis studies demonstrate that WAY100135 results in a concentration-dependent reduction in hippocampal 5-HT levels (Assie and Koek, 1996), a property consistent with 5-HT_1A partial agonist activity at presynaptic autoreceptors.

While the evaluation of 5-HT_1A receptor ligands using in vivo models of presynaptic 5-HT_1A receptor function has proven to be extremely powerful, these techniques are not amenable to the rapid evaluation of novel 5-HT_1A receptor ligands. Accordingly, a number of recombinant cell lines expressing the human 5-HT_1A receptor have been described, with particular emphasis on their potential to evaluate the intrinsic activity of 5-HT_1A receptor ligands Boddeke 1992, Newman-Tancredi 1992, Varrault et al 1992. In this study, we have extended the functional analysis of recombinant human 5-HT_1A receptors to include an evaluation of the Cytosensor microphysiometer, and its potential to detect 5-HT_1A receptor functional agonist and antagonist activity. The microphysiometer continuously monitors the extracellular pH surrounding cells in culture, and reports receptor activation by measuring increases in extracellular acidification rate which occur in response to agonist stimulation. Consequently, the microphysiometer provides a signalling pathway-independent readout of receptor activation. Our objectives were to examine the potential use of the microphysiometer for the characterization of 5-HT_1A receptor ligands and, in particular, to examine its ability to detect intrinsic activity of 5-HT_1A ligands compared with other established methodologies. To this end, the results from this analysis have been compared with those obtained from a parallel study of the inhibition of forskolin-stimulated cAMP accumulation in h5-HT_1A · CHO cells, which represents a more conventional assay of 5-HT_1A receptor function in vitro.

Section snippets

Cell line

The PCR cloning of the human 5-HT_1A receptor subtype from a human genomic library has been described previously (Chanda et al., 1993). A stable Chinese hamster ovary cell line expressing the human 5-HT_1A receptor subtype (h5-HT_1A · CHO cells) was employed throughout this study. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, nonessential amino acids and penicillin/streptomycin. All 5-HT_1A receptor

Radioligand binding studies

A characterization of the h5-HT_1A · CHO cell line was performed to establish expression levels and a radioligand binding profile. Using [³H]8-OH-DPAT as the radioligand, high-affinity saturable binding was observed in h5-HT_1A · CHO cells. Saturation analysis revealed a B_max (maximum site density) of 1.06 pmol/mg protein and a Kd (equilibrium dissociation constant) of 3.56 nM. The binding affinities (Ki) of several 5-HT_1A ligands are depicted in Table 1. The affinities of these compounds

Microphysiometer studies of 5-HT_1A receptor function

This study describes the characterization of the functional activity of a series of 5-HT_1A receptor ligands using the agonist-stimulated increase in extracellular acidification rate, measured with the microphysiometer, as a functional assay. Although still considered to be a relatively new technique, microphysiometry has successfully been applied in the functional analysis of a large number of G-protein coupled receptors (GPCRs) including those that couple to effectors via Gαi (as is the case

Acknowledgements

The authors thank Stanley Nawoschik and Rafal Ochalski for their expert technical assistance.

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Original ArticlesCharacterization of 5-HT1A receptor functional coupling in cells expressing the human 5-HT1A receptor as assessed with the cytosensor microphysiometer

Abstract

Introduction

Section snippets

Cell line

Radioligand binding studies

Microphysiometer studies of 5-HT1A receptor function

Acknowledgements

Trends Neurosci

Eur J Pharmacol

Eur J Pharmacol

Eur J Pharmacol

Eur J Pharmacol

Eur J Pharmacol

Life Sci

Brain Res

Trends Pharmacol Sci

Life Sci

Eur J Pharmacol

Biochem Pharmacol

Pharmacol Ther

Arch Biochem Biophys

Eur J Pharmacol

Eur J Pharmacol

Pharmacology of the novel 5-hydroxytryptamine1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralininhibition of (R)-8-hydroxy-2-(dipropylamino)tetralin-induced effects

J Pharmacol Exp Ther

Agonist/antagonist interactions with cloned human 5-HT1A receptorsvariations in intrinsic activity studied in transfected HeLa cells

Naunyn Schmied Arch Pharmacol

Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor

Mol Pharmacol

Original Articles
Characterization of 5-HT_1A receptor functional coupling in cells expressing the human 5-HT_1A receptor as assessed with the cytosensor microphysiometer

Microphysiometer studies of 5-HT_1A receptor function

Pharmacology of the novel 5-hydroxytryptamine_1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralininhibition of (R)-8-hydroxy-2-(dipropylamino)tetralin-induced effects

Agonist/antagonist interactions with cloned human 5-HT_1A receptorsvariations in intrinsic activity studied in transfected HeLa cells

Identification of residues important for ligand binding to the human 5-hydroxytryptamine_1A serotonin receptor