The selective 5-HT1A antagonist radioligand [3H]WAY 100635 labels both G-protein-coupled and free 5-HT1A receptors in rat brain membranes

doi:10.1016/0922-4106(95)90192-2

European Journal of Pharmacology: Molecular Pharmacology

Volume 288, Issue 2, 16 January 1995, Pages 173-186

https://doi.org/10.1016/0922-4106(95)90192-2 Get rights and content

Abstract

The tritiated derivative of the novel silent 5-HT_1A receptor antagonist WAY 100635 [N-(2-(4-(2-methoxyphenyl)-1-pineperazinyl)ethyl)-N-(2-pridinyl) cyclohexane carboxamide] was tested as a potential radioligand of 5-HT_1A receptors in the rat br brain. Binding assays with membranes from various brain regions showed that [³H]WAY 100635 specifically bound to a homogenous population of sites, with a K_d of 0.10nM. The regional distribution of [³H]WAY 100635 specific binding sites, as assessed in membrane binding assays and by autoradiography of labelled brain sections, superimposed exactly over that of 5-HT_1A receptors specifically labelled by $[^{3} H]8- hydroxy -2-(di -n- propylamino) tetralin$ ([³H]-8-OH-DPAT). Futhermore, the positive correlation (r = 0.96A) between the respective pK_l values of a large series of ligands as inhibitors of the specific binding of [³H]WAY 100635 and [³H]8-OH-DPAT in hippocampal membranes indicated that their pharmacological properties were similar. Nevertheless, marked differences also existed between [³H]8-OH-DPAT and [³H]WAY 100635 specific binding, as the former was inhibited by 1–100 μM GTP and GppNHp, whereas the latter was enhanced by these guanic nucleotides. In contrast, Mn²⁺ (1–10 mM) increased the specific binding of [³H]8-OH-DPAT, but inhibited that of [³H]WAY 100635. Treatment of membranes with N-ethylmalcimide (1–5 mM) markedly reduced their capacity to specifically bind [³H]8-OH-DPAT, but slightly increased (at 1 mM) or did not affect (at 5 mM) their [³H]WAY 100635 specific binding capacity. Finally, the B_max of [³H]WAY 100635 specific binding sites was regularly 50–60% higher than that of [³H]8-OH-DPAT in the same membrane preparations from various brain regions (hippocampus, septum, cerebral cortex). These data are compatible with the idea that whereas [³H]8-OH-DPAT only binds to G-protein-coupled 5-HT_1A receptors, [³H]WAY 100635 is a high affinity ligand of both G-protein-coupled and free 5-HT_1A receptor binding subunits in brain membranes.

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Buspirone generally has anxiolytic and pro-social effects in vertebrates (File and Seth, 2003; Bencan et al., 2009; Gould et al., 2011, 2012; Barba-Escobedo and Gould, 2012; Maaswinkel et al., 2012, 2013). However affinities of zebrafish htr1aa, htr1ab receptors are lower for 8-OH-DPAT, buspirone and WAY100635 than mammalian 5-HT1A receptors, since more 5-HT1A-like receptors and/or other binding sites are involved (Gozlan et al., 1995; Owens et al., 1997; Newman-Tancredi et al., 2001; Barba-Escobedo and Gould, 2012). Given this, we performed behavioral tests of buspirone's anxiolytic effects in the zebrafish light/dark plus maze, in which exploration and preference for a dark background (scototaxis) are explored in the first 5 min of introduction, when the arena is still a novel environment for the fish (Gould, 2011).
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However, 5-HT1AR antagonist ligands bind with equal affinity to high agonist affinity (HA) and low agonist affinity (LA) sites of GPCRs, and cannot detect changes or differences in HA sites that are only a proportion of the total number of receptors.22 In contrast, agonist ligands preferentially bind to the HA receptor conformation thereby providing a more meaningful functional measure of 5-HT1AR transduction capacity.22 The ratio of [3H]WAY100,635: [3H]8-OH-DPAT or antagonist: agonist receptor binding in human neocortex is 1.7–2.7 across different brain regions, using quantitative autoradiography).23
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Most tracers belong to two structural families: (i) compounds with structural similarity to the 5-HT1A antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (e.g. WAY-100635); or (ii) derivatives of the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (Kumar and Mann, 2007; Pike et al., 2001). Among these, [3H] or [11C] labeled WAY-100635 has been the most commonly used 5-HT1AR antagonist ligand (Assem-Hilger et al., 2010; Gozlan et al., 1995; Hall et al., 1997; Parsey et al., 2000; Saijo et al., 2010; Sargent et al., 2010; Stein et al., 2008). WAY-100635 has more than 100-fold selectivity for 5-HT1AR vs. other receptors except for 5-HT2B, adrenergic α1A and dopamine D4 receptors (Chemel et al., 2006).
[¹¹C]CUMI-101 is the first selective serotonin receptor (5-HT_1AR) partial agonist radiotracer for positron emission tomography (PET) tested in vivo in nonhuman primates and humans. We evaluated specific binding of [³H]CUMI-101 by quantitative autoradiography studies in postmortem baboon and human brain sections using the 5-HT_1AR antagonist WAY-100635 as a displacer. The regional and laminar distributions of [³H]CUMI-101 binding in baboon and human brain sections matched the known distribution of [³H]8-OH-DPAT and [³H]WAY-100635. Prazosin did not measurably displace [³H]CUMI-101 binding in baboon or human brain sections, thereby ruling out [³H]CUMI-101 binding to α1-adrenergic receptors. This study demonstrates that [¹¹C]CUMI-101 is a selective 5-HT_1AR ligand for in vivo and in vitro studies in baboon and human brain.

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¹: Present address: INSERM U29, Hôpital Port-Royal, 75674 Paris cedex 14, France

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Regular paperThe selective 5-HT1A antagonist radioligand [3H]WAY 100635 labels both G-protein-coupled and free 5-HT1A receptors in rat brain membranes

Abstract

J. Biol. Chem.

Neuropharmacology

Eur. J. Pharmacol.

Neurosci. Lett.

Biochem. Pharmacol.

Trends Pharmacol. Sci.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Biol. Chem.

Neurochem. Int.

Biochem. Pharmacol.

Biochemical and electrophysiological evidence for an agonist action of CM 57493 at pre- and postsynaptic 5-HT1A receptors in brain

J. Pharmacol. Exp. Ther.

Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction

Biochem. Pharmacol.

S 14506: a novel, potent, high-efficacy 5-HT1A agonist and potential anxiolytic agent

Drug Dev. Res.

3H-TVX Q 7821: identification of 5-HT1 binding sites as target for a novel putative anxiolytic

Naunyn-Schmiedeberg's Arch. Pharmacol.

The genomic clone G-21 which resembles a β-adrenergic receptor sequence encodes the 5-HT1A receptor

Nature

A pharmacological profile of WAY-100655, a potent and selectives 5-HT1A receptor antagonist

Br. J. Pharmacol.

Kinetics and mechanisms. A study of homogenous chemical reactions

Regional studies of catecholamines in the rat brain. I. Disposition of [3H]norepinephrine [3H]dopamine, and [3H]dopa in various regions of the brain

J. Neurochem.

Regular paper
The selective 5-HT_1A antagonist radioligand [³H]WAY 100635 labels both G-protein-coupled and free 5-HT_1A receptors in rat brain membranes

Biochemical and electrophysiological evidence for an agonist action of CM 57493 at pre- and postsynaptic 5-HT_1A receptors in brain

Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC₅₀) of an enzymatic reaction

S 14506: a novel, potent, high-efficacy 5-HT_1A agonist and potential anxiolytic agent

³H-TVX Q 7821: identification of 5-HT₁ binding sites as target for a novel putative anxiolytic

The genomic clone G-21 which resembles a β-adrenergic receptor sequence encodes the 5-HT_1A receptor

A pharmacological profile of WAY-100655, a potent and selectives 5-HT_1A receptor antagonist

Regional studies of catecholamines in the rat brain. I. Disposition of [³H]norepinephrine [³H]dopamine, and [³H]dopa in various regions of the brain