Elsevier

Neuropharmacology

Volume 37, Issue 12, 1 December 1998, Pages 1611-1619
Neuropharmacology

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

https://doi.org/10.1016/S0028-3908(98)00117-8Get rights and content

Abstract

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (±)-pindolol (10 μM)-insensitive [3H]5-CT ([3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 μM) displayed a pharmacological profile similar to the recombinant 5-HT7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (±)-pindolol (10 μM)-insensitive [3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [3H]5-CT binding to residual, possibly, 5-HT1A sites. Competition for this [3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT7 receptor. Saturation studies also indicated that (±)-pindolol (10 μM)/WAY 100635 (100 nM)-insensitive [3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (Bmax=33.2±0.7 fmol mg−1 protein, pKd=8.78±0.05, mean±S.E.M., n=3). The development of this 5-HT7 receptor binding assay will aid investigation of the rat native 5-HT7 receptor.

Introduction

The most recent addition to the 5-HT (5-hydroxytryptamine, serotonin) receptor family is the 5-HT7 receptor for which the cDNA has been sequenced from a number of species (e.g. Xenopus laevis, mouse, rat, guinea pig and human) (Bard et al., 1993, Lovenberg et al., 1993, Meyerhof et al., 1993, Plassat et al., 1993, Ruat et al., 1993, Shen et al., 1993, Tsou et al., 1994, Nelson et al., 1995). More recently, it has been found that alternative mRNA splicing in rat and human tissue produces a number of 5-HT7 receptor isoforms, which display similar pharmacology, signal transduction and distribution (Heidmann et al., 1997, Jasper et al., 1997).

In situ hybridisation histochemistry has demonstrated that 5-HT7 receptor mRNA is localised discretely in rat and guinea pig brain with strong hybridisation signals detected in the thalamus, hypothalamus, hippocampus and cortical brain regions (Meyerhof et al., 1993, Ruat et al., 1993, To et al., 1995, Gustafson et al., 1996). Furthermore, autoradiographic studies report a similar distribution of radiolabelled 5-HT7 receptors (To et al., 1995, Gustafson et al., 1996) (but see below for rat brain).

Whilst the 5-HT7 receptor displays a unique pharmacology (Hoyer et al., 1994), only very recently have the identity of selective ligands been reported (Forbes et al., 1998) although these compounds are not yet widely available. It is interesting, however, that the 5-HT7 receptor displays high affinity for a number of typical and atypical antipsychotic drugs including clozapine (Plassat et al., 1993, Ruat et al., 1993, Shen et al., 1993, Roth et al., 1994, To et al., 1995, Jasper et al., 1997), which together with the expression of this receptor in limbic brain regions (Bard et al., 1993, Lovenberg et al., 1993, Meyerhof et al., 1993, Ruat et al., 1993, Shen et al., 1993, Tsou et al., 1994, To et al., 1995, Waeber and Moskowitz, 1995, Gustafson et al., 1996), has provoked speculation that the 5-HT7 receptor may provide a target for the treatment of affective disorders or in the mediation of side-effects associated with numerous psychotropic agents (Roth et al., 1994).

The development of a radioligand binding assay to selectively label a population of native receptors provides a useful method to study the pharmacology and distribution of the receptor as well as alterations in receptor expression following, for example, pharmacological manipulation. Furthermore, precise definition of the pharmacological profile in native tissue aids classification of receptor mediated responses. Previous studies have used [3H]5-HT (in the presence of the 5-HT1A/1B and β-adrenoreceptor ligand (±)-pindolol to reduce non-5-HT7 receptor binding) in an attempt to label native 5-HT7 receptors in rat hypothalamus. The results from these studies have revealed shallow competition curves for a number of compounds (Sleight et al., 1995, Gobbi et al., 1996; Sleight, personal communication; Stowe and Barnes, unpublished), indicating the labelling of a heterogeneous population of binding sites. Subsequently, the relatively more selective radioligand [3H]5-CT ([3H]5-carboxamidotryptamine; again in the presence of various blocking agents) has been utilised to selectively label a binding site in guinea pig brain which displays a pharmacological profile broadly comparable to the recombinant 5-HT7 receptor (To et al., 1995, Boyland et al., 1996). [3H]5-CT binding in rat brain, however, would appear to be more complex (Boyland et al., 1996) and the selective labelling of rat native 5-HT7 receptors has yet to be reported. Despite these shortcomings, readily acknowledged by the authors, [3H]5-CT (in the presence of PAPP (4-[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl]benzeneamide; 30 nM) and (-)-pindolol (160 nM) has been used in an attempt to demonstrate the autoradiographic distribution of 5-HT7 receptors in rat brain (Gustafson et al., 1996). A similar caveat was reported in an earlier study which also used [3H]5-CT (Waeber and Moskowitz, 1995). In addition, investigation of [3H]5-CT binding (in the presence of cyanopindolol and sumatriptan, both at 1 μM) in rat cerebral cortex homogenates, also revealed shallow competition curves for some competing compounds (e.g. methiothepin), indicating that [3H]5-CT also labels a heterogeneous receptor population in this preparation (Boyland et al., 1996; Middlemiss, personal communication).

In the present study we describe the development of an assay using [3H]5-CT to label an apparently homogenous population of binding sites in rat brain which display the pharmacology of the 5-HT7 receptor. Part of this work has been presented to the British Pharmacological Society (Stowe and Barnes, 1996, Stowe and Barnes, 1998).

Section snippets

Preparation of radioligand binding homogenate

Whole brains devoid of the striatum and cerebellum (these latter two nuclei display low levels of 5-HT7 receptors and the striatum expresses high levels of the 5-HT1B receptor which it was reasoned may increase the non-5-HT7 receptor binding of the radioligand) or hypothalami from male Wistar rats (250–300 g) were frozen at −80°C prior to the preparation of radioligand binding homogenate. To prepare the binding homogenate, brain tissue was defrosted at 4°C and homogenised in 30 volumes of

Pharmacological characterisation of [3H]5-CT binding to whole rat brain (minus cerebellum and striatum) homogenate

(±)-Pindolol (up to 100 μM) competed for approximately 80% of the specific [3H]5-CT (0.5 nM) binding (defined by 5-HT, 10 μM) to rat whole brain homogenate (total binding was approximately 7000 dpm), with half maximal (±)-pindolol competition occurring at 0.14±0.05 μM (Fig. 1; mean±S.E.M., n=6). Near maximal inhibition occurred at a concentration of 10 μM (±)-pindolol which was selected to subsequently use in the incubation buffer to block binding to presumed 5-HT1A and 5-HT1B receptors.

Discussion

The present studies optimised radioligand binding conditions to allow the selective labelling of an apparently homogenous population of sites in rat whole brain homogenate by [3H]5-CT which displayed the pharmacological profile of the 5-HT7 receptor.

Our initial studies attempted to follow a similar protocol to Sleight et al. (1995)who demonstrated that [3H]5-HT (in the presence of (±)-pindolol, 0.1 μM) labelled binding sites in rat hypothalamus homogenates that displayed a pharmacological

Acknowledgements

We are most grateful to Drs Derek N. Middlemiss and Andrew J. Sleight for contributing unpublished information. We would like to thank Glaxo, Janssen, Sandoz and Wyeth for the gifts of drugs. Miss Rebecca L. Stowe is recipient of a Wellcome Trust Prize Studentship.

References (29)

  • E Castro et al.

    Identification and characterisation of a new serotonergic recognition site with high affinity for 5-carboxamidotryptamine in mammalian brain

    Journal of Neurochemistry

    (1997)
  • I.T Forbes et al.

    (R)-3,N-Dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl)propyl]benzenesulfonamide): the first selective 5-HT7 receptor antagonist

    Journal of Medical Chemistry

    (1998)
  • M Gobbi et al.

    Are 5-hydroxytryptamine7 receptors involved in [3H]5-hydroxytryptamine binding to 5-hydroxtryptamine1nonA-nonB receptors in rat hypothalamus?

    Molecular Pharmacology

    (1996)
  • E.L Gustafson et al.

    A receptor autoradiographic and in situ hybridisation analysis of the distribution of the 5-HT7 receptor in rat brain

    British Journal of Pharmacology

    (1996)
  • Cited by (53)

    • Activation and blockade of serotonin<inf>7</inf> receptors in the prelimbic cortex regulate depressive-like behaviors in a 6-hydroxydopamine-induced Parkinson's disease rat model

      2015, Neuroscience
      Citation Excerpt :

      From these results, DA depletion plays an important role in the onset of depression in PD. There are growing data indicating that the PrL is an important brain region involved in the regulation of depression in rats (Hamani et al., 2010a,b; Scopinho et al., 2010; McLaughlin et al., 2013; Hui et al., 2014), and the neocortex, including the PrL, expresses a relatively high density of 5-HT7 receptors (Gustafson et al., 1996; Stowe and Barnes, 1998; Neumaier et al., 2001). Further, several studies have found that intraperitoneal or intrahippocampal administration of SB269970 produces antidepressant-like effects in the FST and tail suspension test in mice and rats (Hedlund et al., 2005; Wesolowska et al., 2006a,b).

    • Hindbrain raphe stimulation boosts cyclic adenosine monophosphate and signaling proteins in the injured spinal cord

      2014, Brain Research
      Citation Excerpt :

      The 5-HT receptor subtypes that stimulate adenylate cyclase (Gs-coupled receptors) are 5-HT4, 5-HT6 and 5-HT7. The 5-HT7 subtype is most strongly present in the spinal cord according to receptor binding studies (Doly et al., 2005; Raymond et al., 2001; Stowe and Barnes, 1998), although the other subtypes also appear (Gerard et al., 1996; Godinez-Chaparro et al., 2012; Waeber et al., 1994). It is associated with anti-nociception and locomotor rhythms (Dogrul et al., 2009; Liu et al., 2009).

    • Serotonin 5-HT<inf>7</inf> receptor agents: Structure-activity relationships and potential therapeutic applications in central nervous system disorders

      2011, Pharmacology and Therapeutics
      Citation Excerpt :

      These results were challenged by a subsequent study (Gobbi et al., 1996) which found that 100 nM pindolol does not completely mask 5-HT1A and 5-HT1B receptors and that the population of pindolol-insensitive [3H]5-HT receptors in rat hypothalamus appeared to be heterogeneous. Also [3H]5-CT failed to define a homogeneous population of 5-HT binding sites in rat hypothalamus homogenates despite the use of a ‘cocktail’ of drugs ((±)-pindolol, sumatriptan, DOI) (Stowe & Barnes, 1998). To et al. (1995) reported that a single population of receptors isolated from guinea pig cerebral cortex membranes could be labeled by [3H]5-CT in the presence of cyanopindolol and sumatriptan as masking agents.

    View all citing articles on Scopus
    View full text