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