[3H] -SB-269970 radiolabels 5-HT7 receptors in rodent, pig and primate brain tissues

Neuropharmacology 42 (2002) 74–81 www.elsevier.com/locate/neuropharm

[3H]-SB-269970 radiolabels 5-HT7 receptors in rodent, pig and primate brain tissues D.R. Thomas *, P.J. Atkinson, P.G. Hastie, J.C. Roberts, D.N. Middlemiss, G.W. Price GlaxoSmithKline Pharmaceuticals, Psychiatry Centre of Excellence for Drug Discovery, New Frontiers Science Park (North), Third Avenue, Harlow, Essex CM19 5AW, UK Received 6 June 2001; received in revised form 29 August 2001; accepted 19 September 2001

Abstract The selective 5-HT7 receptor antagonist radioligand, [3H]-SB-269970, has been reported to radiolabel the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cortex (Thomas et al., 2000). Saturation analysis of [3H]-SB-269970 binding to mouse forebrain, rat cortex, pig cortex, marmoset cortex and human thalamus membranes was consistent with labelling a homogenous population of binding sites in each tissue. KD values for [3H]-SB-269970 binding in these tissues ranged from 0.9 to 2.3 nM, being similar to those reported for the human cloned and guinea pig cortex 5-HT7 receptors (1.3 and 1.7 nM, respectively). Bmax values for [3H]-SB-269970 binding to the mouse, rat, pig, marmoset and human brain membranes were 20, 30, 31, 14 and 68 fmoles mg protein⫺1, respectively. For each species the profile of inhibition of [3H]-SB-269970 binding, using a number of 5HT7 receptor agonists and antagonists, correlated well with that reported for the human cloned 5-HT7(a) receptor (correlation coefficients were 0.95, 0.94, 0.92, 0.95, 0.97 versus the mouse, rat, pig, marmoset and human tissues, respectively). In conclusion, [3H]-SB-269970 has been shown to radiolabel 5-HT7 receptors in rodent, pig and primate brain and represents a valuable tool with which to further characterise the distribution and function of 5-HT7 receptors in native tissues and elucidate their potential role in disease states.  2002 Elsevier Science Ltd. All rights reserved. Keywords: 5-HT7 receptors; Radioligand binding; [3H]-SB-269970; Brain binding; Rodent; Pig; Primate

1. Introduction 5-HT receptors have been sub-divided into seven major classes (5-HT1–7), based on structural, functional and pharmacological criteria (Hoyer et al., 1994). To date, the 5-HT7 receptor has been cloned from mouse (Plassat et al., 1993), rat (Lovenberg et al., 1993; Ruat et al., 1993), guinea pig (Tsou et al., 1994) and human (Bard et al., 1993) and displays a pharmacological profile which appears consistent across species. A number of splice variants of both the human (5-HT7a/b/d) and rat (5-HT7a/b/c) receptor have been identified, which display similar pharmacological and functional characteristics when expressed in cell lines (Heidman et al., 1997; Jasper et al., 1997). 5-HT7 receptors have been shown to

* Corresponding author. Tel.: +44-1279-622468; fax: +44-1279622230. E-mail address: [email protected] (D.R. Thomas).

be positively coupled to adenylyl cyclase (AC) when expressed in cell lines (Bard et al., 1993). 5-HT7 receptor mRNA has been shown to be present in peripheral tissues, including cardiovascular and gastrointestinal smooth muscle and also in brain (Bard et al., 1993; Schoeffter et al., 1996; Hagan et al., 2000). Comparative in situ hybridisation and autoradiography studies in guinea pig and rat brain have revealed a similar distribution pattern for 5-HT7 mRNA and receptor binding sites, with the highest receptor density in thalamic and limbic regions (Branchek et al., 1994; To et al., 1995; Gustafson et al., 1996). As seen in recombinant systems, positive coupling of the receptor to AC has been reported in native (guinea pig hippocampus) tissue (Tsou et al., 1994; Thomas et al., 1999) Based on brain localisation and pharmacological studies with non-selective compounds, it has been suggested that 5-HT7 receptors may play a role in the control of circadian rhythms (Lovenberg et al., 1993; Tsou et al., 1994; Ying and Rusak, 1997) and may have potential

0028-3908/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 1 ) 0 0 1 5 1 - 4

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therapeutic applications in depression (Sleight et al., 1995; Hagan et al., 2000) and schizophrenia (Roth et al., 1994). A majority of radioligand binding studies to characterise 5-HT7 receptors in recombinant and native tissue systems have used the agonist radioligands [3H]-5-HT (Bard et al., 1993; Tsou et al., 1994; Sleight et al., 1995; Clemett et al., 1999) or [3H]-5-carboxamidotryptamine ([3H]-5-CT) (To et al., 1995; Boyland et al., 1996; Stowe and Barnes, 1998; Thomas et al. 1998, 1999). These radioligands have proved useful in characterising the pharmacological profile of cloned and native tissue 5HT7 receptors, as well as providing information on their tissue distribution, particularly in brain. However, neither [3H]-5-HT nor [3H]-5-CT selectively labels 5HT7 receptors and in native tissues it has therefore been necessary to include selective blocking compounds to prevent labelling of non-5-HT7 receptor binding. The antagonist radioligand [3H]-mesulergine has been reported to radiolabel 5-HT7 receptors in rat brain and guinea pig ileum (Hemedah et al., 1999). However, [3H]-mesulergine also lacks selectivity for 5-HT7 receptors, displaying additional affinity for 5-HT2A, 5-HT2C, dopamine D2 and alpha1 and alpha2 adrenergic receptors. These studies have, therefore, also employed selective blocking compounds to prevent labelling of non-5-HT7 receptor binding sites. There has, therefore, been a need for a selective antagonist radioligand to aid in the characterisation of 5-HT7 receptors in native tissues. SB-269970 ((R)-3-(2-(2-(4methyl-piperidin-1-yl)ethyl)-pyrrolidine-1-sulphonyl)phenol) has recently been reported to be a potent 5-HT7 receptor antagonist (Hagan et al., 2000), displaying at least 100-fold selectivity versus all other 5-HT receptor sub-types except the human 5-ht5A receptor (50-fold, Lovell et al., 2000). SB-269970 has therefore been tritiated (specific activity 49 Ci mmol⫺1) and the binding of this radioligand to both the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cerebral cortex has recently been characterised (Thomas et al., 2000). The present study was carried out to investigate [3H]-SB-269970 binding to brain tissue from a number of other mammalian species, namely, mouse, rat, pig, marmoset and human. Some of these data have been presented previously in abstract form (Atkinson et al., 2000a,b).

2. Methods 2.1. Membrane preparations Rat (male, Sprague-Dawley, 200–300 g) cerebral cortex, pig cerebral cortex (obtained from a local abattoir), mouse forebrain (whole brain minus cerebellum, male C57b6/Sv129 hybrid strain, 20–30 g), marmoset cortex

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(from single male, 306 g) and human thalamus (single male subject, age 76, cause of death myocardial infarct, post mortem delay 30 h) were homogenised (Polytron, 15s, setting 5) in 20 volumes (based on wet weight of tissue) of 50 mM Tris (pH 7.4 at 37°C) containing 0.5 mM EDTA. Following centrifugation (50,000×g, 12 min, 4°C) pellets were resuspended in the same medium and incubated at 37°C for 20 min. After three further centrifugation and resuspension steps, membranes were stored at ⫺80°C. 2.2. [3H]-SB-269970 brain membrane binding assay Membranes (150–750 µg protein tube⫺1) were incubated in Tris–HCl buffer (50 mM, pH 7.4 at 37°C) containing CaCl2 (4 mM), pargyline (0.1 mM) and ascorbic acid (1 mM) with [3H]-SB-269970 (1–1.5 nM for competition analysis and six to eight concentrations within the range 0.1–14 nM for saturation analysis) in the presence or absence of test drugs for 60 min at 37°C. For both saturation and competition analyses non-specific binding was determined using 10 µM 5-HT. Incubation was terminated by rapid filtration through Whatman GF/B grade filters (pre-soaked with 0.3% polyethyleneimine) followed by 5×1 ml ice-cold buffer washes and bound radioactivity was determined by liquid scintillation counting. 2.3. Data analysis Analysis of saturation data was performed by non-linear curve fitting using KELL (Biosoft). Saturation binding data were analysed according to the equation

冘

(i)⫽n

Bmax(i)[F] ⫹N[F] K +[F] (i)⫽1 d(i)

[BT]⫽

where [BT] is total observed binding, and N is the ratio of bound/free at infinite free concentration. This analysis provided estimates for KD (equilibrium dissociation constant) and Bmax (maximal binding density) for the radioligand in all brain preparations. The concentration of drug inhibiting specific [3H]-SB-269970 binding by 50% (IC50) and Hill coefficient (nH) was determined by iterative curve fitting (Bowen and Jerman, 1995). pKi values (⫺log of the inhibition constant) were then generated from the IC50 values as described by Cheng and Prusoff (1973). Correlation plots were analysed by linear regression (grafit, Erithacus Software). Data represent the mean±s.e. mean of at least three separate experiments carried out using duplicate (inhibition experiments) or triplicate (saturation experiments) determinations.

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2.4. Chemicals 5-Carboxamidotryptamine (5-CT), 5-hydroxytryptamine (5-HT), 8-hydroxy-dipropylaminotetralin (8-OHDPAT), risperidone, methiothepin mesylate, mesulergine HCl, clozapine, ketanserin tartrate, methysergide maleate and ritanserin were obtained from Sigma-Aldrich (Poole, UK). Methyl-ergometrine maleate was obtained from Tocris Cookson Ltd (Bristol, UK). SB269970-A ((R)-3-(2-(2-(4-methyl-piperidin-1-yl)ethyl)pyrrolidine-1-sulphonyl)-phenol)) HCl and sumatriptan were synthesised at SmithKline Beecham (Harlow, UK). [3H]-SB-269970 (specific activity 49 Ci/mmol) was obtained from the Synthetic Isotope Unit, SmithKline Beecham (Upper Merion, USA). [3H]-5-carboxamidotryptamine (specific activity 50–100 Ci/mmol) was obtained from Amersham, UK. Stock compound dilutions were prepared fresh on the day of assay in DMSO (the final assay concentration of DMSO not exceeding 0.4%). Compound dilutions were prepared in 5 mM Tris Buffer (pH 7.4 at 37°C) containing 0.5 mM ascorbic acid.

3. Results 3.1. Saturation analysis of [3H]-SB-269970 binding to mouse, rat, pig, marmoset and human brain [3H]-SB-269970 (0.1–12 nM) bound saturably and monophasically to mouse forebrain, rat, pig and marmoset cortex and human thalamus membranes, giving KD values which were, in each case, comparable to the KD values reported previously for binding to guinea pig cortex and human recombinant 5-HT7(a) receptors (Thomas et al., 2000) (Table 1). For each tissue, statistical analysis of the saturation binding data (using KELL) did not significantly favour a multi-site versus a singleTable 1 KD and Bmax values from saturation analysis of [3H]-SB-269970 binding to brain tissue homogenates from various species Tissue

KD (nM)

Bmax (fmoles mg protein⫺1)

h5-HT7(a)/HEK293 Guinea pig cortex Mouse forebrain Rat cortex Pig cortex Marmoset cortex Human thalamus

1.3±0.1a 1.7±0.3a 0.9±0.2 0.9±0.1 1.0±0.3 1.8±0.2 2.3±0.4

5800±380a 125±8.2a 20±3.3 30±2.1 31±7.8 14±1.4 68±2.0

a

Data from Thomas et al. (2000). KD (equilibrium dissociation constant) and Bmax (maximal binding density) values were calculated from saturation analysis of [3H]-SB-269970 binding (0.1–12 nM). Data represent the mean±s.e. mean of at least three separate experiments each performed using duplicate determinations.

site interaction. Fig. 1(a)–(e) shows saturation binding data (and Scatchard plots of the same data) from typical experiments for [3H]-SB-269970 binding to mouse forebrain, rat cortex, pig cortex, marmoset cortex and human thalamus membranes. At least three separate saturation experiments were carried out in each case. For each species, non-specific binding (defined in the presence of 10 µM 5-HT) increased linearly with increasing radioligand concentration (data not shown). Specific binding, at a [3H]-SB-269970 concentration at or close to 1 nM, represented 44.8±3.1, 44.5±3.2, 49.6±4.5, 37±5.2 and 61±3.4% of total binding to the mouse, rat, pig, marmoset and human brain tissues, respectively. Bmax values for [3H]-SB-269970 binding to mouse forebrain and rat, pig and marmoset cortex membranes were between fourand nine-fold lower than previously reported for [3H]SB-269970 binding to guinea pig cortex membranes (Thomas et al., 2000) (Table 1). 3.2. Pharmacological characterisation of [3H]-SB269970 binding to mouse, rat, pig, marmoset and human brain The pharmacological profile of [3H]-SB-269970 binding to mouse forebrain, rat, pig and marmoset cortex and human thalamus membranes was investigated using a range of 5-HT7 receptor agonists and antagonists. In each case the pharmacological profile was consistent with selective labelling of 5-HT7 receptors. For example, SB-269970-A and 5-CT potently inhibited [3H]-SB269970 binding in each species, whilst mesulergine and clozapine were moderately potent inhibitors (Table 2). Fig. 2(a)–(e) shows representative data for compound inhibition of [3H]-SB-269970 binding to each of the above tissues. Hill slopes for inhibition of [3H]-SB269970 binding were, in each species, not significantly different to 1, consistent with binding to a single population of receptors. In addition, for each species, the profile of inhibition of [3H]-SB-269970 binding correlated well with that reported for the human cloned 5-HT7(a) receptor. Linear regression analysis of affinity (pKi) values determined in the native tissue and cloned receptor systems gave correlation coefficients of 0.95, 0.94, 0.92, 0.95 and 0.97, respectively, for mouse, rat, pig, marmoset and human brain tissues compared to the human cloned 5-HT7(a) receptor (Fig. 3(a)–(e)). Corresponding slope factors were, respectively, 0.95, 0.97, 0.95, 0.94 and 1.2 for mouse, rat, pig, marmoset and human tissue.

4. Discussion Radioligand binding studies to characterise 5-HT7 receptors in recombinant and native tissues have, to date, mainly utilised non-selective radioligands, including the

D.R. Thomas et al. / Neuropharmacology 42 (2002) 74–81

Fig. 1. Saturation analysis and corresponding Scatchard plot of [3H]-SB-269970 binding to (a) mouse forebrain, (b) rat cerebral cortex (c) pig cerebral cortex (d) marmoset cortex and (e) human thalamus membranes. Data points show specific binding, calculated by subtracting the non-specific binding (defined in the presence of 10 µM 5HT) from total binding. Data are from typical experiments performed using triplicate determinations.

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agonist radioligands [3H]-5-HT (Bard et al., 1993; Tsou et al., 1994; Sleight et al., 1995; Clemett et al., 1999) and [3H]-5-CT (To et al., 1995; Boyland et al., 1996; Stowe and Barnes, 1998; Thomas et al. 1998, 1999) and the antagonist radioligand [3H]-mesulergine (Hemedah et al., 1999). Although such studies have provided useful information on the pharmacological profile and tissue localisation of 5-HT7 receptors, the use of non-selective radioligands has necessitated the inclusion of blocking drugs to mask binding to non-5-HT7 sites. [3H]-SB269970 has recently been identified as a selective antagonist radioligand, which has been used to radiolabel both the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cortex membranes (Thomas et al., 2000). The present study was carried out to investigate [3H]SB-269970 binding to brain tissue homogenates from a number of other species, namely, mouse, rat, pig, marmoset and human. [3H]-SB-269970 bound saturably to mouse forebrain and rat, pig and marmoset cortex, in each case labelling an apparently homogenous population of sites. KD values were, in each case, similar to those reported for binding to human recombinant 5HT7(a) receptors and 5-HT7 receptors in guinea pig cerebral cortex (Thomas et al., 2000). In contrast, Bmax values for [3H]-SB-269970 binding to the mouse forebrain and rat, pig and marmoset cortex tissues (Table 1) were at least four-fold lower than that reported in guinea pig cortex (Thomas et al., 2000). The Bmax determined in the present study in rat cortex is similar to the value of 33 fmoles mg protein⫺1 reported in rat whole brain membranes by Stowe and Barnes (1998) using [3H]-5CT (in the presence of 10 µM pindolol and 100 nM WAY-100635). Preliminary characterisation of [3H]-SB-269970 binding to human cerebral cortex membranes suggested a very low level of 5-HT7 receptor expression that could not be accurately quantified. Since 5-HT7 mRNA has been reported to be expressed in human cortex (Hagan et al., 2000), it is likely that the 5-HT7 receptor is expressed in this tissue, but that the level is too low to quantify using [3H]-SB-269970. Instead, human brain [3H]-SB-269970 binding studies were carried out using thalamic membranes. This region was chosen since autoradiographic studies in guinea pig and rat brain had reported relatively high 5-HT7 receptor levels in this region (To et al., 1995; Gustafson et al., 1996). Furthermore, a relatively high level of 5-HT7 mRNA has been reported in human thalamus, in addition to hypothalamus, hippocampus and amygdala (Hagan et al., 2000). [3H]-SB-269970 bound saturably and monophasically to human thalamic membranes. As seen for the mouse, rat, pig and marmoset tissues, the KD for [3H]-SB-269970 binding to human thalamic membranes was comparable to that reported for [3H]-SB-269970 binding to the human cloned 5-HT7(a) receptor (Table 1). The maximal binding density (Bmax) for [3H]-SB-269970 binding to

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Table 2 Potency of compounds to inhibit [3H]-SB-269970 binding to brain tissue from various species Compound

Mouse forebrain

Rat cortex

pKi

nH

pKi

nH

pKi

1.0±0.1 1.0±0.4 0.8±0.2 1.2±0.3

9.4±0.1 8.5±0.1 7.1±0.1 6.3±0.1

1.1±0.1 0.9±0.1 1.0±0.1 0.9±0.1

1.2±0.1 1.3±0.2 0.9±0.1 1.1±0.1 1.2±0.4 1.2±0.4 1.1±0.1 1.1±0.3

9.2±0.1 7.9±0.1 8.4±0.1 7.5±0.1 7.4±0.1 6.9±0.1 7.1±0.2 6.3±0.1

1.0±0.1 1.2±0.1 1.1±0.2 1.2±0.1 1.2±0.1 1.2±0.1 1.3±0.2 1.1±0.2

Agonists 5-CT 9.3±0.1 5-HT 8.5±0.1 8-OH-DPAT 7.2±0.1 Sumatriptan 6.3±0.1 Antagonists SB-269970 9.1±0.1 Methiothepin 8.0±0.2 Risperidone 8.4±0.1 Methysergide 7.5±0.1 Mesulergine 7.9±0.1 Clozapine 7.1±0.1 Ritanserin 6.8±0.1 Ketanserin 6.3±0.3

Pig cortex

Marmoset cortex

Human thalamus

nH

pKi

nH

pKi

nH

9.4±0.1 8.7±0.1 6.9±0.1 6.3±0.1

0.9±0.1 0.9±0.2 0.9±0.2 0.9±0.1

9.5±0.1 8.8±0.1 7.1±0.2 –

0.9±0.1 1.0±0.2 1.2±0.1 –

9.4±0.2 8.7±0.1 7.1±0.1 –

1.0±0.1 0.9±0.1 0.9±0.1 –

9.0±0.1 7.8±0.1 8.6±0.1 7.5±0.1 7.7±0.1 6.8±0.1 6.9±0.1 6.2±0.2

1.1±0.2 1.1±0.1 1.1±0.1 1.1±0.1 1.1±0.1 0.9±0.1 1.3±0.2 1.0±0.1

8.9±0.1 8.2±0.2 – – 7.4±0.1 7.0±0.1 – 6.3±0.1

0.9±0.1 1.1±0.2 – – 1.2±0.2 1.2±0.3 – 1.1±0.1

8.8±0.1 – – – 7.7±0.2 7.0±0.1 7.0±0.2 –

1.0±0.1 – – – 1.0±0.2 1.2±0.1 1.1±0.1 –

pKi (⫺log inhibition constant) and nH (Hill coefficient) values from [3H]-SB-269970 binding experiments. Data are the mean±s.e. mean from at least three separate experiments each performed using duplicate determinations; (–) not determined.

human thalamus of 68 fmoles mg protein⫺1 was higher than that reported in a previous study of [3H]-SB-269970 binding to human thalamus (33 fmoles mg protein⫺1, Atkinson et al., 2000b). It is possible that differences in post mortem delay, tissue handling and storage could give rise to differences in tissue viability and in turn could explain the differences in Bmax values. The profile of inhibition of [3H]-SB-269970 binding to the mouse, rat, pig, marmoset and human brain tissues, defined using a range of 5-HT7 receptor agonists and antagonists, correlated well with that reported for [3H]-SB-269970 binding to the human cloned 5-HT7(a) receptor (Thomas et al., 2000). For example, 5-CT and SB-269970-A potently inhibited binding in each species, whereas 8-OH-DPAT and clozapine showed lower affinity. Also, in each species, Hill slopes for compound inhibition of [3H]-SB-269970 binding were close to unity, consistent with binding to a single population of receptors. Furthermore, correlation plots comparing compound potencies to inhibit [3H]-SB-269970 binding to the human cloned 5-HT7(a) receptor versus the mouse, rat, pig, marmoset and human brain tissues revealed correlation coefficients between 0.92 and 0.97, consistent with the similar pharmacological profile in the cloned and native tissue systems. Overall, these data are consistent with [3H]-SB-269970 radiolabelling 5-HT7 receptors in mouse, rat, pig, marmoset and human brain and suggest that the pharmacological profile is consistent across these species. SB-269970-A has been shown to display at least 100fold selectivity for human 5-HT7 receptors versus a

range of other human 5-HT and non-5-HT receptor subtypes, apart from the 5-ht5A receptor, over which it displays approximately 50-fold selectivity (Lovell et al., 2000). This 50-fold or greater selectivity profile suggests that, at the radioligand concentrations used in the present study, [3H]-SB-269970 would be unlikely to radiolabel 5-ht5A or other non-5-HT7 receptor sub-types in the brain tissues investigated. In support of this, [3H]-SB-269970 binding to mouse, rat, pig, marmoset and human brain appeared monophasic in profile, consistent with binding to a single population of binding sites. In addition, as already mentioned, in each tissue Hill slopes for drug inhibition of [3H]-SB-269970 binding were not significantly different from 1, consistent with inhibition of binding to a single population of binding sites. Furthermore, available literature data suggest that the density of 5-ht5A receptor binding sites in brain appears to be no higher than that reported for 5-HT7 receptors (Grailhe et al., 1999). Taken together these findings suggest that [3H]-SB-269970 radiolabels, selectively, 5-HT7 receptors in the brain tissues investigated in the present study. A number of splice variants of both the human (5-HT7a/b/d) and rat (5-HT7a/b/c) receptors have been identified. Studies to date suggest that these splice variants display similar pharmacological and functional characteristics when expressed in cell lines (Heidman et al., 1997; Jasper et al., 1997). Although the relative affinity of [3H]-SB-269970 for the different human and rat splice variants has not been investigated to date, the apparently monophasic saturation binding profile seen in both rat cortex and human thalamus appears to suggest

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Fig. 2. Inhibition of 1 nM [3H]-SB-269970 binding to (a) mouse forebrain, (b) rat cerebral cortex, (c) pig cerebral cortex (d) marmoset cortex and (e) human thalamus by 5-CT, SB-269970-A, 5-HT, mesulergine and clozapine. Data points represent the mean±s.e. mean of at least three separate experiments. Results are expressed as per cent of specific binding where non-specific binding was defined using 10 µM 5-HT.

Fig. 3. Correlation plots of drug potencies (expressed as pKi; values shown in Table 2) to inhibit [3H]-SB-269970 binding to (a) mouse forebrain, (b) rat cerebral cortex, (c) pig cerebral cortex (d) marmoset cortex and (e) human thalamus membranes. Data points shown are for agonists (앩) and antagonists (쐌).

that [3H]-SB-269970 does not distinguish between the rat and human splice variants. The brain regional distribution of 5-HT7 receptors has previously been investigated in both guinea pig and rat using [3H]-5-CT autoradiography (To et al., 1995; Waeber and Moskowitz, 1995; Gustafson et al., 1996). Since [3H]-5-CT does not selectively label 5-HT7 receptors, it has been necessary in these studies to include selective blocking compounds to prevent labelling of non-5-HT7 binding. We have recently reported initial studies suggesting that [3H]-SB-269970 labels, autoradiographically, 5-HT7 receptors in guinea pig brain (Roberts et al., 2000). Specific [3H]-SB-269970 binding was localised mainly in cerebral cortex, hippocampus (CA regions) and thalamus, with lower levels seen in

caudate nucleus and cerebellum. This distribution pattern is consistent with previous [3H]-5-CT autoradiography studies and mRNA localisation studies in guinea pig brain (To et al., 1995). [3H]-SB-269970 autoradiographic studies in rat brain have, to date, been unsuccessful, due to a low level of specific [3H]-SB-269970 binding in this species. This is consistent with the relatively low 5-HT7 expression levels measured in the present study in rat cortex, compared to the levels previously reported in guinea pig cortex (Thomas et al., 2000). The above findings suggest that the guinea pig displays markedly higher brain 5-HT7 receptor expression levels compared to rat, as well as mouse and pig. The relevance of this apparent species difference is unclear. To date, there have been no reports of species differences in the function of brain

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5-HT7 receptors. The data presented in the present study suggest that, in brain regions where the receptor density is very low, a higher specific activity, selective 5-HT7 receptor radioligand is required to further investigate the distribution and pharmacology of 5-HT7 receptors. In conclusion, [3H]-SB-269970 has been shown to radiolabel, with high affinity, 5-HT7 receptors in rodent, pig, marmoset and human brain. [3H]-SB-269970 is the first selective antagonist radioligand for 5-HT7 receptors which represents a valuable tool with which to further characterise 5-HT7 receptors in native tissues.

Acknowledgement The authors would like to thank Professor Norman Bowery for supplying human brain tissue.

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