The 5-Hydroxytryptamine6 Receptor-Selective radioligand [H]Ro 63–0563 Labels 5-Hydroxytryptamine Receptor Binding Sites in Rat and Porcine Striatum

Ro 63–0563 [4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)benzene sulfonamide] is a high affinity 5-hydroxytryptamine6 (HT6) receptor antagonist with more than 100-fold selectivity for the 5-HT6 receptor compared with 69 other receptors and binding sites. The present study describes the properties of [H]Ro 63–0563, the first selective 5-HT6 receptor radioligand. Specific binding of [H]Ro 63–0563 (nonspecific binding defined in the presence of 10 mM methiothepin) to recombinant rat and human 5-HT6 receptors was saturable, rapid, and reversible with equilibrium dissociation constants (Kd) of 6.8 nM and 4.96 nM, respectively. The pharmacological profile of the rat 5-HT6 receptor labeled with [ H]Ro 63–0563 (methiothepin . D-lysergic acid diethylamide . clozapine ; Ro 63–0563 . lisuride . ergotamine ; Ro 04–6790 . 5-HT . amitriptyline ; metergoline ; mianserin ; ritanserin . methysergide . mesulergine) was similar to that obtained by using either [H]Dlysergic acide diethylamide or [H]5-HT as radioligand. In equilibrium binding studies with rat striatal membranes, [H]Ro 63–0563 labeled a single binding site with Kd and Bmax values of 11.7 nM and 175 fmol/mg protein, respectively. In porcine striatal membranes, [H]Ro 63–0563 also labeled a single binding site with Kd and Bmax values of 8.0 nM and 130 fmol/mg protein, respectively. The affinities of 14 5-HT6 receptor ligands at this binding site were similar to those found for the recombinant rat and human 5-HT6 receptor, which suggested the presence of 5-HT6 receptors in porcine striatum. The effects of the neurotransmitter serotonin (5-HT) are mediated by at least 14 different receptors: one ligand-gated ion channel (the 5-HT3 receptor) and 13 G protein-coupled receptors (Boess and Martin, 1994; Hoyer and Martin, 1997). Of these, at least five are coupled to inhibition of adenylyl cyclase (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F), three are linked to phosphoinositide hydrolysis (5-HT2A, 5-HT2B, 5-HT2C), and three have been shown to stimulate adenylyl cyclase activity (5-HT4, 5-HT6, 5-HT7). Unlike the classical 5-HT receptors, the 5-HT6 receptor was not recognized as a pharmacological entity in physiological or radioligand binding experiments before its cloning from rat striatal cDNA (Monsma et al., 1993; Ruat et al., 1993). The highest levels of 5-HT6 receptor mRNA were detected in the olfactory tubercle, nucleus accumbens, striatum, and hippocampus (Monsma et al., 1993; Ruat et al., 1993; Ward et al., 1995; Gérard et al., 1996). To determine the distribution of 5-HT6 receptor protein, Gérard et al. (1997) raised polyclonal antibodies against a synthetic peptide corresponding to part of the carboxyl-terminal domain of the 5-HT6 receptor. They observed high levels of 5-HT6 receptor-like immunoreactivity in olfactory tubercle, piriform cortex, nucleus accumbens, islands of Calleja, striatum, hippocampus (CA1 and dentate gyrus), and the molecular layer of the cerebellum. Many nonselective compounds, including several tricyclic antidepressant drugs, antipsychotic agents, and tryptamine and ergoline derivatives interact with the 5-HT6 receptor, as was shown in binding studies on recombinant rat and human receptors using [H]LSD, I-LSD, and [H]5-HT as radioligands (Monsma et al., 1993; Roth et al., 1994; Boess et al., 1997; reviewed in Sleight et al., 1997). Putative 5-HT6-like receptors positively coupled to adenylyl cyclase have been described in the mouse neuroblastomaderived cell lines N18TG2 and NCB20 (Berry-Kravis and Dawson, 1983; Conner and Mansour, 1990; Unsworth and Molinoff, 1994). In addition, responses with a 5-HT6 receptorlike profile have been observed in pig caudate membranes (Schoeffter and Waeber, 1994) and in mouse embryonic striatal neurons (Sebben et al., 1994). Until recently, the physiological role of the 5-HT6 receptor was not known, because of the lack of selective ligands. However, the functional significance of this receptor has been investigated by using intracerebroventricular injections of 5-HT6 receptor-specific antisense oligonucleotides. This ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; LSD, D-lysergic acid diethylamide; Ro 63–0563, 4-amino-N-(2,6-bis-methylamino-pyridin-4-yl)benzene sulfonamide; Ro 04–6790, 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzene sulfonamide); HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 0026-895X/98/030577-07$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:577–583 (1998). 577 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from treatment, which should abolish or reduce the expression of 5-HT6 receptor protein, produced a behavioral syndrome consisting of yawning, stretching, and chewing (Bourson et al., 1995; Sleight et al., 1996). Recently, we have described two selective 5-HT6 receptor antagonists, Ro 04–6790 and Ro 63–0563, and demonstrated the ability of Ro 04–6790 to produce behavioral effects similar to those observed after antisense treatment (Sleight et al., 1998). In the current study, we present the first selective 5-HT6 receptor radioligand [ H]Ro 63–0563 and demonstrate that it labels recombinant rat and human 5-HT6 receptors as well as 5-HT6 receptor binding sites in rat and porcine striatal membranes. Experimental Procedures Materials. HeLa cells expressing human 5-HT6 receptors were obtained from Dr. David Sibley (National Institutes of Health, Bethesda, MD) under licensing agreement. 5-HT was purchased from Fluka (Buchs, Switzerland); ergotamine from Sigma (Buchs, Switzerland); mesulergine, metergoline, methysergide, lisuride, methiothepin, clozapine, amitriptyline, ritanserin, mianserin, and pargyline from Research Biochemicals (Natick, MA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, streptomycin, and geneticin were obtained from Gibco Life Technologies (Basel, Switzerland). Ro 20–1724, Ro 63–0563 (Fig. 1), Ro 04–6790 (Fig. 1), and LSD were synthesized at F. Hoffmann-La Roche AG (Basel, Switzerland). [H]Ro 63–0563 (specific activity, 29 Ci/mmol) was kindly prepared by Dr. Philipp Huguenin at F. Hoffmann-La Roche (Basel). Preparation of membranes from cells expressing recombinant 5-HT6 receptor. Membranes were prepared from HEK 293 cells stably transfected with the rat 5-HT6 receptor (Boess et al., 1997) or HeLa cells stably expressing a human 5-HT6 receptor clone (Kohen et al., 1996). HEK 293 cells were grown in Dulbecco’s modified Eagle medium 1 10% fetal bovine serum containing penicillin (100 IU/ml), streptomycin (100 mg/ml), and 0.5 mg/ml geneticin in a humidified atmosphere (5% CO2). HeLa cells were grown in exactly the same conditions except that geneticin was omitted from the media. The cells were detached with phosphate-buffered saline containing 1 mM EDTA, washed with phosphate-buffered saline (Gibco Life Technologies) by two centrifugations (10 min, 500 3 g) and the resulting pellet was resuspended in ice-cold 50 mM TriszHCl, pH 7.4, containing 10 mM MgCl2 and 0.5 mM EDTA by using a Polytron homogenizer (15 sec at maximal speed) at a concentration corresponding to 2 3 10 cells/ml. This homogenate was centrifuged at 100,000 3 g for 60 min, the resulting pellet was resuspended in the same buffer to obtain a concentration corresponding to 4 3 10 cells/ml, and aliquots were stored at 280°. Preparation of rat and porcine striatal membranes. Before dissection, rat brains (male Ibm:RoRo; Biological Research Laboratories, Füllinsdorf, Switzerland) and porcine brains (from a local abattoir) were kept on ice. Striatal tissue (in 3-g aliquots) was homogenized in 26 ml of 0.32 M sucrose with a glass/teflon homogenizer and centrifuged for 10 min at 1000 3 g (4°). The pellet was discarded and the supernatant centrifuged for 30 min at 25,000 3 g. The pellet was resuspended in 26 ml of 50 mM TriszHCl, pH 7.4, centrifuged for 15 min at 25,000 3 g, resuspended in 50 mM TriszHCl, incubated for 15 min at 37° and centrifuged for 15 min at 25,000 3 g. The pellet was washed two times by resuspension in 50 mM TriszHCl and centrifugation for 15 min at 25,000 3 g and the final pellet was stored frozen at 280°. [H]Ro 63–0563 binding assays. Membranes prepared from cells expressing recombinant rat and human 5-HT6 receptor were resuspended in assay buffer (50 mM TriszHCl, 10 mM pargyline, 5 mM MgCl2, 0.5 mM EDTA, and 0.1% ascorbic acid, pH 7.4). Binding assays consisted of 100 ml of membrane suspension (corresponding to 4 3 10 cells per assay tube), 50 ml of [H]Ro 63–0563 (specific activity, 29 Ci/mmol), and 50 ml of displacing drug or assay buffer (final assay volume, 200 ml). Nonspecific binding was measured in the presence of 10 mM methiothepin. Saturation experiments were performed using eight concentrations of [H]Ro 63–0563 (final concentrations of 0.31 nM to 40 nM). In competition assays, seven to 14 concentrations of the displacing ligands were tested (3 3 10 to 10 M) at a final [H]Ro 63–0563 concentration of 5 nM. At this concentration, specific binding corresponded to 70% of total binding. Incubations were performed at room temperature for 80 min. Association and dissociation experiments were conducted in the presence of 5 nM [H]Ro 63–0563. Association experiments were carried out by incubating samples with [H]Ro 63–0563 in the absence or presence of 10 mM methiothepin for 1 min to 120 min. Dissociation was initiated by addition of 10 mM methiothepin after equilibration with [H]Ro 63–0563 for 120 min and allowed to proceed for 1 to 120 min. For binding experiments with rat and porcine striatal membranes, the pellets were resuspended in 26 ml of assay buffer. Binding assays were performed using 0.5 ml of the membrane suspension (corresponding to 1.5 mg of protein per assay tube) in a final assay volume of 1 ml using a shaking incubator. Otherwise, the assay conditions and the ligand concentrations examined were similar to the experiments with recombinant 5-HT6 receptors with the exception of the final concentration of [H]Ro 63–0563 used in the competition and kinetic experiments (2 nM). At this concentration, specific binding corresponded to 20% of total binding. The incubations were terminated by rapid filtration through Whatmann GF/B filters pretreated with polyethyleneimine (0.3%). The filters were washed with 3 3 2 ml of cold TriszHCl (50 mM, pH 7.4) and the radioactivity retained on the filters was measured by liquid scintillation counting in 2 ml of scintillation fluid. All experiments were performed in triplicate and repeated at least three times (unless indicated otherwise). Values are given as mean 6 standard error. Data were analyzed using the programs EBDA and LIGAND (Munson and Rodbard, 1980; McPherson, 1985). Protein concentrations were determined using either the bicinchoninic acid method (Pierce, Munich, Germany) or a Coomassie Brilliant Blue G-250 based assay (Biorad).