[H]Methoxymethyl-3-[(2-methyl-1,3-thiazol-4- yl)ethynyl]pyridine Binding to Metabotropic Glutamate Receptor Subtype 5 in Rodent Brain: In Vitro and in Vivo Characterization

The binding of [H]methoxymethyl-3-[(2-methyl-1,3-thiazol-4yl)ethynyl]pyridine (methoxymethyl-MTEP), a potent and selective antagonist for metabotropic glutamate (mGlu)5 receptors, was characterized in rat brain both in vitro and in vivo. Nonspecific binding, as defined with 10 M 2-methyl-6-(phenylethynyl)-pyridine (MPEP), was less than 10% of total binding in rat brain membranes. The binding of [H]methoxymethyl-MTEP was of high affinity (Kd 20 2.7 nM), saturable (Bmax 487 48 fmol/mg protein), and to a single site. The mGlu5 antagonists methoxymethyl-MTEP and MPEP displaced [H]methoxymethyl-MTEP binding with IC50 values of 30 and 15 nM, respectively. In vivo administration of [H]methoxymethylMTEP (50 Ci/kg i.v.) revealed 12-fold higher binding in hippocampus (an area enriched in mGlu5 receptors) relative to cerebellum (an area with few mGlu5 receptors) in rats. Similarly, administration of [H]methoxymethyl-MTEP to mGlu5-deficient mice demonstrated binding at background levels in forebrain, whereas wild-type littermates exhibited 17-fold higher binding in forebrain relative to cerebellum. Systemic administration of unlabeled mGlu5 antagonists methoxymethyl-MTEP and MPEP to rats reduced the binding of [H]methoxymethyl-MTEP with ID50 values of 0.8 and 2 mg/kg i.p., respectively, 1 h posttreatment. The mGlu5 agonist 2-chloro-5-hydroxyphenylglycine (CHPG) (0.3, 1, and 3 mol) dose-dependently increased phosphoinositide (PI) hydrolysis in the hippocampus after i.c.v. administration in rats. CHPG-evoked increases in PI hydrolysis were blocked with MPEP at a dose (10 mg/kg i.p.) that markedly reduced [H]methoxymethyl-MTEP binding in vivo. These results indicate that [H]methoxymethyl-MTEP is a selective radioligand for labeling mGlu5 and is useful for studying the binding of mGlu5 receptors in rat brain in vitro and in vivo. Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors that are activated by glutamate and can modulate fast excitatory responses evoked by glutamatergic stimulation of ionotropic receptors (Conn and Pin, 1997). There are eight mGluR subtypes, which are subdivided into three groups principally based on sequence homology, but also on signal transduction pathways and agonist selectivity (Nakanishi, 1992; Pin and Duvoisin, 1995). Group I mGluRs initiate cell responses through Gq/11 protein coupling to phospholipase C and stimulation of phosphoinositide hydrolysis. In contrast, group II and group III mGluRs are negatively coupled via Gi/Go to adenylyl cyclase and reduce forskolinstimulated increases in cAMP in recombinant expression systems. Group I receptors are selectively activated by dihydroxyphenylglycine (DHPG), group II receptors can be stimulated by (2S,2 R,3 R)-2-(2 ,3 -dicarboxycyclopropyl)glycine and ( )-2-aminobicyclo[3.1.0]-hexane-2,6-dicarboxylate monohydrate (LY354740), and group III receptors are selectively stimulated by L-( )-2-amino-4-phosphonobutyric acid and (R,S)-4-phosphonophenylglycine (Schoepp et al., 1999). Group I mGluRs include the mGlu1 and mGlu5 subtypes. These two receptors exhibit a regional pattern of expression in the central nervous system, suggesting distinct, functional roles for each receptor (Spooren et al., 2001). For example, expression of mGlu5 receptor protein is high-to-moderate in frontal cortex, caudate putamen, nucleus accumbens, olfacArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.040618. ABBREVIATIONS: mGluR, metabotropic glutamate receptor; DHPG, 3,5-dihydroxyphenylglycine; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; MTEP, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine; CHPG, 2-chloro-5-hydroxyphenylglycine; PBS, phosphate-buffered saline; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]propanesulfonate; PI, phosphoinositide. 0022-3565/02/3033-1044–1051$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 40618/1025748 JPET 303:1044–1051, 2002 Printed in U.S.A. 1044 at A PE T Jornals on July 0, 2017 jpet.asjournals.org D ow nladed from tory tubercle, hippocampus, and dorsal surface of the spinal cord, whereas low levels of expression are observed in cerebellum (Shigemoto et al., 1993; Romano et al., 1995). In contrast, mGlu1 receptors are present in high density in the cerebellum and low-to-moderate expression is found in frontal cortex, caudate putamen, nucleus accumbens, and olfactory tubercle (Martin et al., 1992). Given the high level of expression of mGlu5 receptors in the limbic forebrain, these receptors are positioned to play key roles in emotional and behavioral processing. Selective and systemically available mGlu5 antagonists have recently been developed (Gasparini et al., 1999; Varney et al., 1999), and insight into the function of mGlu5 receptors in the brain is emerging. The selective mGlu5 antagonist MPEP produced anxiolytic effects in several rodent models of anxiety, including conditioned response tests (Vogel test, the four-plate test, fear-potentiated startle, and Geller-Seifter test), unconditioned response tests (social exploration, marble burying, and elevated plus-maze), and in stress-induced hyperthermia (Spooren et al., 2000, 2001; Tatarczynska et al., 2001; Brodkin et al., 2002). MPEP also blocked both the acquisition and expression of fear in rats (Schulz et al., 2001). These findings suggest a role for mGlu5 receptors in the generation of fear and anxiety and point to the therapeutic potential of mGlu5 antagonists in the treatment of anxiety. mGlu5 receptors may also be important in nociceptive processing because these receptors are localized to key points along the pain neuraxis (Jia et al., 1999; Walker et al., 2001b). In addition, intrathecal application of DHPG induces spontaneous nociceptive behaviors in rats (Fisher and Coderre, 1996) and mice (Karim et al., 2001) and also produces thermal hyperalgesia and allodynia in rats (Fisher and Coderre, 1998). mGlu5 antagonists have antinociceptive properties because MPEP blocked the responses of nociceptive neurons in the ventroposterolateral nucleus of the thalamus to pressure stimuli (Bordi and Ugolini, 2000) and reversed mechanical hyperalgesia in an inflammatory pain model (Walker et al., 2001a). Taken together, these results support a role for spinal and central mGlu5 receptors in modulating nociception and indicate that mGlu5 antagonists may be useful in the treatment of pain. Although the use of selective mGlu5 antagonists is becoming widespread in basic research, little is known about the in vitro binding affinities of these ligands (Gasparini et al., 2002) and the appropriate in vivo receptor occupancy required for efficacy in behavioral models. The present investigation describes the use of a novel radiolabeled mGlu5 antagonist, [H]methoxymethyl-MTEP, for determining the binding of compounds to mGlu5 receptors in brain both in vitro and in vivo. In vivo phosphoinositide hydrolysis was also used to compare in vivo receptor occupancy with the functional efficacy of the mGlu5 antagonists. Materials and Methods Male Sprague-Dawley rats (175–225 g) were purchased from Harlan (San Diego, CA) and mGlu5 knockout mice (20–25 g) (Lu et al., 1997) were purchased from Jackson Laboratories (Bar Harbor, ME). Wildtype littermates served as controls. All animals were group housed on a 12-h light/dark cycle with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee in accordance with The Guide for the Care and Use of Laboratory Animals. [H]Myo-inositol (17 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ). CHPG was obtained from Tocris Cookson (Ballwin, MO). All other compounds, including [H]methoxymethylMTEP (Fig. 1), were synthesized at Merck Research Laboratories (Cos-