Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism.

The metabotropic glutamate receptors (mGluRs) are a family of G-protein linked receptors that can be divided into three groups (group I, II and III). A number of studies have implicated group I mGluR activation in acute neuronal injury, but until recently it was not possible to pharmacologically differentiate the roles of the two individual subunits (mGluR1 and mGluR5) in this group. We investigated the role of mGluR5 in acute NMDA and glutamate mediated neurodegeneration in cultured rat cortical cells using the mGluR5 antagonists MPEP and SIB-1893, and found that they provide significant protection at concentrations of 20 or 200 μM. These compounds act as effective mGluR5 antagonists in our cell culture system, as indicated by the ability of SIB-1893 to prevent phosphoinositol hydrolysis induced by the specific mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG). However, they also significantly reduce NMDA evoked current recorded from whole cells voltage clamped at −60 mV, and significantly decrease the duration of opening of NMDA channels recorded in the outside out patch configuration. This suggests that although MPEP and SIB-1893 are effective mGluR5 antagonists, they also act as noncompetitive NMDA receptor antagonists. Therefore, the neuroprotective effects of these compounds are most likely mediated through their NMDA receptor antagonist action, and caution should be exercised when drawing conclusions about the roles of mGluR5 based on their use. Keywords: Group I mGluRs, MPEP, SIB-1893, NMDA receptors, NMDA-induced toxicity, neuroprotection, patch clamp recording Introduction The metabotropic glutamate receptors (mGluRs) are a family of G-protein linked glutamate receptors, of which eight different subtypes have been described to date (Knopfel et al., 1995; Pin & Duvoisin, 1995; Riedel et al., 1996; Saugstad et al., 1997). These eight subtypes can be divided into three groups (group I, II and III) based on sequence homology, signal transduction mechanisms and pharmacological profile. Group I mGluR, which comprises subtype mGluR1 and mGluR5, are coupled via phospholipase C to the inositol triphosphate/Ca2+ pathway and show sensitivity to (RS)-3,5-dihydroxyphenylglycine [(RS)-DHPG]: (Schoepp et al., 1994). Group II and III receptors are negatively coupled to the adenylyl cyclase second messenger system, and are sensitive to agonists such as (+)-1S,2S,5R,6S-2-aminobicyclo {3.1.0]hexane-2,6-dicarboxylic acid (LY354740) and L-2-amino-4-phosphonobutyric acid (L-AP4) respectively (Knopfel et al., 1995; Pin & Duvoisin, 1995; Riedel et al., 1996; Saugstad et al., 1997; Schoepp et al., 1999). A wide range of physiological roles has been ascribed to the mGluRs, including interactions with Ca2+ and K+ channels, and regulation of ionotropic glutamate receptors (for review see Anwyl, 1999). As a result, the mGluRs have been implicated in a number of pathophysiological and neurodegenerative disorders, including neuronal injury. A number of studies have shown that group I mGluR activation can exacerbate neuronal injury both in vitro (Bruno et al., 1995; 1996; 1999; Mukhin et al., 1996; 1997a,1997b; Agrawal et al., 1998; Strasser et al., 1998) and in vivo (Gong et al., 1995; Mukhin et al., 1996; Bruno et al., 1999). It has been suggested that mGluR1 is involved in this exacerbation of injury, as antisense-oligodeoxynucleotide (AS-ODN) directed to mGluR1 reduced injury, whereas AS-ODN directed to mGluR5 did not (Mukhin et al., 1996). However, the elucidation of the role of the individual subtypes has been hampered by the lack of availability of subtype-specific compounds. Recently, a number of mGluR5 specific antagonists, such as (E)-2-methyl-6-styryl-pyridine (SIB-1893) and its structural derivative 2-methyl-6- (phenylethynyl)-pyridine (MPEP) have become available. These compounds have been reported to be potent, selective and systemically active antagonists of the mGluR5 subtype, with no appreciable agonist or antagonist activity at recombinant mGluR1b, group II or III mGluRs or ionotropic glutamate receptors (Gasparini et al., 1999). It has been previously reported that MPEP may cause a small reduction (22%) in glutamate evoked responses in Xenopus oocytes expressing recombinant hNMDA1A/2B receptors, but this reduction was not considered significant (Gasparini et al., 1999). Here we report that both MPEP and SIB-1893 provide neuroprotection against glutamate- or NMDA-induced toxicity in cultured rat cortical neurones in vitro. However, although these compounds act as effective mGluR5 antagonists in our system, they also appear to significantly modulate NMDA receptor activity. These observations raise questions about the receptor specificity of these purported mGluR5 selective receptor antagonists and suggest caution in drawing conclusions about the role of mGluRs based on the use of such compounds.