Neuroprotective activity of the mGluR5 antagonists MPEP and MTEP against acute excitotoxicity differs and does not reflect actions at mGluR5 receptors

Neuroprotection has been reported after either activation or blockade of the group I metabotropic glutamate receptor subtype 5 (mGluR5). However, some recent evidence suggests that protection provided by mGluR5 antagonists may reflect their ability to inhibit N-methyl-D-aspartate (NMDA) receptor activity. Here, in both rat and mouse cortical neurons, we compare the neuroprotective actions of two mGluR5 antagonists: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), which has been commonly used and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), a more recently developed compound believed to have greater mGluR5 selectivity. We have previously shown that MPEP directly reduces single-channel NMDA receptor open time at the same concentrations (20 μM or greater) that show neuroprotection, whereas MPEP antagonizes mGluR5 agonist ((RS)-2-chloro-5-hydroxyphenylglycine (CHPG))-induced changes in inositol phosphates (IP) at concentrations as low as 0.2 μM. In the present studies, MTEP significantly inhibited CHPG-mediated IP hydrolysis at concentrations as low as 0.02 μM. In contrast to MPEP, which significantly reduced glutamate- or NMDA-mediated cell death in primary rat neuronal cultures at a concentration of 20 μM, small neuroprotective effects were observed with MTEP only at a concentration of 200 μM. Neither MPEP- nor MTEP-mediated mGluR5 inhibition had any effect on etoposide-induced apoptotic cell death. In rat cortical neurons, the neuroprotective effects of MTEP at very high concentrations, like those of MPEP, reflect ability to directly reduce NMDA receptor peak and steady-state currents. We also compared the effects of MPEP and MTEP in primary cortical neuronal cultures from parental and mGluR5 knockout mice. Both agents were neuroprotective, at high concentrations in normal as well as in the knockout cultures. In contrast to rat cortical neurons, neither MPEP nor MTEP appears to directly alter NMDA receptor activity. Combined, these studies support the conclusion that MTEP has greater mGluR5 selectivity than MPEP, and that neuroprotection provided by either antagonist in neuronal cultures does not reflect inhibition of mGluR5 receptors. Keywords: mGluR5, NMDA receptors, MTEP, MPEP, neuroprotection Introduction Metabotropic glutamate receptors (mGluRs) are known to modulate ionotropic glutamate receptors (iGluRs), various calcium and potassium channels, and neurotransmitter release (Anwyl, 1999). Because many of these systems are involved in the pathophysiology of acute CNS injury, drugs that activate or inhibit mGluR have been examined with regard to post-traumatic or postischemic cell death (Bruno et al., 1998; Lea & Faden, 2003b). The mGluRs have been categorized into three groups, on the basis of structure, signal transduction mechanisms, and pharmacological sensitivities (Conn & Pin, 1997; Schoepp et al., 1999). Group I mGluRs include mGluR1 and mGluR5. Activation of mGluR1 appears to exacerbate necrotic cell death after trauma or ischemia, in part by activating N-methyl-D-aspartate (NMDA) receptors (Allen et al., 2000; 2001). However, the role of mGluR5 has been more controversial because of the lack of truly selective antagonists. Several purportedly selective mGluR5 antagonists have been developed, such as 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (Gasparini et al., 1999) and (E)-2-methyl-6-(2-phenylethenyl)-pyridine (SIB-1893) (Varney et al., 1999). Although both compounds show some neuroprotective activity, such actions occur only at concentrations that inhibit NMDA receptors and are substantially higher than that required to inhibit agonist-induced phosphoinositide (PI) hydrolysis (O'Leary et al., 2000; Movsesyan et al., 2001a). These findings are consistent with a prior study using antisense oligonucleotides (ODN) directed against mGluR1 or mGluR5 receptors; at concentrations that equally reduced agonist-induced IP3, only inhibition of mGluR1 was neuroprotective (Mukhin et al., 1996). Recently, a noncompetitive mGluR5-specific antagonist was developed. 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) appears to have greater selectivity at mGluR5 than other antagonists (Cosford et al., 2003a). In vivo and in vitro characterization of MTEP (Anderson et al., 2002; 2003; Cosford et al., 2003a) indicates that it is highly selective for mGluR5 over mGluR1, has no effect on other mGluR subtypes, and has fewer off target effects than MPEP (Cosford et al., 2003a). In the present studies, we compared the selectivity and neuroprotective actions of MPEP and MTEP against glutamate-, NMDA-, and etoposide-induced toxicity in primary rat cortical neuronal cultures. We also compared the actions of these compounds in cortical neuronal cultures derived from mGluR5-deficient mice and parental (mGluR5 positive) animals.