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

Abstract

1 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. 2 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 microM 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 microM. 3 In the present studies, MTEP significantly inhibited CHPG-mediated IP hydrolysis at concentrations as low as 0.02 microM. In contrast to MPEP, which significantly reduced glutamate- or NMDA-mediated cell death in primary rat neuronal cultures at a concentration of 20 microM, small neuroprotective effects were observed with MTEP only at a concentration of 200 microM. 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. 4 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. 5 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.

Figure 1

Treatment with MTEP blocks mGluR5 agonist-induced PI hydrolysis in rat cortical neuronal cultures. MTEP at indicated concentrations was administered to 7 DIV rat cortical neuronal cultures 20 min prior to stimulation with 1 mM of CHPG. PI hydrolysis was measured by IP accumulation within 40 min after addition of CHPG as described in Methods. Histograms represent IP levels as percentage of control±s.d.; n=6 cultures per condition. ^*P<0.05, ^**P<0.001 versus cultures treated with CHPG alone compared by ANOVA, followed by the Student–Newman–Keuls test.

Figure 2

Comparison of effects of MTEP, MPEP, and MK801 on NMDA- or glutamate-induced cell death in 14 DIV rat cortical neuronal cultures. MPEP, MTEP, or MK801 at indicated concentrations was added to cultures 20 min prior to administration of NMDA (a, b) or Na-glutamate (c, d) (each at 150 μM). Cell viability was assessed by LDH release (a, c) or calcein AM assay (b, d) after 24 h of treatment. Histograms indicate LDH release or calcein AM fluorescence as percentage of that in intact controls±s.d.; n=8–16 cultures per condition. ^*P<0.05, ^**P<0.01 versus injured cultures as shown by ANOVA, followed by the Student–Newman–Keuls test.

Figure 3

Comparison of effects of MTEP, MPEP, and MK801 on etoposide-induced apoptotic cell death in rat cortical neuronal cultures. MPEP, MTEP, or MK801 at indicated concentrations was added to cultures 20 min prior to administration of etoposide (50 μM). Cell viability was assessed by LDH release after 24 h of treatment. Histograms indicate LDH release as percentage of that in intact controls±s.d.; n=8–16 cultures per condition. ^*P<0.05, ^**P<0.01 versus injured cultures as shown by ANOVA, followed by the Student–Newman–Keuls test.

Figure 4

Effects of MTEP on NMDA receptor activity in rat cortical neurons. (a) NMDA-evoked peak currents were directly reduced by both 20 and 200 μM MTEP. (b) NMDA-evoked steady-state currents were directly reduced by 200 but not 20 μM MTEP. Data are expressed as average±s.e.m. ^*P<0.05 versus NMDA alone compared by Student's two-tailed t-test.

Figure 5

Comparison of effects of MTEP, MPEP, and MK801 on NMDA-induced cell death in wild-type (a, c) or mGluR5-deficient (b, d) mouse cortical neuronal cultures. MPEP, MTEP, or MK801 at indicated concentrations was added to cultures 20 min prior to administration of NMDA (150 μM). Cell death was measured by LDH release after 24 h of treatment. Histograms indicate LDH release as percentage of that in intact controls±s.d.; n=8–16 cultures per condition. ^*P<0.05, ^**P<0.01 versus injured cultures as shown by ANOVA, followed by the Student–Newman–Keuls test.

Figure 6

Effects of MPEP and MTEP on NMDA receptor activity in mouse cortical neurons. (a) In wild-type mouse cortical neurons, neither 200 μM MPEP nor MTEP had any effect on NMDA-evoked peak currents. (b) In mGluR5 (−/−) mouse cortical neurons, neither MTEP (20 and 200 μM) nor MPEP (200 μM) had direct effects on NMDA-evoked peak currents. (c) In wild-type mouse cortical neurons, neither 200 μM MPEP nor MTEP had any effect on NMDA-evoked steady-state currents. (d) In mGluR5 (−/−) mouse cortical neurons, neither MTEP (20 and 200 μM) nor MPEP (200 μM) had direct effects on NMDA-evoked steady-state currents. Data are expressed as average±s.e.m. ^*P<0.05 versus NMDA alone compared by ANOVA followed by Fisher's PLSD.