TY - JOUR T1 - Quantitative Analysis Reveals Multiple Mechanisms of Allosteric Modulation of the mGlu5 Receptor in Rat Astroglia JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.110.068882 SP - mol.110.068882 AU - Sophie J Bradley AU - Christopher J Langmead AU - Jeannette M Watson AU - R.A. John Challiss Y1 - 2011/01/01 UR - http://molpharm.aspetjournals.org/content/early/2011/02/24/mol.110.068882.abstract N2 - Positive and negative allosteric modulators (PAMs and NAMs, respectively) of the type 5 metabotropic glutamate (mGlu5) receptor have demonstrable therapeutic potential in an array of neurological and psychiatric disorders. Here we have used rat cortical astrocytes to investigate how PAMs and NAMs mediate their activity and reveal marked differences between PAMs with respect to their modulation of orthosteric agonist affinity and efficacy. Affinity cooperativity factors (α) were assessed using [3H]MPEP-PAM competition binding in the absence and presence of orthosteric agonist, while efficacy cooperativity factors (β) were calculated from net affinity/efficacy cooperativity parameters ((αβ) obtained from analyses of the abilities of PAMs to potentiate [3H]inositol phosphate accumulation in astrocytes stimulated with a sub-maximal (EC20) concentration of orthosteric agonist. We report that while DFB (3,3'-difluorobenzaldazine) and CDPPB (3-cyano-N-(1,3-diphenyl-1H-prazol-5-yl)benzamide) primarily exert their allosteric modulatory effects through modifying the apparent orthosteric agonist affinity at the astrocyte mGlu5 receptor, the effects of ADX47273 (S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone) are mediated primarily via efficacy-driven modulation. In [3H]MPEP-NAM competition binding assays, both MPEP (2-methyl-6-(phenylethynyl)-pyridine) and M-5MPEP (2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine) defined similar specific binding components, with affinities that were unaltered in the presence of orthosteric agonist, indicating wholly negative efficacy-driven modulations. Interestingly, while M-5MPEP only partially inhibited orthosteric agonist-stimulated [3H]inositol phosphate accumulation in astrocytes, it could completely suppress Ca2+ oscillations stimulated by quisqualate or (S)-3,5-dihydroxyphenylglycine. In contrast, MPEP was fully inhibitory with respect to both functional responses. The finding that M-5MPEP has different functional effects depending on the endpoint measured is discussed as a possible example of permissive allosteric antagonism. ER -