Characterization of 3H-(2S2′R3′R)-2-(2′3′-dicarboxy- cyclopropyl)glycine (3H-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes

摘要

class="enumerated" style="list-style-type:decimal">The binding of the new selective group II metabotropic glutamate receptor radioligand, [³H]-(2>S,2′>R,3′>R)-2-(2′,3′-dicarboxycyclopropyl)glycine ([³H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes.[³H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg⁻¹ protein. Binding was not sensitive to Na⁺-dependent glutamate uptake blockers or Cl⁻-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (>S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [³H]-DCG IV binding.Of the compounds observed to inhibit [³H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5′-O-(3-thiotriphosphate) (GTPγS) inhibited [³H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nM.A highly significant correlation was observed between the potencies of compounds able to inhibit [³H]-DCG IV binding and potencies obtained for agonist activity in a GTPγ³⁵S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((>RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (>R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (>RS)-α-methylserine-O-phosphate (MSOP).