title: In vitro characterization of [3H]MethoxyPyEP, an mGluR5 selective radioligand.
abstract: We have characterized the in vitro properties of 3-[3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([3H]MethoxyPyEP), an analogue of the mGluR(5) receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 +/- 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k(on) and k(off) were determined as 2.9 x 10(7) M(-1) min(-1) and 0.11 min(-1) respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEP > trans-azetidine-2,4-dicarboxylic acid congruent with (S)-4-carboxyphenylglycine congruent with (+)MK801 congruent with CP-101,606 congruent with clozapine congruent with atropine congruent with ketanserin congruent with yohimbine congruent with benoxathian). Autoradiographic studies with [3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.
authors.affiliation: Department of Pharmacology and Imaging, WP44C-2, Merck Research Laboratories, West Point, PA 19486, USA. shailendra_patel@merck.com
authors.forename: Shil
authors.initials: S
authors.lastname: Patel
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Krause
authors.forename: Terence
authors.initials: T
authors.lastname: Hamill
authors.forename: Ashok
authors.initials: A
authors.lastname: Chaudhary
authors.forename: Donald H
authors.initials: DH
authors.lastname: Burns
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Gibson
mesh_terms: D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008024:Ligands; D008297:Male; D011725:Pyridines / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013788:Thalamus / Q000378:metabolism
chemical_list: C474798:3-(3H)methoxy-5-(pyridin-2-ylethynyl)pyridine; C578937:Grm5 protein, rat; D008024:Ligands; D011725:Pyridines; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C121465:6-methyl-2-(phenylethynyl)pyridine