title: Pharmacological characterization of ligands at recombinant NMDA receptor subtypes by electrophysiological recordings and intracellular calcium measurements.
abstract: Generation of in vitro cellular assays using fluorescence measurements at heterologously expressed NMDA receptors would speed up the process of ligand characterization and enable high-throughput screening. The major drawback to the development of such assays is the cytotoxicity caused by Ca(2+)-flux into the cell via NMDA receptors upon prolonged activation by agonists present in the culture medium. In the present study, we established four cell lines with stable expression of NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D in BHK-21 cells. To assess the usefulness of the stable cell lines in conjunction with intracellular calcium ([Ca(2+)](i)) measurements for evaluation of NMDA receptor pharmacology, several ligands were characterized using this method. The results were compared to parallel data obtained by electrophysiological recordings at NMDA receptors expressed in Xenopus oocytes. This comparison showed that agonist potencies determined by [Ca(2+)](i) measurements and electrophysiological recordings correlated well, meaning that the stable cell lines in conjunction with [Ca(2+)](i) measurements provide a useful tool for characterization of NMDA receptor ligands. The agonist series of conformationally constrained glutamate analogues (2S,3R,4S)-alpha-(carboxycyclopropyl)glycine (CCG), 1-aminocyclobutane-r-1,cis-3-dicarboxylic acid (trans-ACBD), and (+/-)-1-aminocyclopentane-r-1,cis-3-dicarboxylic acid (cis-ACPD), as well as the highly potent agonist tetrazolylglycine were among the characterized ligands that were assessed with respect to subtype selectivity at NMDA receptors. However, none of the characterized agonists displays more than 2-3 fold selectivity towards a specific NMDA receptor subtype. Thus, the present study provides a broad pharmacological characterization of structurally diverse ligands at recombinant NMDA receptor subtypes.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark. kbh@farma.ku.dk
authors.forename: Kasper B
authors.initials: KB
authors.lastname: Hansen
authors.forename: Hans
authors.initials: H
authors.lastname: Bräuner-Osborne
authors.forename: Jan
authors.initials: J
authors.lastname: Egebjerg
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004594:Electrophysiology / Q000379:methods; D005260:Female; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D008024:Ligands; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D021122:Protein Subunits / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection; D014981:Xenopus
chemical_list: D008024:Ligands; C473649:NR1A NMDA receptor, rat; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; C432823:NR2C NMDA receptor; C464436:NR2D NMDA receptor; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; D002118:Calcium; D005998:Glycine