Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system.
Pharmacological characterization of N-methyl-D-aspartate (NMDA) receptors has been hampered by the difficulty to outwit cytotoxicity after functional expression in recombinant systems. In this study a muristerone-inducible expression system for the NNMDA-R1 subunit was used. This was combined with constitutive expression of NMDA-R2A, 2B, 2C and 2D in different cell clones. After establishment of the cell lines, quantitative RT-PCR demonstrated the inducibility of the NNMDA-R1 subunit, and verified the expression of the NMDA-R2 subunits in the different cell clones. Functional responses were characterized using calcium influx through the ion channel as a robust assay system. Stimulation of the NMDA-receptor subtypes in the different cell lines led to calcium transients which were rising gradually, peaked after 30-160 s and declined thereafter very slowly. The expression of the four different NMDA-receptor subtypes in the same cellular background allowed a direct pharmacological comparison of the different receptors. Glutamate showed the highest potency at the NMDA-R1-2D. NMDA displayed at all subtypes a lower potency compared to glutamate and was a partial agonist except at the NMDA-R1-2D. 20 antagonists were tested in this study and the pharmacological characterization of the inhibition of glutamate-evoked elevation of intracellular free Ca(2+) revealed a distinct rank order of antagonist potency for each receptor subtype. These data illustrate that assessment of calcium transients upon receptor stimulation in the same cellular background is a powerful tool to compare the functional effects of compounds acting at the different NMDA-R2 receptors.
Feuerbach
Dominik
D
Neuroscience Research, Novartis Institutes for BioMedical Research, Basel, Switzerland. dominik.feuerbach@novartis.com
Loetscher
Erika
E
Neurdin
Stephanie
S
Koller
Manuel
M
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D005470:Fluorometry; D015870:Gene Expression; D006801:Humans; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002118:Calcium; D004441:Ecdysterone; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; C055348:muristerone A