Modulation of 45Ca2+ influx into cells stably expressing recombinant human NMDA receptors by ligands acting at distinct recognition sites.
A 45Ca2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45Ca2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45Ca2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45Ca2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45Ca2+ influx into cells expressing NR1a/NR2A receptors (IC50 = 408 microM) and NR1a/NR2B receptors (EC50 = 37.3 microM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC50 = 0.099 microM) compared with NR1a/NR2A receptors (IC50 = 164 microM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg2+ and Zn2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45Ca2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.
Grimwood
S
S
Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, England.
Gilbert
E
E
Ragan
C I
CI
Hutson
P H
PH
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes / Q000378:metabolism; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D007739:L Cells / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D051379:Mice; D009498:Neurotoxins / Q000494:pharmacology; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D002132:Calcium Radioisotopes; D018690:Excitatory Amino Acid Agonists; D008024:Ligands; D009498:Neurotoxins; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; D013096:Spermine; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D004228:Dithionitrobenzoic Acid; D007608:Kainic Acid; D002118:Calcium; D004229:Dithiothreitol; D005998:Glycine