title: Presence of functional NMDA receptors in a human neuroblastoma cell line.
abstract: Data are presented that provide convincing evidence for the expression of structurally normal and functional NMDA receptors by acetylcholine-producing human LA-N-2 neuroblastoma cells in culture. Reverse transcription and polymerase chain reaction (RT-PCR), followed by cloning and DNA sequencing, revealed the presence in these cells of mRNA representing the key subunit, NMDAR1, of the receptor. This mRNA was further demonstrated by Northern analysis to be the same size as that described for human neurons. The neutral red cytotoxicity assay was utilized to examine the influence on these neuroblastoma cells of a 48-h incubation with either L-glutamic acid or the specific NMDA agonist N-phthalamoyl-L-glutamic acid (NPG). Cell cytotoxicity was shown by this assay to be increased through incubation with glutamate at 1 and 10 mM by 27 and 37%, and through incubation with NPG at 0.1 and 1 mM by 28 and 46%. A possible mechanism of these toxic effects was further evaluated using the whole-cell configuration of the patch-clamp technique and the specific NMDA agonists (+/-)1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA) and NPG. Using this procedure, a voltage-dependent tetrodotoxin-sensitive inward sodium current was found to be increased (x 1.5) by L-glutamic acid and by both NMDA agonists in the presence of glycine. Another voltage-gated inward current, probably carried by calcium ions, was increased three- to fourfold. Hence, these glutamate activities observed in human LA-N-2 neuroblastoma cells appear to occur through the activation of functional NMDA receptors in much the same way as reported for neurons, and both glutamate and NMDA agonists can be toxic to these neuroblastoma cells. Our findings, therefore, suggest this cell line will provide a model suitable for investigating the mechanisms of NMDA-related long-term potentiation (LTP) in neurons and of the NMDA-related neurotoxic effects of glutamate in disease states that involve a reduction in cholinergic function.
authors.affiliation: Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA.
authors.forename: W G
authors.initials: WG
authors.lastname: North
authors.forename: M J
authors.initials: MJ
authors.lastname: Fay
authors.forename: J
authors.initials: J
authors.lastname: Du
authors.forename: M
authors.initials: M
authors.lastname: Cleary
authors.forename: J D
authors.initials: JD
authors.lastname: Gallagher
authors.forename: F V
authors.initials: FV
authors.lastname: McCann
mesh_terms: D015152:Blotting, Northern; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002795:Choline O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003001:Cloning, Molecular; D017931:DNA Primers; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid / Q000633:toxicity; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009447:Neuroblastoma / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D005971:Glutamates; D046911:Macromolecular Substances; D009498:Neurotoxins; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D013757:Tetraethylammonium Compounds; C066302:2,4-methanoglutamate; D018698:Glutamic Acid; C034741:N-phthaloylglutamic acid; D013779:Tetrodotoxin; D019789:Tetraethylammonium; D002795:Choline O-Acetyltransferase; D005998:Glycine