PRESENCE OF FUNCTIONAL NMDA RECEPTORS IN A HUMAN NEUROBLASTOMA CELL-LINE

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 transc ription and polymerase chain reaction (RT-PCR), followed by cloning an d DNA sequencing, revealed the presence in these cells of mRNA represe nting the key subunit, NMDAR1, of the receptor. This mRNA was further demonstrated by Northern analysis to be the same size as that describe d for human neurons. The neutral red cytotoxicity assay was utilized t o examine the influence on these neuroblastoma cells of a 48-h incubat ion with either L-glutamic acid or the specific NMDA agonist N-phthala moyl-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 u sing the whole-cell configuration of the patch-clamp technique and the specific NMDA agonists (+/-)1-aminocyclobutane-cis-1,3-dicarboxylic a cid (ACDA) and NPG. Using this procedure, a voltage-dependent tetrodot oxin-sensitive inward sodium current was found to be increased (x1.5) by L-glutamic acid and by both NMDA agonists in the presence of glycin e. Another voltage-gated inward current, probably carried by calcium i ons, was increased three- to fourfold. Hence, these glutamate activiti es observed in human LA-N-2 neuroblastoma cells appear to occur throug h the activation of functional NMDA receptors in much the same way as reported for neurons, and both glutamate and NMDA agonists can be toxi c to these neuroblastoma cells. Our findings, therefore, suggest this cell line will provide a model suitable for investigating the mechanis ms of NMDA-related long-term potentiation (LTP) in neurons and of the NMDA-related neurotoxic effects of glutamate in disease states that in volve a reduction in cholinergic function.