Wg. North et al., PRESENCE OF FUNCTIONAL NMDA RECEPTORS IN A HUMAN NEUROBLASTOMA CELL-LINE, Molecular and chemical neuropathology, 30(1-2), 1997, pp. 77-94
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.