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 eitherl-glutamic acid or the specific NMDA agonistN-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 (×1.5) byl-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.
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Altschul S. F., Warren G., Miller W., Myers E. W., and Lipman D. J. (1990) Basic local alignment search tool.J. Mol. Biol. 215, 410–430.
Amano S., Ohashi M., Ishiko T., and Hazama F. (1994) Humoral factors derived from glial cells protect cultured neuroblastoma cells against glutamate toxicity.Brain Res. 645, 347–350.
Badley J. E., Bishop G. A., St. John T., and Frelinger J. A. (1988) A simple, rapid method for the purification of poly A + RNA.Biotechniques 6, 114–116.
Barinaga, M. (1995) A new face for the glutamate receptor.Science 267, 177–178.
Bartus R. T., Dean R. L. III, Beer B., and Lippa A. S. (1982) The cholinergic hypothesis of geriatric memory dysfunction.Science 217, 408–417.
Bartus R. T., Flicker C., Dean R. L., Pontecorvo M., Figueredo J. C., and Fisher S. K. (1985) Selective memory loss following nucleus basalis lesions: long term behavioral recovery despite persistent cholinergic deficiencies.Pharmacol. Biochem. Behav. 23, 125–135.
Behi C., Hovey L. III, Krajewski S., Schubert D., and Reed J. C. (1993) BCL-2 prevents killing of neuronal cells by glutamate but not by amyloid beta protein.Biochem. Biophys. Res. Commun. 197, 949–956.
Boudraeu N., Sympson C. J., Werb Z., and Bissell M. J. (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix.Science 267, 891–893.
Choi D. W., Maulucci-Gedde M., and Kriegstein A. R. (1987) Glutamate neurotoxicity in cortical cell culture.J. Neurosci. 7, 357–368.
Collingridge G. L. and Bliss T. V. P. (1987) NMDA receptors-their role in long-term potentiation.Trends Neurosci. 10, 288–293.
Collingridge G. L. and Lester R. A. J. (1989) Excitatory amino acid receptors in the vertebrate central nervous system.Pharmacol. Rev. 40, 143–210.
Corasaniti M. T., Melino G., Tartaglia R. L., Finazzi-Agro A., and Nistico G. (1994)N-Methyl-d-aspartate-induced excessive formation of nitric oxide in CHP100 neuroblastoma cells produces death of BMEL melanoma cells in coculture.Neuropharmacology 33, 1071–1077.
Dayanithi G., Rage F., Richard P., and Tapia-Arancibia L. (1995) Characterization of spontaneous andN-methyl-d-Aspartate-induced calcium rise in rat cultured hypothalamic neurons.Neuroendocrinology 61, 243–255.
Durham H. D., Dahrouge S., and Cashman N. R. (1993) Evaluation of the spinal chord neuron x neuroblastoma hybrid cell line NSC-34 as a model for neurotoxicity testing.Neurotoxicity 14, 387–395.
Garthwaite G. and Garthwaite J. (1987) Receptor-linked ionic channels mediateN-methyl aspartate neurotoxicity in rat cellebellar slices.Neurosci. Lett. 83, 241–246.
Ginsborg B. L., Martin R. J., and Patmore L. (1991) On the sodium and potassium currents of a human neuroblastoma cell line.J. Physiol. 434, 121–149.
Haring R., Zukin R. S., and Zukin S. R. (1990) Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line.Neurosci. Lett. 112, 92–98.
Henneberry R. C., Novelli A., Cox J. A., and Lysko P. G. (1989) Neurotoxicity at theN-methyl-d-aspartate receptor in energy-compromised neurons: An hypothesis for cell death in aging and disease.Ann. NY Acad. Sci. 225–233.
Johnson M. E., Gores G. J., Uhl C. B., and Sill J. C. (1994) Cytosolic free calcium and cell death during metabolic inhibition in a neuronal cell line.J. Neurosci. 14, 4040–4049.
Karp S. J., Masu M., Eki T., Ozawa K., and Nakanishi S. (1993) Molecular cloning and chromosomal localization of the key subunit of theN-methyl-d-aspartate receptor.J. Biol. Chem. 268, 3728–3733.
Kopke A. K., Bonk I., Sydow S., Menke H., and Spiess J. (1993) Characterization of the NR1, NR2A, and NR2C receptor proteins.Protein Sci. 12, 2066–2076.
Kure S., Tominaga T., Yoshimoto T., Tada K., and Narisawa K. (1991) Glutamate triggers internucleosomal DNA cleavage in neuronal cells.Biochem. Biophys. Res. Commun. 179, 39–45.
Lerma J., Kushner L., Spray D. C., Bennett M. V. L., and Zukin R. S. (1989) mRNA from NCB-20 cells encodes theN-methyl-d-aspartate/phencyclidine receptor: A xenopus oocyte expression study.Proc. Natl. Acad. Sci. USA 86, 1708–1711.
Malouf A. T., Schnaar R. L., and Coyle J. T. (1984a) Characterization of a glutamic acid neurotransmitter binding site on neuroblastoma hybrid cells.J. Biol. Chem. 259, 12,756–12,762.
Malouf A. T., Coyle J. T., and Schaar R. L. (1984b) Agonists and cations regulate the glutamic acid receptors on intact neuroblastoma hybrid cells.J. Biol. Chem. 259, 1263–1268.
McCann F. V., McCarthy D. C., Keller T. M., and Noelle R. J. (1988) Characterization of large conductance non-selective anion channels in B lymphocytes.Cell. Signalling 1, 31–44.
McCann F. V., McCarthy D. C., and Noelle R. J. (1990) Patch-clamp profile of ion channels in resting murine B lymphocytes.J. Membr. Biol. 112, 175–188.
McCann F. V., McCarthy D. C., and Noelle R. J. (1991) Interleukin-4 activates ion channels in B lymphocytes.Cell. Signalling 3, 483–490.
Monyer H., Sprengel R., Schoepfer R., Herb A., Higuchi M., Lomeli H., Burnashev N., Sakman B., and Seeburg P. H. (1992) Heteromeric NMDA receptors: Molecular and functional distinction of subtypes.Science 256, 1217–1221.
Moriyoshi K., Masu M., Ishii T., Shigemoto R., Mizuno N., and Nakanishi S. (1991) Molecular cloning and characterization of the rat NMDA receptor.Nature 354, 31–37.
Murphy T. H., Malouf A. T., Sastre A., Schaar R., and Coyle J. T. (1988) Calcium-dependent glutamate cytotoxicity in a neuronal cell line.Brain Res. 444, 325–332.
Murphy T. H., Miyamoto M., Sastre A., Schnaar R. L., and Coyle J. T. (1989) Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress.Neuron 2, 1547–1558.
Naarala J., Nykvist P., Tuomala M., and Savolainen K. (1993) Excitatory amino acid-induced slow biphasic responses of free intracellular calcium in human neuroblastoma cells.FEBS Lett. 330, 222–226.
North W. G. and Yu X-M. (1993) Vasopressin mRNA and neurophysin-related cell-surface antigen (NRSA) of small-cell carcinoma.Peptides 14, 303–307.
Ohkuma S., Katsura M., Chen D. Z., Chen S. H., and Kuriyama K. (1994) Presence ofN-methyl-d-aspartate (NMDA) receptors in neuroblastoma x glioma hybrid NG108-15 cells-analysis using [45Ca2+] influx and [3H]MK-801 binding as functional measures.Mol. Brain Res. 22, 166–172.
Prehn J. H. M., Lippert K., and Krieglstein J. (1995) Are NMDA or AMPA/kainate receptor antagonists more efficacious in the delayed treatment of excitotoxic neuronal injury?Eur. J. Pharmacol. 292, 179–189.
Richardson U. I., Liscovitch M., and Blusztajn J. K. (1989) Acetylcholine synthesis and secretion by LA-N-2 human neuroblastoma cells.Brain Res. 476, 323–331.
Seeger R. C., Rayner S. A., Banerjee A., Chung H., Laug W. E., Neustein H. B., and Benedict W. F. (1977) Morphology, growth, chromosomal pattern, and fibrinolytic activity of two new neuroblastoma cell lines.Cancer Res. 37, 1364–1371.
Singh I. N., Sorrentino G., Massarelli R., and Kanfer J. N. (1991) The metabolic fate of [3H-methyl]choline in cultured human neuroblastoma cell lines, LA-N-1 and LA-N-2.Mol. Chem. Neuropathol. 14, 53–66.
Uney J. B., Thomas S., Anderton B. H., and Campbell I. C. (1992) C1300 neuroblastoma cells possess quisqualate sensitive glutamate binding sites.Biochem. Soc. Trans. 20, 365S.
Van der Valk J. B. F. and Vijverberg H. P. M. (1990) Glutamate-induced inward current in a clonal neuroblastoma cell line.Eur. J. Pharmacol. 189, 99–102.
Vendrell M., Curan T., and Morgan J. I. (1993) Glutamate, immediate-early genes, and cell death in the nervous system.Ann. NY Acad. Sci. 679, 132–141.
Weiss R. E. and Sidell N. (1991) Sodium currents during differentiation in a human neuroblastoma cell line.J. Gen. Physiol. 97, 521–539.
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North, W.G., Fay, M.J., Du, J. et al. Presence of functional NMDA receptors in a human neuroblastoma cell line. Molecular and Chemical Neuropathology 30, 77–94 (1997). https://doi.org/10.1007/BF02815151
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DOI: https://doi.org/10.1007/BF02815151