Abstract
The conversion of choline in cultures of the human neuroblastoma cell lines, LA-N-1 and LA-N-2 cells, was investigated in order to identify potential precursors in acetylcholine (AcCho) synthesis. LA-N-1, a catecholaminergic and LA-N-2, a cholinergic, cell line were incubated with [3H-methyl]choline (Cho) for varying periods of time up to 72h. The radioactivity present in lipids and water-soluble metabolites increased linearly up to 24h in both cell lines. Approximately 20% of the radioactivity associated with the water-soluble metabolites in both control (untreated) and retinoic acid-induced differentiated (RA-treated cells) LA-N-2 cells was present as Cho and AcCho. There was no detectable AcCho in the catecholaminergic cell line, LA-N-1.
The untreated and RA-treated LA-N-1 and LA-N-2 cells were labeled for 24h with [3H-methyl]Cho, followed by a chase in growth medium containing 100 μM unlabeled choline. The distribution of radioactivity in the LA-N-2 cells was 6–10% of AcCho, 84–89% as phosphocholine (PCho), 1–3% as glycerophosphocholine (GroPCho), and 2–4% as Cho. The distribution of radioactivity in the LA-N-1 cells was similar except for the absence of AcCho. The distribution of radioactivity in the culture medium of LA-N-1 cells was 70–80% as Cho, 20–30% asPCho, and 1–3% as GroPCho. In contrast, the radioactivity was equally distributed between Cho (50%) andPCho (50%), with only 1–3% as GroPCho in the medium of LA-N-2 cells.
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Abbreviations
- Cho:
-
Choline
- AcCho:
-
Acetylcholine
- RA:
-
Retinoic acid
- PCho:
-
Phosphocholine
- GroPCho:
-
Glycerophosphocholine
- L-15:
-
Leibovitz’s L-15 medium
References
Ansell G. B. and Spanner S. (1979) Sources of choline for acetylcholine synthesis in the brain, inNutrition and Brain, vol. 5 (Barbeau A., Growdon J. H., and Wurtman R. J., eds.), Raven, NY, pp. 35–46.
Barker L. A. and Mittag T. W. (1974) Comparative studies of substances and inhibitors of choline transport and cholineacetyltransferase.J. Pharmacol. Exp. Ther. 192, 86–94.
Blusztajn J. K., Holbrook P. G., Lakher M., Liscovitch M., Maire J. C., Mauron C., Richardson U. I., Tacconi M. T., and Wurtman R. J. (1986) Relationships between acetylcholine release and membrane phosphatidylcholine turnover in brain and in cultured cholinergic neurons, inPhospholipids in the Nervous System: Biochemical and Molecular Pharmacology (Horrocks L. A., Freysz L., and Toffano G., eds.), Liviana, Padua, Italy, pp. 283–290.
Bluszatjn J. K. and Wurtman R. J. (1981) Choline biosynthesis by a preparation enriched in synaptosomes from rat brain.Nature,290, 417–418.
Cohen E. L. and Wurtman R. J. (1976) Brain acetylcholine: Control by dietary choline.Science 191, 561–562.
Folch J., Lees M., and Sloane Stanley G. H. (1957) A simplified method for the isolation of total lipides from animal tissues.J. Biol. Chem. 226, 497–509.
Fonnum F. (1975) A rapid radiochemical method for the determination of cholineacetyltransferase.J. Neurochem. 24, 407–409.
Jenden D. J., Roch M., and Booth R. A. (1973) Simultaneous measurement of endogenous and deuterium-labeled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography/mass spectrometry.Anal. Biochem. 55, 438–448.
Jope R. S. (1982) Effects of phosphatidylcholine administration to rats on choline in blood and choline and acetylcholine in brain.J. Pharmacol. Exp. Ther. 220, 322–328.
Jope R. S. and Jenden D. J. (1979) Choline and phospholipid metabolism and the synthesis of acetylcholine in rat brain.J. Neurosci. Res. 4, 69–82.
Kato A. C., Lefresne P., Berwald-Netter Y., Beaujouan J. C., Glowinski J., and Gros F. (1977) Choline stimulates the synthesis of acetylcholine from acetate and accumulation of acetate in a cholinergic neuroblastoma clone.Biochem. Biophys. Res. Commun. 78, 350–256.
Keller F., Rimvall K., and Waser P. G. (1987) Choline acetylcholine metabolism in slice cultures of the new born rat septum.Brain Res. 405, 305–312.
Lanks K., Somers L., Papirmeister B., and Yamamura H. (1974) Choline transport by neuroblastoma cells in tissue culture.Nature 252, 476–478.
Lowry O. H., Rosebrough N. J., Farr A. L., and Randall R. J. (1951) Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193, 265–275.
Massarelli R., Sensenbrenner M., Ebel A., and Mandel P. (1974a) Choline uptake in nerve cell cultures.Neurobiology 4, 293–300.
Massarelli R., Ciesielski-Treska J., Ebel A., and Mandel P. (1974b) Kinetics of choline uptake in neuroblastoma clones.Biochem. Pharmacol. 23, 2857–2865.
Massarelli R. and Wong T. Y. (1981) Choline uptake in nerve cell cultures and in synaptosomal preparations is regulated by the endogenous pool of choline,Cholinergic Mechanisms (Pepeu G. and Landinsky H., eds.), Plenum, NY, pp. 511–520.
Morash S. C., Cook H. W., and Spence M. W. (1988) Phosphatidylcholine metabolism in cultured cells: Catabolism via glycerophosphocholine.Biochim. Biophys. Acta 961, 194–202.
Morel N., Israel, M., Manaranche R., and Mastour-Frachon P. (1977) Isolation of pure cholinergic nerve endings from Torpedo electric organ. Evaluation of their metabolic properties.J. Cell. Biol. 75, 43–55.
O’Regan S., Collier B., and Israel M. (1982) Studies on presynaptic cholinergic mechanisms using analogues of choline and acetate.J. Physiol. (Paris) 78, 454–460.
Richelson E. and Thompson E. J. (1973) Transport of neurotransmitter precursors into cultured cells.Nature New Biol. 241, 201–204.
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., Lang W. E., Neustein H. B. and Benedict W. F. (1977) Morphology, growth, chromosomal pattern, and fibrinolytic activity of two new human neuroblastoma cell lines.Cancer Res. 37, 1364–1371.
Schmidt D. E. and Wecker L. (1981) CNS effects of choline administration: Evidence for temporal dependence.Neuropharmacology 20, 535–539.
Singh I. N., Sorrentino G., McCartney D. G., Massarelli R., and Kanfer J. N. (1990) Enzymatic activities during differentiation of the human neuroblastoma cell, LA-N-1 and LA-N-2.J. Neurosci. Res. 25, 476–485.
Vance D. E., Trip E. M., and Paddon H. B. (1980) Polio virus increases phosphatidylcholine biosynthesis in HeLa cells by stimulation of the rate-limiting reaction catalyzed by CTP: Phosphocholine cytidyltransferase.J. Biol. Chem. 255, 1064–1069.
West G., Uki J., Herschman H. R., and Seeger R. C. (1977) Adrenergic, cholinergic, and inactive human neuroblastoma cell lines with the action-potential Na+ ionophore.Cancer Res. 37, 1372–1376.
Wong T. Y., Mandel P., and Massarelli R. (1983) Choline fluxes in primary nerve cell cultures. Correlation with the endocellular metabolism of choline.Neurochem. Int. 5, 73–79.
Yavin E. (1976) Regulation of phospholipid metabolism in differentiating cells from rat brain hemispheres in culture: Patterns of acetylcholine, phosphocholine and choline phosphoglyceride labeling from [methyl-14C]choline.J. Biol. Chem. 251, 1392–1397.
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Singh, I.N., Sorrentino, G., Massarelli, R. et al. The metabolic fate of [3H-methyl]choline in cultured human neuroblastoma cell lines, LA-N-1 and LA-N-2. Molecular and Chemical Neuropathology 14, 53–66 (1991). https://doi.org/10.1007/BF03160997
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DOI: https://doi.org/10.1007/BF03160997