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. 1989 Sep;86(18):7233–7237. doi: 10.1073/pnas.86.18.7233

Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.

P Horellou 1, B Guibert 1, V Leviel 1, J Mallet 1
PMCID: PMC298031  PMID: 2571152

Abstract

Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resistance gene was used to infect a fibroblast (NIH 3T3), a neuroblastoma (NS20 Y), and a neuroendocrine (AtT-20) cell line. After selection in the presence of neomycin and in tyrosine-free medium, high levels of exogenous tyrosine hydroxylase activity were detected in extracts of the three cell lines. High-performance liquid chromatography of cell extracts and culture supernatants confirmed that the three cell lines hydroxylated tyrosine to form L-DOPA and released this metabolite into the culture medium. Interestingly, the neuroendocrine cell line AtT-20 synthesized not only L-DOPA but also dopamine. Evoked secretion studies established that AtT-20 cells released the transmitter upon depolarization in a regulated, calcium-dependent way. We discuss the implication of this approach for the analyses of neurotransmitter release as well as in the context of degenerative disorders such as Parkinson disease.

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Selected References

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  1. ANTON A. H., SAYRE D. F. A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J Pharmacol Exp Ther. 1962 Dec;138:360–375. [PubMed] [Google Scholar]
  2. Amano T., Richelson E., Nirenberg M. Neurotransmitter synthesis by neuroblastoma clones (neuroblast differentiation-cell culture-choline acetyltransferase-acetylcholinesterase-tyrosine hydroxylase-axons-dendrites). Proc Natl Acad Sci U S A. 1972 Jan;69(1):258–263. doi: 10.1073/pnas.69.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Björklund A., Stenevi U. Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res. 1979 Nov 30;177(3):555–560. doi: 10.1016/0006-8993(79)90472-4. [DOI] [PubMed] [Google Scholar]
  4. Bradbury A. F., Finnie M. D., Smyth D. G. Mechanism of C-terminal amide formation by pituitary enzymes. Nature. 1982 Aug 12;298(5875):686–688. doi: 10.1038/298686a0. [DOI] [PubMed] [Google Scholar]
  5. Breakefield X. O., Nirenberg M. W. Selection for neuroblastoma cells that synthesize certain transmitters. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2530–2533. doi: 10.1073/pnas.71.6.2530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brundin P., Strecker R. E., Lindvall O., Isacson O., Nilsson O. G., Barbin G., Prochiantz A., Forni C., Nieoullon A., Widner H. Intracerebral grafting of dopamine neurons. Experimental basis for clinical trials in patients with Parkinson's disease. Ann N Y Acad Sci. 1987;495:473–496. doi: 10.1111/j.1749-6632.1987.tb23695.x. [DOI] [PubMed] [Google Scholar]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clarke D. J., Brundin P., Strecker R. E., Nilsson O. G., Björklund A., Lindvall O. Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry. Exp Brain Res. 1988;73(1):115–126. doi: 10.1007/BF00279666. [DOI] [PubMed] [Google Scholar]
  9. Dunant Y., Israël M. The release of acetylcholine. Sci Am. 1985 Apr;252(4):58–66. doi: 10.1038/scientificamerican0485-58. [DOI] [PubMed] [Google Scholar]
  10. Emson P. C., Fonnum F. Choline acetyltransferase, acetylcholinesterase and aromatic L-amino acid decarboxylase in single identified nerve cell bodies from snail Helix aspersa. J Neurochem. 1974 Jun;22(6):1079–1088. doi: 10.1111/j.1471-4159.1974.tb04340.x. [DOI] [PubMed] [Google Scholar]
  11. Eveleth D. D., Gietz R. D., Spencer C. A., Nargang F. E., Hodgetts R. B., Marsh J. L. Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants. EMBO J. 1986 Oct;5(10):2663–2672. doi: 10.1002/j.1460-2075.1986.tb04549.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fritz L. C., Haidar M. A., Arfsten A. E., Schilling J. W., Carilli C., Shine J., Baxter J. D., Reudelhuber T. L. Human renin is correctly processed and targeted to the regulated secretory pathway in mouse pituitary AtT-20 cells. J Biol Chem. 1987 Sep 15;262(26):12409–12412. [PubMed] [Google Scholar]
  13. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  14. Greene L. A., Rein G. Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheo-chromocytoma cells. Brain Res. 1977 Jul 1;129(2):247–263. doi: 10.1016/0006-8993(77)90005-1. [DOI] [PubMed] [Google Scholar]
  15. Grima B., Lamouroux A., Boni C., Julien J. F., Javoy-Agid F., Mallet J. A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics. Nature. 1987 Apr 16;326(6114):707–711. doi: 10.1038/326707a0. [DOI] [PubMed] [Google Scholar]
  16. Gumbiner B., Kelly R. B. Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):318–322. doi: 10.1073/pnas.78.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gumbiner B., Kelly R. B. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell. 1982 Jan;28(1):51–59. doi: 10.1016/0092-8674(82)90374-9. [DOI] [PubMed] [Google Scholar]
  18. Hirsch E., Graybiel A. M., Agid Y. A. Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson's disease. Nature. 1988 Jul 28;334(6180):345–348. doi: 10.1038/334345a0. [DOI] [PubMed] [Google Scholar]
  19. Horellou P., Le Bourdellès B., Clot-Humbert J., Guibert B., Leviel V., Mallet J. Multiple human tyrosine hydroxylase enzymes, generated through alternative splicing, have different specific activities in Xenopus oocytes. J Neurochem. 1988 Aug;51(2):652–655. doi: 10.1111/j.1471-4159.1988.tb01088.x. [DOI] [PubMed] [Google Scholar]
  20. Kelly R. B. Pathways of protein secretion in eukaryotes. Science. 1985 Oct 4;230(4721):25–32. doi: 10.1126/science.2994224. [DOI] [PubMed] [Google Scholar]
  21. Kirschmeier P. T., Housey G. M., Johnson M. D., Perkins A. S., Weinstein I. B. Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences. DNA. 1988 Apr;7(3):219–225. doi: 10.1089/dna.1988.7.219. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Le Bourdellès B., Boularand S., Boni C., Horellou P., Dumas S., Grima B., Mallet J. Analysis of the 5' region of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms. J Neurochem. 1988 Mar;50(3):988–991. doi: 10.1111/j.1471-4159.1988.tb03009.x. [DOI] [PubMed] [Google Scholar]
  24. Leviel V., Chéramy A., Glowinski J. Role of the dendritic release of dopamine in the reciprocal control of the two nigro-striatal dopaminergic pathways. Nature. 1979 Jul 19;280(5719):236–239. doi: 10.1038/280236a0. [DOI] [PubMed] [Google Scholar]
  25. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  26. Matsuuchi L., Buckley K. M., Lowe A. W., Kelly R. B. Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells. J Cell Biol. 1988 Feb;106(2):239–251. doi: 10.1083/jcb.106.2.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. NAGATSU T., LEVITT M., UDENFRIEND S. A RAPID AND SIMPLE RADIOASSAY FOR TYROSINE HYDROXYLASE ACTIVITY. Anal Biochem. 1964 Sep;9:122–126. doi: 10.1016/0003-2697(64)90092-2. [DOI] [PubMed] [Google Scholar]
  28. Perlow M. J., Freed W. J., Hoffer B. J., Seiger A., Olson L., Wyatt R. J. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science. 1979 May 11;204(4393):643–647. doi: 10.1126/science.571147. [DOI] [PubMed] [Google Scholar]
  29. Ritchie A. K. Catecholamine secretion in a rat pheochromocytoma cell line: two pathways for calcium entry. J Physiol. 1979 Jan;286:541–561. doi: 10.1113/jphysiol.1979.sp012636. [DOI] [PMC free article] [PubMed] [Google Scholar]

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