Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.
Identifieur interne : 000306 ( Pmc/Curation ); précédent : 000305; suivant : 000307Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.
Auteurs : P. Horellou ; B. Guibert ; V. Leviel ; J. MalletSource :
- Proceedings of the National Academy of Sciences of the United States of America [ 0027-8424 ] ; 1989.
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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PubMed: 2571152
PubMed Central: 298031
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Proc Natl Acad Sci U S A</journal-id><journal-title>Proceedings of the National Academy of Sciences of the United States of America</journal-title><issn pub-type="ppub">0027-8424</issn><issn pub-type="epub">1091-6490</issn></journal-meta><article-meta><article-id pub-id-type="pmid">2571152</article-id><article-id pub-id-type="pmc">298031</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Horellou</surname><given-names>P</given-names></name></contrib><contrib contrib-type="author"><name><surname>Guibert</surname><given-names>B</given-names></name></contrib><contrib contrib-type="author"><name><surname>Leviel</surname><given-names>V</given-names></name></contrib><contrib contrib-type="author"><name><surname>Mallet</surname><given-names>J</given-names></name></contrib></contrib-group><aff>Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.</aff><pub-date pub-type="ppub"><month>9</month><year>1989</year></pub-date><volume>86</volume><issue>18</issue><fpage>7233</fpage><lpage>7237</lpage><abstract><p>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.</p></abstract></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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