Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.
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.
Horellou
P
P
Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Guibert
B
B
Leviel
V
V
Mallet
J
J
D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism; D002395:Catecholamines / Q000096:biosynthesis / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D004298:Dopamine / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D010911:Pituitary Neoplasms / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism
D002395:Catecholamines; D004247:DNA; D014446:Tyrosine 3-Monooxygenase; D001142:Aromatic-L-Amino-Acid Decarboxylases; D004298:Dopamine