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l ‐DOPA and glia‐conditioned medium have additive effects on tyrosine hydroxylase expression in human catecholamine‐rich neuroblastoma NB69 cells
Author(s) -
RodríguezMartín E.,
Canals S.,
Casarejos M. J.,
de Bernardo S.,
Handler A.,
Mena M. A.
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00440.x
Subject(s) - tyrosine hydroxylase , aromatic l amino acid decarboxylase , endocrinology , ascorbic acid , biology , glutathione , medicine , lactate dehydrogenase , biochemistry , chemistry , microbiology and biotechnology , dopamine , enzyme , food science
The aim of this study was to investigate the effect of l ‐DOPA and glia‐conditioned medium (GCM) on cell viability, tyrosine hydroxylase (TH) expression, dopamine (DA) metabolism and glutathione (GSH) levels of NB69 cells. l ‐DOPA (200 µ m ) induced differentiation of NB69 cells of more than 4 weeks in vitro, as shown by phase‐contrast microscopy and TH immunocytochemistry, and decreased replication, as shown by 5‐bromodeoxyuridine immunostaining. l ‐DOPA did not increase the number of necrotic or apoptotic cells, as shown by morphological features, Trypan Blue, lactate dehydrogenase activity, bis‐benzimide staining and TUNEL assay. Furthermore, l ‐DOPA (200 µ m ) increased Bcl‐xL protein expression. Incubation of cells with l ‐DOPA (50, 100, 200 µ m ) for 24 h resulted in an increase in TH protein levels (174, 196 and 212% versus control). Neither carbidopa, an inhibitor of l ‐aromatic amino acid decarboxylase enzyme, nor l ‐buthionine sulfoximine, which inhibits GSH synthesis, or ascorbic acid, an antioxidant, blocked the l ‐DOPA‐induced effect on TH protein expression. l ‐DOPA (0, 50, 100 and 200 µ m ) plus GCM further increased the amount of TH protein (346, 446, 472 and 424%). l ‐DOPA (200 µ m ) increased TH protein levels to 132, 191 and 245% of controls after incubation for 24, 48 and 72 h. DA metabolism in NB69 cells was increased in cultures treated with either l ‐DOPA (200–300 µ m ) or GCM and these two agents had a synergistic effect on DA metabolism. In addition, l ‐DOPA (200 µ m ) or/and GCM‐treated cells increased their GSH extracellular levels (223, 257, 300% of controls) after 48 h of treatment. The l ‐DOPA‐induced increase of TH protein expression in NB69 cells was independent of DA production, free radicals and GSH up‐regulation.