l‐DOPA and glia‐conditioned medium have additive effects on tyrosine hydroxylase expression in human catecholamine‐rich neuroblastoma NB69 cells

• Published in: Journal of neurochemistry 2001-08, Vol.78 (3), p.535-545
• Main Authors: Rodríguez‐Martín, E., Canals, S., Casarejos, M. J., de Bernardo, S., Handler, A., Mena, M. A.
• Format: Article
• Language: English
• Subjects: antioxidants; Antioxidants - pharmacology; Apoptosis - drug effects; Ascorbic Acid - pharmacology; bcl-X Protein; Bcl‐2 proteins; Biological and medical sciences; Buthionine Sulfoximine - pharmacology; Carbidopa - pharmacology; Cell Differentiation - drug effects; Culture Media, Conditioned; Culture Media, Serum-Free; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Dopamine - metabolism; Dopamine Agents - pharmacology; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; glia; Glutathione - metabolism; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Levodopa - pharmacology; l‐DOPA; Medical sciences; Neuroblastoma; Neuroglia - metabolism; Neurology; Neurons - cytology; Neurons - enzymology; Parkinson's disease; Proto-Oncogene Proteins c-bcl-2 - metabolism; Time Factors; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - metabolism; tyrosine hydroxylase
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2001.00440.x

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 µm) 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 µm) increased Bcl‐xL protein expression. Incubation of cells with l‐DOPA (50, 100, 200 µm) 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 µm) plus GCM further increased the amount of TH protein (346, 446, 472 and 424%). l‐DOPA (200 µm) 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 µm) or GCM and these two agents had a synergistic effect on DA metabolism. In addition, l‐DOPA (200 µm) 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.