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

The aim of this study was to investigate the effect Of L-DOPA and glia-cond itioned medium (GCM) on cell viability, tyrosine hydroxylase (TH) expressio n, dopamine (DA) metabolism and glutathione (GSH) levels of NB69 cells. L-D OPA (200 muM) induced differentiation of NB69 cells of more than 4 weeks in vitro, as shown by phase-contrast microscopy and TH immunocytochemistry, a nd 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 muM) increase d Bcl-xL protein expression. Incubation of cells with L-DOPA (50, 100, 200 muM) for 24 h resulted in an increase in TH protein levels (174, 196 and 21 2% versus control). Neither carbidopa, an inhibitor Of L-aromatic amino aci d decarboxylase enzyme, nor L-buthionine sulfoximine, which inhibits GSH sy nthesis, or ascorbic acid, an antioxidant, blocked the L-DOPA-induced effec t on TH protein expression. L-DOPA (0, 50, 100 and 200 muM) plus GCM furthe r increased the amount of TH protein (346, 446, 472 and 424%). L-DOPA (200 Vm) increased TH protein levels to 132, 191 and 245% of controls after incu bation for 24, 48 and 72 h. DA metabolism in NB69 cells was increased in cu ltures treated with either L-DOPA (200-300 muM) or GCM and these two agents had a synergistic effect on DA metabolism. In addition, L-DOPA (200 muM) o r/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.