Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line.
The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 microM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 microM SNAP. In addition, a decrease in (3)H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50-125 microM), SNAP induced an increase in catecholamine levels, (3)H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 microM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2, 4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on (3)H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.
Rodríguez-Martín
E
E
Departamento de Investigación, Servicio de Neurobiología, Hospital Ramón y Cajal, Carretera de Colmenar Viejo, Km.9, 28034 Madrid, Spain.
Casarejos
M J
MJ
Bazán
E
E
Canals
S
S
Herranz
A S
AS
Mena
M A
MA
D002395:Catecholamines / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D006152:Cyclic GMP / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D002395:Catecholamines; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; D010396:Penicillamine; D006152:Cyclic GMP