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), i nduced differentiation of human neuroblastoma NB69 cells to a dopamine phen otype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 mu M SNAP in se rum-free-defined medium for 24 h. SNAP treatment did not increase the numbe r of necrotic or apoptotic cells. However, a decrease in the number of viab le cells was observed at 750 mu M SNAP. In addition, a decrease in H-3-thym idine uptake was detected at the highest dose of SNAP. An increase in the a ntiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapopt otic Pax and Bcl-xS protein levels were also detected by Western blot analy sis after SNAP treatment. At low doses (50-125 mu M), SNAP induced an incre ase in catecholamine levels, H-3-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 mu M). The TH protein content, analyzed by Western blot, rem ained unchanged in SNAP-treated cells throughout the range of doses studied , when compared with the control group. SNAP produced a dose-dependent decr ease in the glutathione (GSH) content of the culture medium, without alteri ng intracellular GSH. In addition, cGMP levels and nitrite concentration, m easured in the supernatant of SNAP-treated cells, increased in a dose-depen dent manner, as compared to control levels. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxaline-1-one (ODQ) did not revert the SNAP- induced effect on H-3-dopamine uptake to control values. These results sugg est that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the posttranscriptional level through a cGMP-indepe ndent mechanism. (C) 2000 Elsevier Science Ltd. All rights reserved.