Rodríguez-Martín E, Casarejos M J, Bazán E, Canals S, Herranz A S, Mena M A
Departamento de Investigación, Servicio de Neurobiología, Hospital Ramón y Cajal, Carretera de Colmenar Viejo, Km.9, 28034 Madrid, Spain.
Neuropharmacology. 2000 Aug 23;39(11):2090-100. doi: 10.1016/s0028-3908(00)00049-6.
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.
一氧化氮（NO）供体S-亚硝基-N-乙酰-D,L-青霉胺（SNAP）可诱导人神经母细胞瘤NB69细胞分化为多巴胺表型，相差显微镜和酪氨酸羟化酶（TH）免疫细胞化学结果表明了这一点。在无血清限定培养基中，用50至750微摩尔/升的SNAP处理NB69细胞24小时。SNAP处理并未增加坏死或凋亡细胞的数量。然而，在750微摩尔/升的SNAP处理下，观察到活细胞数量减少。此外，在最高剂量的SNAP处理下，检测到[3H]胸腺嘧啶核苷摄取减少。SNAP处理后，通过蛋白质免疫印迹分析还检测到抗凋亡蛋白Bcl-2和Bcl-xL水平升高，促凋亡蛋白Bax和Bcl-xS水平降低。在低剂量（50 - 125微摩尔/升）时，SNAP可诱导儿茶酚胺水平、[3H]多巴胺摄取、TH活性和单胺代谢增加，而在高剂量（250 - 750微摩尔/升）时，所有这些参数均降低。与对照组相比，在整个研究剂量范围内，通过蛋白质免疫印迹分析的SNAP处理细胞中的TH蛋白含量保持不变。SNAP使培养基中的谷胱甘肽（GSH）含量呈剂量依赖性降低，但不改变细胞内GSH。此外，与对照水平相比，在SNAP处理细胞的上清液中测得的cGMP水平和亚硝酸盐浓度呈剂量依赖性增加。鸟苷酸环化酶抑制剂1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮（ODQ）并未使SNAP诱导的对[3H]多巴胺摄取的影响恢复到对照值。这些结果表明，SNAP释放的NO可诱导NB69细胞分化，并通过非cGMP依赖机制在转录后水平调节TH蛋白。
