Koyama Tetsuya, Kimura Chiwaka, Park Sung Jin, Oike Masahiro, Ito Yushi
Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, 812-82, Fukuoka, Japan.
Life Sci. 2002 Dec 20;72(4-5):511-20. doi: 10.1016/s0024-3205(02)02246-4.
We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.
我们利用fura - 2和一种对一氧化氮（NO）敏感的染料二氨基荧光素 - 2（DAF - 2），在牛主动脉内皮细胞（BAEC）中研究了钙离子动员与细胞内一氧化氮生成之间的关系。高浓度的ATP（100微摩尔）或毒胡萝卜素（1微摩尔）通过单个钙离子瞬变耗尽细胞内钙离子储存位点，甚至在无钙溶液中也能诱导DAF - 2荧光增加，从而表明储存耗尽会导致一氧化氮生成。低浓度的ATP（1微摩尔）能引发相同程度的DAF - 2荧光增加，这种低浓度ATP诱导钙离子振荡但不耗尽储存位点，不过只有在细胞外钙离子存在的情况下才会如此。此外，用镧离子抑制ATP（1微摩尔）诱导的钙离子内流会抑制DAF - 2荧光。在无钙、含锰的溶液中施加ATP（0.3微摩尔）会诱导与锰离子内流偶联的fura - 2淬灭，在每个振荡峰值后不久重复出现。这些结果表明，一氧化氮优先由进入的钙离子生成，并且由低水平刺激诱导的钙离子振荡通过强烈调节钙离子内流在一氧化氮生成中起重要作用。
