Meini Antonella, Garcia Julian Blanco, Pessina Gian Paolo, Aldinucci Carlo, Frosini Maria, Palmi Mitri
Dipartimento di Scienze Biomediche, Universita di Siena, via A. Moro 2, 53100 Siena, Italy.
Eur J Neurosci. 2006 Apr;23(7):1690-700. doi: 10.1111/j.1460-9568.2006.04705.x.
To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 microm) determined a gradual, moderate elevation in [Ca2+]i (46.8 +/- 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 +/- 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 +/- 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50-300 microm) of DETA/NO negatively regulated cell proliferation via a Ca2+-independent mechanism.
为阐明一氧化氮（NO）对细胞生长调节的机制以及Ca2+在其中所起的作用，我们研究了暴露于不同水平NO的细胞系中细胞内Ca2+浓度（[Ca2+]i）、丝裂原活化蛋白激酶[细胞外信号调节蛋白激酶（ERK）]与细胞增殖之间的关系。数据显示，低浓度[(Z)-1-[2-氨乙基]-N-[2-氨乙基]氨基]重氮-1,2-二醇盐（DETA/NO）（10微摩尔）释放的NO使[Ca2+]i逐渐适度升高（比对照升高46.8±7.2%），这与ERK的激活以及细胞分裂的增强相平行。功能性阻断Ca2+或抑制钙调蛋白或丝裂原活化蛋白激酶激酶活性可阻止ERK激活，并拮抗NO的促有丝分裂作用。有利于Ca2+进入细胞的实验条件导致[Ca2+]i升高（189.5±4.8%）、ERK激活和细胞分裂。NO增强了Ca2+升高（358±16.8%）和ERK激活，导致p21Cip1表达并抑制细胞增殖。此外，功能性阻断Ca2+可下调ERK激活，并逆转NO的抗增殖作用。NO诱导的促有丝分裂和抗有丝分裂反应均被一种环鸟苷酸（cGMP）类似物模拟，而它们被选择性cGMP抑制剂完全拮抗。这些结果首次证明，低水平NO对细胞增殖的调节是cGMP依赖性的，并且通过Ca2+/钙调蛋白/丝裂原活化蛋白激酶激酶/ERK途径发生。在这种效应中，Ca2+信号的幅度可能通过调节ERK激活的强度来决定对NO增殖反应的特异性。与低水平相反，高水平（50 - 300微摩尔）的DETA/NO通过一种不依赖Ca2+的机制对细胞增殖产生负调节作用。
