Nitric oxide release follows endothelial nanomechanics and not vice versa
Nitric oxide release follows endothelial nanomechanics and not vice versa
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DOI：
10.1007/s00424-010-0871-8
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发表时间：
2010-10-01
影响因子：
4.5
通讯作者：
Oberleithner, Hans
中科院分区：
文献类型：
--
作者：
Fels, Johannes;Callies, Chiara;Oberleithner, Hans
在血管内皮细胞中，机械细胞刚性(De)和一氧化氮(NO)的释放是紧密耦合的。“软”细胞比“硬”细胞释放更多的NO。然而，目前尚不清楚NO本身是否是软化细胞的主要因素，或者NO释放是否是细胞软化的结果。为了解决这个问题，使用混合荧光/原子力显微镜来同时测量活的血管内皮细胞中DE和NO释放的变化。应用醛固酮来暂时软化细胞，并触发NO释放。然后，抑制或刺激NO的合成，同时测量De。细胞压痕实验用于评估De，而NO的释放则通过细胞内NO依赖的荧光指示剂(DAF-FM/DA)或靠近细胞表面的非选择性电极来测量。应用醛固酮后，De在10分钟内下降至对照组(100%)的80.5+/-1.7%。荧光强度同时增加到132.9+/-2.2%，表明内皮型一氧化氮合酶(ENOS)活性显著增强。N(Omega)-硝基-L-精氨酸甲酯抑制eNOS可阻断NO的释放，但不影响醛固酮引起的去甲肾上腺素的变化。应用eNOS非依赖的NO供体(NONOate/AM)可提高细胞内NO浓度，但同样不影响De。数据分析表明，DE值降低约10%就足以诱导eNOS活性显着增加。总之，血管内皮细胞的这些纳米力学特性决定了NO的释放，反之亦然。
In the vascular endothelium, mechanical cell stiffness (De) and nitric oxide (NO) release are tightly coupled. "Soft" cells release more NO compared to "stiff" cells. Currently, however, it is not known whether NO itself is the primary factor that softens the cells or whether NO release is the result of cell softening. To address this question, a hybrid fluorescence/atomic force microscope was used in order to measure changes in De and NO release simultaneously in living vascular endothelial cells. Aldosterone was applied to soften the cells transiently and to trigger NO release. NO synthesis was then either blocked or stimulated and, simultaneously, De was measured. Cell indentation experiments were performed to evaluate De, while NO release was measured either by an intracellular NO-dependent fluorescence indicator (DAF-FM/DA) or by NO-selective electrodes located close to the cell surface. After the application of aldosterone, De decreases, within 10 min, to 80.5 +/- 1.7% of control (100%). DAF-FM fluorescence intensity increases simultaneously to 132.9 +/- 2.2%, which indicates a significant increase in the activity of endothelial NO synthase (eNOS). Inhibition of eNOS (by N (omega)-nitro-l-arginine methyl ester) blocks the NO release, but does not affect the aldosterone-induced changes in De. Application of an eNOS-independent NO donor (NONOate/AM) raises intracellular NO concentration, but, again, does not affect De. Data analysis indicates that a decrease of De by about 10% is sufficient to induce a significant increase of eNOS activity. In conclusion, these nanomechanic properties of endothelial cells in vascular endothelium determine NO release, and not vice versa.