Acute mechanical stretch promotes eNOS activation in venous endothelial cells mainly via PKA and Akt pathways.
Acute mechanical stretch promotes eNOS activation in venous endothelial cells mainly via PKA and Akt pathways.
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DOI：
10.1371/journal.pone.0071359
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Luo J
中科院分区：
文献类型：
--
作者：
Hu Z;Xiong Y;Han X;Geng C;Jiang B;Huo Y;Luo J
在脉管系统中，生理水平的一氧化氮（NO）保护免受各种应激源，包括机械拉伸。虽然内皮细胞对各种刺激的NO产生已被广泛研究，但静脉内皮细胞中牵张诱导的NO产生的确切机制仍不完全清楚。使用一个模型的连续细胞拉伸，我们发现，拉伸促进磷酸化的内皮NO合酶（eNOS）在Ser 1177，Ser 633和Ser 615和NO生产在人脐静脉内皮细胞。尽管牵张激活了激酶AMPKα、PKA、Akt和ERK 1/2，但牵张诱导的eNOS激活仅被PKA和PI 3 K/Akt的激酶特异性抑制剂抑制，而不被AMPKα和ERK 1/2抑制。通过靶向PKA和Akt基因的shRNA敲低获得了类似的结果。此外，PKA的抑制优先衰减eNOS激活的早期阶段，而PI 3 K/Akt通路的抑制减少eNOS激活的晚期阶段，这表明PKA和PI 3 K/Akt通路发挥不同的作用，在一个时间依赖性的方式。最后，我们研究了这些途径在牵张诱导的内皮细胞胞吐和白细胞粘附中的作用。有趣的是，我们发现，抑制PI 3 K/Akt通路增加牵张诱导的韦伯-帕拉德体胞吐和白细胞粘附，而抑制PKA通路具有相反的效果，这表明PKA的胞吐促进作用优于PKA介导的NO产生的抑制作用。总之，这些结果表明，PKA和Akt是重要的调控eNOS激活的静脉内皮细胞在机械拉伸下，而发挥不同的作用，在调节拉伸诱导的内皮细胞胞吐和白细胞粘附。
In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser1177, Ser633 and Ser615 and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.
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