Bi-phasic activation of eNOS in response to uni-axial cyclic stretch is mediated by differential mechanisms in BAECs.
Bi-phasic activation of eNOS in response to uni-axial cyclic stretch is mediated by differential mechanisms in BAECs.
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DOI：
10.1016/j.lfs.2005.12.051
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发表时间：
2006-06
期刊：
影响因子：
6.1
通讯作者：
Hideo Takeda;K. Komori;N. Nishikimi;Y. Nimura;M. Sokabe;K. Naruse
中科院分区：
文献类型：
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作者：
Hideo Takeda;K. Komori;N. Nishikimi;Y. Nimura;M. Sokabe;K. Naruse
我们研究了牵张诱导牛动脉内皮细胞（BAECs）产生NO（一氧化氮）的信号机制。BAEC上培养的弹性硅胶室涂有纤维连接蛋白进行单轴循环拉伸（1 Hz，20%的长度）和产生的NO的量进行了测量cGMP测定。NO的产生呈双相增加，在牵张开始后5 min和20 min达到峰值。相应地，内皮型一氧化氮合酶（eNOS）和Akt/PKB（分别通过丝氨酸1177和丝氨酸473的磷酸化测定）的活性随时间推移显示出两个峰值。应用Gd 3+，一种有效的SA通道阻断剂，和外部Ca 2+的耗竭只抑制eNOS和Akt活性的第一个峰，但对第二个峰几乎没有影响。另一方面，PI 3 K抑制剂Wortmannin，LY 294002几乎完全抑制第二峰，但不抑制第一峰。这些结果表明，eNOS响应周期性牵张的上调是由两个不同的途径介导的，[Ca 2 +] i增加通过SA通道在早期阶段（部分Akt/PKB）和PI 3 K-Akt/PKB途径在晚期阶段。
We investigated the signaling mechanism of stretch-induced NO (Nitric oxide) production in bovine arterial endothelial cells (BAECs). BAECs cultured on an elastic silicone chamber coated with fibronectin were subjected to uni-axial cyclic stretch (1 Hz, 20% in length) and the amount of produced NO was measured by a cGMP assay. NO production increased in a bi-phasic manner and peaked at 5 min and 20 min after stretch onset. Correspondingly, the activities of endothelial nitric oxide synthase (eNOS) and Akt/PKB (measured by phosphorylation at serine 1177 and serine 473, respectively), showed two peaks over time. Application of Gd3+, a potent SA channel blocker, and depletion of external Ca2+exclusively inhibited the first peaks of eNOS and Akt activity, but exerted little effect on the second peak. On the other hand, the PI3K inhibitors, Wortmannin, LY294002, almost completely inhibited the second peak but not the first. These results suggest that up-regulation of eNOS in response to cyclic stretch was mediated by two distinct pathways, [Ca2+]iincreases via the SA channel in an early phase (partially Akt/PKB), and PI3K–Akt/PKB pathways in a late phase.