Takeda Hideo, Komori Kimihiro, Nishikimi Naomichi, Nimura Yuji, Sokabe Masahiro, Naruse Keiji
Division of Vascular Surgery, Department of Surgery, Nagoya 466-8550, Japan.
Life Sci. 2006 Jun 13;79(3):233-9. doi: 10.1016/j.lfs.2005.12.051. Epub 2006 Feb 3.
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 1,177 and serine 473, respectively), showed two peaks over time. Application of Gd(3+), a potent SA channel blocker, and depletion of external Ca(2+) 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, Ca(2+) increases via the SA channel in an early phase (partially Akt/PKB), and PI3K-Akt/PKB pathways in a late phase.
我们研究了牛主动脉内皮细胞（BAECs）中拉伸诱导一氧化氮（NO）产生的信号传导机制。将培养在涂有纤连蛋白的弹性硅酮腔室上的BAECs进行单轴循环拉伸（1Hz，长度增加20%），并通过cGMP测定法测量产生的NO量。NO的产生呈双相增加，并在拉伸开始后5分钟和20分钟达到峰值。相应地，内皮型一氧化氮合酶（eNOS）和Akt/PKB的活性（分别通过丝氨酸1177和丝氨酸473的磷酸化来测量）随时间呈现两个峰值。应用强力SA通道阻滞剂Gd(3+)和耗尽细胞外Ca(2+)仅抑制了eNOS和Akt活性的第一个峰值，但对第二个峰值影响很小。另一方面，PI3K抑制剂渥曼青霉素、LY294002几乎完全抑制了第二个峰值，但对第一个峰值没有影响。这些结果表明，响应循环拉伸时eNOS的上调是由两种不同的途径介导的，早期通过SA通道使[Ca(2+)]i增加（部分通过Akt/PKB），后期通过PI3K-Akt/PKB途径。
