Thomas Shane R, Chen Kai, Keaney John F
Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118. USA.
J Biol Chem. 2002 Feb 22;277(8):6017-24. doi: 10.1074/jbc.M109107200. Epub 2001 Dec 13.
Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.
内皮型一氧化氮合酶（eNOS）是血管稳态的重要组成部分。在血管疾病期间，内皮细胞会暴露于过量的活性氧中，这些活性氧可通过诱导各种信号通路改变细胞表型。在本研究中，我们检测了过氧化氢（H₂O₂）诱导的信号传导对猪主动脉内皮细胞中eNOS磷酸化状态和活性的影响。我们发现，通过l-[(³)H]精氨酸向l-[(³)H]瓜氨酸的转化以及细胞中环鸟苷酸（cGMP）含量测定，H₂O₂处理增强了eNOS活性和一氧化氮（NO）生物活性。与eNOS激活同时发生的是，H₂O₂还激活了蛋白激酶B（Akt），增加了eNOS在丝氨酸1177位点的磷酸化，并降低了eNOS在苏氨酸495位点的磷酸化。Src激酶、表皮生长因子受体（ErbB）家族受体和磷脂酰肌醇3激酶（PI 3-K）的选择性抑制剂显著减弱了H₂O₂诱导的eNOS活性促进以及eNOS在丝氨酸1177和苏氨酸495位点磷酸化状态的调节。我们发现，Akt激活、eNOS丝氨酸1177磷酸化以及H₂O₂诱导的eNOS激活是钙依赖性的，而eNOS在苏氨酸495位点的去磷酸化则不是，这表明在PI 3-K下游的信号级联反应中有一个分支点。与此一致的是，Akt显性负性异构体的过表达抑制了H₂O₂诱导的eNOS在丝氨酸1177位点的磷酸化，但不抑制eNOS在苏氨酸495位点的去磷酸化。总之，这些数据表明，H₂O₂通过Src和ErbB受体依赖性PI 3-K激活介导的酶磷酸化状态的协同变化促进钙依赖性eNOS活性。反过来，PI 3-K通过钙和Akt依赖性途径介导eNOS丝氨酸1177磷酸化，而eNOS苏氨酸495去磷酸化不涉及钙或Akt。这种反应可能代表内皮细胞在氧化应激增强的条件下维持NO生物活性的一种尝试。
