Division of Cardiovascular Medicine, Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University, 473 W. 12th Ave, Columbus, OH 43210, USA.
Cardiovasc Res. 2013 Jan 1;97(1):33-43. doi: 10.1093/cvr/cvs287. Epub 2012 Sep 12.
The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC.
Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOS(Ser1176), Akt(Ser473), and PKA(Thr197) phosphorylation was increased following IPC. I/R decreased eNOS(Ser1176) phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOS(Ser1176) phosphorylation and quantitative Western blots showed ∼24% modification of eNOS(Ser1176) following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt(Ser473) or PKA(Thr197) phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOS(Ser1176) phosphorylation as well as cardioprotection.
Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.
内皮型一氧化氮合酶（eNOS）/NO 信号在晚期缺血预处理（IPC）中作用已得到充分证实；然而，eNOS 在早期 IPC 中的作用及其激活仍存在争议。本研究旨在探讨 eNOS 在早期 IPC 中的作用，以及调节早期 IPC 中 eNOS 激活的信号通路和分子相互作用。
在存在或不存在 NOS 抑制剂 l-NAME、磷脂酰肌醇 3-激酶（PI3K）抑制剂 LY294002（LY）和蛋白激酶 A（PKA）抑制剂 H89 的情况下，大鼠心脏经历 30 分钟的整体缺血再灌注（I/R），并进行 IPC（三个 5 分钟的 I 和 5 分钟的 R 周期）。在 IPC 诱导期间或内皮通透性增加之前，给予 LY 或 H89。与 I/R 相比，IPC 改善了缺血后收缩功能并减少了梗死，而 l-NAME 或内皮通透性的增加则消除了这种作用。IPC 后，eNOS（Ser1176）、Akt（Ser473）和 PKA（Thr197）磷酸化增加。I/R 降低了 eNOS（Ser1176）磷酸化，而 IPC 增加了它。质谱分析证实了 eNOS（Ser1176）磷酸化，定量 Western blot 显示 IPC 后 eNOS（Ser1176）修饰约 24%。免疫沉淀显示 eNOS、Akt 和 PKA 复合物形成。免疫组织化学显示 IPC 诱导的 Akt 和 PKA 在心肌细胞和内皮细胞中的磷酸化。随着 eNOS 的激活，IPC 通过电子顺磁共振自旋捕获和荧光显微镜增加了 NO 的产生。LY 或 H89 不仅分别降低了 Akt（Ser473）或 PKA（Thr197）磷酸化，而且还消除了 IPC 诱导的 eNOS 保护和 eNOS（Ser1176）磷酸化以及心脏保护作用。
因此，Akt 和 PKA 介导的 eNOS 激活，伴有 C 端附近的磷酸化，对于早期 IPC 诱导的心脏保护至关重要，内皮细胞来源的 eNOS 衍生的 NO 发挥着关键作用。
