Laboratory of Exercise Biochemistry and Physiology, Postgraduate Program in Health Sciences, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil.
Int J Cardiol. 2013 Sep 10;167(6):2983-8. doi: 10.1016/j.ijcard.2012.08.050. Epub 2012 Sep 16.
Acute exercise increases reactive oxygen species (ROS) levels, including hydrogen peroxide (H2O2). H2O2 promotes endothelial nitric oxide synthase (eNOS) activation and phosphorylation in endothelial cells. With this in mind, the present study was designed to evaluate ex vivo eNOS phosphorylation in rat aortas incubated with H2O2 and to test this hypothesis in vivo in the aortas of rats submitted to acute exercise.
For ex vivo studies, six groups of aortic tissue were formed: control, H2O2, N-acetylcysteine (NAC), LY294002, compound C, and LY294002 plus compound C. While incubation with H2O2 increased Akt, AMPK and eNOS phosphorylation, pre-incubation with NAC strongly reduced the phosphorylation of these enzymes. For in vivo studies, male Wistar rats were divided into four groups: control, cont+NAC, exercise, and exer+NAC. After a 3h swimming session, animals were decapitated and aortas were excised for biochemical and immunoblotting analysis.
Acute exercise increased superoxide levels and dichlorofluorescein (DCF) concentrations, and this increase was related to phosphorylation of Akt, AMPK and eNOS. On the other hand, use of NAC reduced superoxide levels and DCF concentration. Reduced superoxide levels and DCF in the exer+NAC group were associated with decreased Akt, AMPK and eNOS phosphorylation. These results appear to be connected with vascular function because VASP phosphorylation increased in acute exercise and decreased in exer+NAC.
Our results indicate that ROS induced by acute exercise play the important role of activating eNOS, a process apparently mediated by Akt and AMPK.
急性运动增加活性氧物种（ROS）水平，包括过氧化氢（H2O2）。H2O2 促进内皮型一氧化氮合酶（eNOS）在血管内皮细胞中的激活和磷酸化。有鉴于此，本研究旨在评估 H2O2 孵育的大鼠主动脉中 eNOS 磷酸化的离体研究，并在急性运动大鼠的主动脉中体内检验这一假说。
对于离体研究，形成了六组主动脉组织：对照组、H2O2 组、N-乙酰半胱氨酸（NAC）组、LY294002 组、化合物 C 组和 LY294002 加化合物 C 组。虽然 H2O2 孵育增加了 Akt、AMPK 和 eNOS 的磷酸化，但 NAC 的预孵育强烈降低了这些酶的磷酸化。对于体内研究，雄性 Wistar 大鼠分为四组：对照组、CONT+NAC 组、运动组和 EXER+NAC 组。游泳 3 小时后，动物断头取主动脉进行生化和免疫印迹分析。
急性运动增加了超氧化物水平和二氯荧光素（DCF）浓度，这种增加与 Akt、AMPK 和 eNOS 的磷酸化有关。另一方面，NAC 的使用降低了超氧化物水平和 DCF 浓度。EXER+NAC 组中减少的超氧化物水平和 DCF 与 Akt、AMPK 和 eNOS 磷酸化减少有关。这些结果似乎与血管功能有关，因为急性运动使 VASP 磷酸化增加，而 EXER+NAC 则使 VASP 磷酸化减少。
我们的结果表明，急性运动诱导的 ROS 发挥了激活 eNOS 的重要作用，这一过程显然由 Akt 和 AMPK 介导。
