Zhang Quan-Jiang, McMillin Shawna L, Tanner Jason M, Palionyte Milda, Abel E Dale, Symons J David
College of Health, University of Utah, Salt Lake City, UT 84132, USA.
J Physiol. 2009 Aug 1;587(Pt 15):3911-20. doi: 10.1113/jphysiol.2009.172916. Epub 2009 Jun 8.
The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB - a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by approximately 50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition.
在培养的内皮细胞或分离的血管中，细胞内信号激酶Akt/蛋白激酶B（Akt）、蛋白激酶A（PKA）和单磷酸腺苷激活的蛋白激酶（AMPK）会因机械力增加或灌注速率提高而发生磷酸化。这三种激酶均使内皮型一氧化氮合酶（eNOS）的丝氨酸（S）1177位点磷酸化，而Akt和PKA还分别使eNOS的S617和S635位点磷酸化。尽管这些激酶可能在动态运动过程中促成eNOS的后续激活，但运动诱导的eNOS在体内磷酸化和激活的具体介质尚不清楚。我们测定了在存在或不存在使用渥曼青霉素对PI3激酶（PI3K）和Akt信号进行药理学抑制的情况下，50分钟跑步机跑步对Akt、AMPK、环磷酸腺苷反应元件结合蛋白（CREB - PKA的一个靶点）和eNOS（S 1177、635和617以及苏氨酸（T）495）磷酸化的影响。与久坐小鼠的动脉相比，跑步机跑步动物血管中的eNOS酶活性更高，且与Akt（S473）、CREB（S133）、AMPK（T172）以及eNOS的S1177和S617位点磷酸化增加相关，但与S635或T495位点的磷酸化无关。这些数据表明Akt信号是eNOS激活的主要介质。为证实这一点，在存在溶剂（二甲基亚砜）或PI3K抑制的情况下进行跑步机跑步。与久坐小鼠的结果相比，渥曼青霉素而非溶剂处理可阻止跑步机跑步期间血管Akt磷酸化和eNOS的S617位点磷酸化，而两组间运动相关的AMPK和CREB磷酸化增加相似。与久坐动物相比，渥曼青霉素处理后运动期间动脉eNOS的S1177位点磷酸化增加，但与溶剂处理小鼠的结果相比，升高幅度约降低了50%。这些发现表明，在跑步机跑步期间，Akt和AMPK对血管eNOS的S1177位点磷酸化有重要作用，并且在存在PI3K抑制的情况下，AMPK足以激活p - eNOS S1177。
