Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases
Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases
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DOI：
10.1113/jphysiol.2009.172916
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发表时间：
2009-08-01
影响因子：
5.5
通讯作者：
Symons, J. David
中科院分区：
文献类型：
--
作者：
Zhang, Quan-Jiang;McMillin, Shawna L.;Symons, J. David
细胞内信号激酶Akt/蛋白激酶B （Akt）、蛋白激酶A （PKA）和单磷酸腺苷活化蛋白激酶（AMPK）在培养的内皮细胞或离体血管中随着机械力或灌注率的增加而磷酸化。这三种激酶磷酸化内皮一氧化氮合酶（eNOS）丝氨酸(S) 1177位点，而Akt和PKA分别磷酸化eNOS的S617和S635位点。尽管这些激酶可能在动态运动中促进eNOS的激活，但运动诱导eNOS磷酸化和激活的具体介质在体内尚不清楚。我们用wortmannin测定了50分钟跑步机对Akt、AMPK、环腺苷单磷酸反应元件结合蛋白（CREB - PKA的靶标）和eNOS （S 1177、635、617和苏氨酸(T) 495）磷酸化的影响，无论是否存在PI3K和Akt信号的药理抑制。与久坐小鼠的动脉相比，跑步动物血管中的eNOS酶活性更高，并且与Akt （S473）、CREB （S133）、AMPK （T172）和eNOS在S1177和S617的磷酸化增加有关，而与S635或T495的磷酸化无关。这些数据表明Akt信号是eNOS激活的主要介质。为了证实这一点，在DMSO或PI3K抑制存在的情况下进行跑步机跑步。与久坐小鼠的结果相比，wortmannin可阻止跑步机运动期间血管Akt磷酸化和eNOS S617磷酸化，但不影响载药治疗，而AMPK和CREB磷酸化的运动相关增加在两组之间相似。与久坐动物相比，wortmannin治疗后运动期间动脉eNOS S1177位点的磷酸化水平升高，但与药物治疗小鼠相比，升高幅度降低了50%。这些发现表明，Akt和AMPK在跑步过程中对血管eNOS S1177的磷酸化起重要作用，并且AMPK足以在PI3K抑制的情况下激活p-eNOS S1177。
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 similar to 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.