AMPK调节血管内皮钙激活钾离子通道的作用与机制

Endothelium-dependent hyperpolarization (EDH) is predominantly observed in resistance vessels and plays a role in the regulation of arterial blood pressure and cardiovascular homeostasis, such as flow-induced vasodilatation, reactive hyperemia, conducted vasodilation. Activation of endothelial intermediate- and small-conductance calcium-activated potassium channels (IKCa or KCa3.1 and SKCa, or KCa2.3 respectively) is characteristic of the EDH pathway. KCa3.1- and KCa2.3-mediated EDH is reduced with endothelial dysfunction, which develops with ageing, hypertension, and type 2 diabetes. Vascular adenosine monophosphate-activated protein kinase (AMPK) is now recognized as an attractive target in the treatment of vascular complications of obesity. However, whether KCa channels in endothelium are regulated by AMPK remains unclear. Primal study from our group indicates that AMPK is likely implicated in the regulation of KCa channels in endothelium. We hypothesize that the expression of KCa channels in endothelium would be upregulated by the activation of AMPK through ERK5-MEF2-KLF2/4 cascade, and the activity of KCa channels would be enhanced with direct phospholation by AMPK. The present project is designed to explore the modulation of KCa channels by AMPK and the underlying mechanism in mesenteric resistance arteries from normal mice, high fat diet induced obesity model as well as AMPKα1-/- and AMPKα2-/- mice via observing flow-mediated dilation in Pressure Myograph System and isometric tension in Multi Wire Myograph System. The effect of AMPK on KCa channels will be also examined in culture of endothelial cells from branches of the superior mesenteric artery (SMAECs), human umbilical vein endothelial cells (HUVECs) and HEK293 cells stably expressing KCa3.1 or KCa2.3 channels by using techniques of real-time PCR, Western blotting, immunoprecipitation, whole cell patch clamp, site-specific mutagenesis, respectively. The results will elucidate the role and mechanism of AMPK in the regulation of KCa channels, provide evidence for endothelial dysfunction-mediated by KCa channels in obesity, and support the clinical use of dugs acting through AMPK activation to restore impaired function of KCa channels.

血管内皮钙激活钾离子通道（KCa）介导的内皮依赖性超极化参与血流引起的血管舒张等生理适应反应和疾病时内皮功能障碍。AMPK对内皮KCa通道的调节不清楚。预实验结果提示刺激AMPK可能上调KCa通道的表达及活性。假设：血管内皮AMPK激活通过ERK5-MEF2-KLF2/4通路上调内皮KCa通道的表达，并通过直接磷酸化调节通道的功能。拟在正常和高脂饲料诱导的肥胖小鼠及AMPKα1或α2敲除小鼠的肠系膜阻力动脉上，以血流介导的舒张和血管张力变化为KCa通道功能指标，结合培养小鼠肠系膜动脉分支内皮细胞、人脐静脉内皮细胞和稳定表达人KCa通道的HEK293细胞系，应用膜片钳和分子生物学技术，阐明AMPK激活对血管内皮KCa通道表达的转录活化作用以及对通道蛋白的磷酸化激活作用与机制，为肥胖状态下KCa通道介导的舒张功能障碍提供依据，为临床应用AMPK激活作用的药物改善KCa通道功能障碍提供理论支持。

血管内皮钙激活钾离子通道（KCa）介导的内皮依赖性超极化（EDH）参与血流引起的血管舒张等生理适应反应，但AMPK对内皮KCa通道的调节不清楚。本研究发现激活血管内皮AMPK可通过ERK5-MEF2-KLF2/4信号通路上调KCa2.3通道的表达，而血清中含量最丰富的饱和脂肪酸软脂酸可通过激活Nox/ROS/p38-MAPK/NF-κB信号通路下调内皮细胞KCa2.3的表达，导致内皮舒张功能障碍。高脂饮食降低小鼠肠系膜二级动脉KCa2.3基因和蛋白表达、抑制KCa2.3介导的EDH反应并引起小鼠收缩压升高。AMPK激动剂AICAR可逆转上述变化。激活血管内皮AMPK使KCa2.3磷酸化（p-KCa2.3Thr-111）水平升高，通道电流增加。在Ang II灌注引起的小鼠高血压模型，肠系膜二级动脉内皮p-KCa2.3Thr-111水平明显降低，KCa2.3介导的EDH反应受损，激活AMPK可逆转Ang II引起的p-KCa2.3Thr-111水平、EDH反应的变化并改善高血压。在野百合碱腹腔注射引起的小鼠肺动脉高压模型，富尔顿指数（RV/LV+S）和肺中小动脉壁厚度增加，肺组织KCa2.3、p-CREBSer133和p-AMPKThr172蛋白水平显著降低，二甲双胍治疗可减轻肺动脉高压小鼠的上述变化。在培养的小鼠肺内皮细胞，AMPK通过激活CREB上调内皮细胞KCa2.3通道蛋白表达。给予AAV-KCNN3使内皮过表达KCa2.3通道可降低缺氧所致肺动脉高压小鼠右心室压、富尔顿指数和舒张期右心室游离壁厚度，改善肺动脉血流加速时间/肺动脉射血时间比。以上结果表明激活AMPK或靶向内皮KCa2.3通道均可作为代谢性高血压和肺动脉高压的治疗靶点。这为AMPK激动剂（二甲双胍等）老药新用治疗代谢性高血压和肺动脉高压提供了依据，也为以内皮KCa2.3通道激动剂为靶点开发新药指明了方向。