MEGJ调节缺氧后血管平滑肌细胞低反应性的cAMP-PKA机制

The c AMP-PKA mechanism involved in the regulation of MEGJ on vascular reactivity after hypoxia

目的观察肌内皮缝隙连接(MEGJ)参与血管生成素-2(Ang2)调节缺氧血管平滑肌细胞(VSMCs)中诱导型一氧化氮合酶(i NOS)高表达和血管低反应性的机制,探讨其是否与c AMP-PKA途径有关。方法采用大鼠血管内皮细胞(VECs)和VSMCs双面共培养模型,分别采用western blotting、phalloidin荧光法和染料Sulforhodamine B转运技术,观察缝隙连接蛋白43(CX43)亚型表达、MEGJ形成及通讯功能,同时检测VSMCs中i NOS表达,并利用FITC-BSA荧光透过率反映VSMCs收缩反应性。结果在双面共培养模型中,PKA抑制剂H-89可削弱外源性Ang2作用下缺氧VSMCs的i NOS表达和VSMC收缩反应性(P<0.05);缺氧后VECs中c AMP浓度,以及PKA活性显著增高(P<0.05),其增高可被Ang2进一步增强,而被Tie2的SiRNA削弱(P<0.05)。在双面共培养系统上腔(VECs面)给予c AMP和PKA抑制剂,均可显著抑制外源性Ang2作用下缺氧后增高的MEGJ中Cx43蛋白表达、MEGJ形成和通讯功能(P<0.05)。结论缺氧使VSMCs中i NOS表达增高和收缩性降低,是通过c AMP-PKA信号途径上调Cx43蛋白表达、MEGJ形成和通讯功能而实现的。

Objective To observe the the c AMP-PKA mechanism involved in the regulation of myoendothelial gap junctions（ MEGJ） on vascular hyporeactivity after hypoxia in rats. Methods The double-sided cells coculture model of vascular endothelial cells（ VECs） and vascular smooth muscle cells（ VSMCs） were set up,the protein expression of inducible nitric oxide synthase（ i NOS） and Cx43 was detected via Western blotting,the VSMCs contraction was detected via the leakage of FITC-BSA,MEGJ formation was observed by immunocytochemistry of phalloidin,and MEGJ communication was reflected by the dye transfer of Sulforhodamine B. Results（ 1） In the double-sided cells co-culture model,the inhibitor of PKA added to VECs could weaken the increased i NOS expression in VSMCs and VSMCs hyporeactivity after exogenous angiopoietin-2（ Ang2） treatment following hypoxia;（ 2）The c AMP concentration and PKA activity in VECs increased after hypoxia（ P〈0. 05）,further enhanced by exogenous Ang2 and weakened by Tie2 siRNA（ P〈0. 05）; the antagonists of c AMP or PKA added to VECs could inhibit the increase of Cx43 expression in MEGJ,and weaken the increase of MEGJ formation and communication after exogenous Ang2 treatment following hypoxia（ P〈0. 05）. Conclusion After hypoxia,c AMP-PKA system up-regulates Cx43 protein expression,MEGJ formation and communication functions,leading to high expression of i NOS and low reactivity in VSMCs.