TRPV4通道和BK通道在caveolae中的相互作用对剪切力诱导内皮依赖性血管舒张的影响

Shear stress-induced endothelium dependent vasodilation is concerned with caveolae in endothelium. Caveolae are postulated to be mechanosensors and function as mechanotransducers in the vascular endothelial cell (EC). We found that shear stress-induced vasodilation was attenuated significantly in mouse coronary arteries in caveolin-1 null mice. These effects were through decreasing production of endothelium dependent relaxation factor. These results suggested that shear stress-induced vasodilation was endothelium and caveolae dependent. Shear stress-induced intracellular Ca2+ increasing also was diminished in caveolin-1 null mice, this demonstrated that Ca2+ influx was correlative with caveolae. Intracellular Ca2+ level in vascular EC facilitates the modulation of multiple signaling pathways. TRPV4 channel is the main influx pathway in vascular EC, and was postulated to be mechanosensors. Another important ionic channel is BK channel in vascular EC. Our primary data showed that shear stress activated BK current in EC. TRPV4 and BK are co-localization with caveolin-1 in EC. Whether TRPV4 and BK cannels interact with each other in EC has not been determined. The goal of this study will focus an integrated scheme in understanding of caveolae-TRPV4-BK channels on endothelial function, provide the pathogenesis of coronary artery disease caused by endothelium disfunction.

剪切力诱导血管内皮依赖性舒张与内皮细胞上caveolae相关。Caveolae在血管内皮细胞上被认为是一种感受剪切力的机械感受器。我们研究发现剪切力诱导内皮依赖性血管舒张降低与caveolae相关,并且caveolae基因敲除可使内皮细胞Ca2+内流减少。Ca2+在血管内皮细胞内具有调节多种信号通路的功能，TRPV4通道目前被认为是一种剪切力感受器，是内皮细胞主要的Ca2+内流通道。BK通道存在于caveolae内，并且我们预实验发现剪切力可使其明显被激活，说明BK通道在剪切力引起的血管舒张中发挥作用。TRPV4通道和BK通道在caveolae内都与caveolin-1共定位，三者之间是否有相互作用目前尚不清楚。本研究旨在观察caveolae,TRPV4和BK通道三者之间的相互作用，对三者影响内皮功能作用提供整合性的理解。为由内皮细胞功能损伤引起的冠状动脉疾病的发病机理提供理论基础。

剪切力诱导血管内皮依赖性舒张与内皮细胞上Caveolae相关，其在血管内皮细胞上被认为是一种感受剪切力的机械感受器。本课题研究发现：（1）在Caveolin-1基因敲除小鼠（CAV-1 KO）的冠状动脉剪切力诱导的血管舒张较正常小鼠（WT）明显减弱，去除血管内皮后CAV-1 KO较WT小鼠的舒张减弱明显减少。应用血管内皮细胞一氧化氮合成酶（eNOS）抑制剂L-NAME后，剪切力诱导的WT血管舒张明显减弱而CAV-1 KO却没有变化，说明这种减弱是与血管内皮细胞一氧化氮（NO）释放减少相关。 在培养的小鼠主动脉内皮细胞研究发现应用Caveolin-1 SiRNA后总eNOS表达没有变化，但是磷酸化eNOS表达减弱。（2）由于血管内皮细胞NO释放与细胞内Ca2+的浓度相关，进一步在培养的小鼠主动脉内皮细胞研究发现，剪切力可引起细胞内Ca2+浓度明显升高，但应用Caveolin-1 SiRNA使正常小鼠主动脉内皮细胞Caveolin-1基因减弱后发现剪切力诱导的内皮细胞Ca2+浓度并无明显升高，说明剪切力诱导的内皮细胞Ca2+浓度增高与Caveolin-1密切相关。（3）由于血管内皮细胞Ca2+浓度与细胞膜上的TRPV4通道有关，TRPV4 通道介导Ca2+内流，在应用TRPV4 通道抑制剂RN-1734后 剪切力诱导的WT舒张明显减弱而在CAV-1 KO却没有变化。应用SiRNA使TRPV4通道基因减弱后剪切力诱导的内皮细胞Ca2+浓度无明显升高。在WT主动脉内皮细胞应用TRPV4通道激活剂4α-PDD后细胞内Ca2+浓度明显增高，而在Caveolin-1基因减弱细胞却没有增高。结果显示Caveolin-1基因与TRPV4通道介导细胞内Ca2+浓度相关。同时研究发现应用Caveolin-1基因减弱后内皮细胞TRPV4通道蛋白表达明显减弱，而免疫共沉淀结果显示Caveolin-1蛋白与TRPV4通道蛋白存在共定位。说明Caveolae与TRPV4通道存在相互作用。（4）剪切力诱导的血管舒张在WT冠状动脉应用大电导钙激活钾通道（BK通道）阻断剂IBTX后减弱要明显大于在CAV-1 KO的减弱，且内皮细胞发现Caveolin-1基因因减弱后BK通道的α与β亚基（BKα和BKβ）的蛋白表达均减弱。免疫共沉淀研究发现Caveolin-1，TRPV4和BKα和BKβ存在共定位现象。