a Department of Cardiovascular Surgery, Xiehe Hospital, Huazhong University of Science and Technology, Wuhan, 430022, China.
Biochem Cell Biol. 2014 Apr;92(2):137-44. doi: 10.1139/bcb-2013-0042. Epub 2014 Jan 8.
The objective was to investigate whether M3 muscarinic acetylcholine receptor (mAChR) dysfunction disrupts the linkage between the vascular endothelial (VE)-cadherin in the adherens junctional complex and the actin-based cytoskeleton, increasing vascular permeability in atherosclerosis. Western blotting revealed that a selective M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and M3 receptor siRNA decrease VE-cadherin and β-catenin in Triton X-100-insoluble fractions, indicating that M3 receptor inhibition weakens the linkage between the VE-cadherin/β-catenin complex and the actin cytoskeleton. Co-immunoprecipitation assays showed that M3 receptor inhibition reduces Rac1 activity and the association of IQ motif-containing GTPase-activating protein 1 (IQGAP1) with Ras-related C3 botulinum toxin substrate 1 (Rac1), while increasing the interaction between IQGAP1 and β-catenin. Using IQGAP1 siRNA, we found that IQGAP1 is required for stable interaction between VE-cadherin/β-catenin and the actin cytoskeleton in quiescent endothelial cells; IQGAP1 siRNA augments the M3 receptor inhibition-induced dissociation between them. Moreover, S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, attenuates this disassociation and Rac1 activity inhibition. The M3 receptor facilitates interaction of the VE-cadherin-based adherens junctional complex and the actin-based cytoskeleton by maintaining Rac1 activity, which regulates the interaction between IQGAP1/Rac1 and IQGAP1/β-catenin, and may contribute to endothelial barrier function under physiological conditions.
目的在于研究 M3 毒蕈碱型乙酰胆碱受体（mAChR）功能障碍是否会破坏血管内皮（VE）-钙粘蛋白在黏附连接复合体与肌动蛋白细胞骨架之间的连接，从而增加动脉粥样硬化中的血管通透性。Western blot 分析显示，一种选择性 M3 受体拮抗剂 4-二苯基乙氧基-N-甲基哌啶碘甲烷（4-DAMP）和 M3 受体 siRNA 减少了 Triton X-100 不溶性级分中的 VE-钙粘蛋白和β-连环蛋白，表明 M3 受体抑制减弱了 VE-钙粘蛋白/β-连环蛋白复合物与肌动蛋白细胞骨架之间的连接。免疫共沉淀实验表明，M3 受体抑制降低了 Rac1 活性和 IQ motif 含有 GTPase 激活蛋白 1（IQGAP1）与 Ras 相关 C3 肉毒梭菌毒素底物 1（Rac1）的结合，同时增加了 IQGAP1 与β-连环蛋白的相互作用。使用 IQGAP1 siRNA，我们发现 IQGAP1 是静止内皮细胞中 VE-钙粘蛋白/β-连环蛋白与肌动蛋白细胞骨架之间稳定相互作用所必需的；IQGAP1 siRNA 增强了 M3 受体抑制诱导的它们之间的解离。此外，一氧化氮（NO）供体 S-亚硝基-N-乙酰青霉胺（SNAP）可减弱这种解离和 Rac1 活性抑制。M3 受体通过维持 Rac1 活性促进 VE-钙粘蛋白为基础的黏附连接复合体与肌动蛋白为基础的细胞骨架之间的相互作用，从而调节 IQGAP1/Rac1 和 IQGAP1/β-连环蛋白之间的相互作用，这可能有助于生理条件下内皮屏障功能。
