血管平滑肌细胞中膜钙泵PMCA与肌球蛋白轻链磷酸酶引导亚单位MYPT1的互作在血管张力调节及高血压病理发生中的作用

原发性高血压病因不明。经血管平滑肌膜钙泵PMCA降低胞内钙浓度及经肌球蛋白轻链磷酸酶（MLCP）对MLC脱磷是血管舒张的最后通路。GWAS发现PMCA是高血压致病基因，但其调节异常对高血压发病可能更重要。申请人前期报告了血管内皮细胞一氧化氮和B细胞CD22激活PMCA的降钙机制（2000 JBC；2004 Nature Immunol）。为探索血管平滑肌上类似降钙机制，我们用质谱分析PMCA蛋白复合体，找出MLCP分子中引导亚单位MYPT1与PMCA互作于静息时，血管紧张素刺激后消失。免疫共沉淀证实其完整分子三亚单位都在复合体中，恶性高血压时互作减弱。接下来要研究二者直接互作、还是间接通过Grb2、互作中的MLCP是否感知PMCA状态而对MLC脱磷、对复合体中的哪个成员进行脱磷从而抑制PMCA功能、互作异常或断裂与高血压发病的关系。从而为血管张力调节提供新机制及为高血压药物研发提供新靶标。

In the most cases, the etiology of hypertension is unknown and is thus called as primary hypertension. Increased vascular tone caused by dysregulation of contraction and relaxation of vascular smooth muscle cells (VSMCs) forms the basis of hypertension. Decreasing intracellular calcium concentration through plasma membrane Ca2+ ATPase (PMCA) and dephosphorylation of myosin light chain (MLC) by myosin light chain phosphatase (MLCP) are the two final common pathways for vasorelaxation. GWAS found that PMCA is a pathogenic gene for hypertension, but our understanding for its regulatory mechanism is very limited. The applicant has previously reported the mechanisms of PMCA activation to reduce the intracellular calcium concentration ([Ca2+]i) in vascular endothelial cells by endogenous nitric oxide (2000 JBC) and in B cells by CD22 (2004 Nature Immunol). In order to explore a similar [Ca2+]i-lowering mechanism through PMCA in VSMCs, we analyzed the PMCA protein complex by mass spectrometry and found a novel interaction between the myosin phosphatase target subunit 1 (MYPT1) and PMCA, which occurred at resting state but disappeared after Ang-II stimulation. Co-immunoprecipitation found that all three subunits of the MLCP molecule (PP1c-MYPT1-M20) are all included in the complex. Furthermore, in Dahl rats with high-salt-induced malignant hypertension, the in vivo interaction between MLCP and PMCA in the aortic tissue turned to be looser as compared with it of control rats. Next, we need to study whether the interaction between the two is direct or indirect, if indirect whether the third one is Grb2 or other yet-identified protein sandwiched between MLCP and PMCA, whether the interacting MLCP can sense the PMCA’s functional state and then dephosphorizes MLC, whether the MLCP can dephosphorizes other member(s) of the PMCA protein complex, which member of the complex is dephosphorized to regulate PMCA function, and the causal relationship between an abnormal/broken interactions of the two and the pathogenesis of hypertension. The results of this study will provide novel mechanisms for the regulation of vascular tone and new molecular targets for the development of anti-hypertensive medicine.