Converge of Pathways Regulating SMC Contractility

本项目的总体目标是了解在生理和病理生理条件下调节血管平滑肌收缩力的复杂过程，这些过程导致高血压、动脉粥样硬化、冠状动脉再狭窄、休克或脑血管痉挛。平滑肌（SM）的收缩性通过肌球蛋白的调节轻链（RLC）的磷酸化而开启，并且磷酸化水平取决于Ca 2 +/钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）和肌球蛋白轻链磷酸酶的相反活性，这两者都可以通过上游信号传导途径调节。我们在项目2中已经产生了MLCK缺失小鼠，其是胚胎致死的，从E15.5开始，具有一些延伸期，但在此之前，胚胎胎盘或脐血管显示RLC磷酸化和响应于Ca 2+的正常力发育。我们将检验这样的假设，即普遍表达的SMMLCK分别对平滑肌和心肌的收缩、迁移、细丝和肌节形成以及A404 SMC“祖”细胞至关重要。（项目2，核心A）和转化的前心外膜细胞（项目3，核心A）;或者，补偿激酶在MLCK缺失胚胎中解释MLCK活性，或者另一种激酶在胚胎发育期间通常占优势。初步研究表明，MLCK无效胚胎中冠状血管的形成是有缺陷的，因此，我们将使用项目3检验MLCK对于心外膜器官的心外膜细胞（冠状血管的前体）迁移至关重要的假设。肌球蛋白马达的细胞迁移和收缩性也受到调节，并有助于SM收缩表型。这些机制将探讨使用动力学分析与一个新的 最近合成的ATP的荧光3 '-氨基衍生物。我们将检验AM的假设。ADP强结合的横桥在低肌动球蛋白活性、慢缩短速度和低水平的RLC磷酸化（SM肌球蛋白的特征性特征）下维持强直力中起重要作用。拟议的研究需要与其他项目和核心A进行广泛的互动，并与本项目规划的中心主题相匹配。

The overall goal of this Project is to understand the complex processes that regulate contractility in vascular smooth muscle under physiological and pathophysiological conditions, which lead to high blood pressure, atherosclerosis, coronary restenosis, shock or cerebral vasospasm. Contractility is switched on in smooth muscle (SM) via phosphorylation of the regulatory light chain of myosin (RLC) and the level of phosphorylation is dependent upon the opposing activities of the Ca2+/calmodulin dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase, both of which can be regulated by upstream signaling pathways. We, with Project 2, have generated MLCK null mice, which are embryonic lethal, starting at E15.5 with some reaching term, but prior to this embryonic aortae or umbilical vessels display RLC phosphorylation and normal force development in response to Ca 2+. We will test the hypothesis that ubiquitously expressed SM MLCKs are critical for contraction, migration, filament and sarcomere formation in smooth and cardiac muscle respectively, as well as A404SMC "progenitor" cells (Project 2, Core A) and transformed proepicardial cells (Project 3, Core A); or alternatively that a compensatory kinase(s) accounts for the MLCK activity in MLCK null embryos or that another kinase normally predominants during embryonic development. Preliminary studies suggest that formation of the coronary vessels is defective in MLCK null embryos, thus, we will test the hypothesis that MLCKs are critical for migration of the epicardial cells of the proepicardial organ, the precursors of the coronary vessels with Project 3. The myosin motors underlying cell migration and contractility are also regulated and contribute to the SM contractile phenotype. These mechanisms will be explored using kinetic analysis with a novel recently synthesized fluorescent 3'-amino derivative of ATP. We will test the hypothesis that AM. ADP strongly bound crossbridges play a significant role in maintaining tonic force at low actomyosin activity, slow shortening velocity and low levels of RLC phosphorylation, characteristic features of SM myosins. The proposed studies require extensive interactions with the other projects and Core A and match the central theme of this PPG.