SIGNAL TRANSDUCTION OF SMOOTH MUSCLE

这一多学科计划将阐明调节血管平滑肌发育、肥大、基因表达和收缩的信号转导途径--这些过程容易导致高血压、动脉粥样硬化、血管痉挛、血管成形术后再狭窄和休克。这些项目是相互依存的，集中在对了解平滑肌细胞收缩和生长的共同主题的研究上，并且需要在分子生物学(项目3)、生物化学(项目2)、生理学(项目1)和生物物理学和新型探针的生成(核心A)方面的互补专业知识。新开发的工具和方法将被用来识别和表征蛋白激酶和磷酸酶，它们的上游和下游目标，以及交叉桥状态动力学。新的试剂和方法包括用于蛋白激酶亲和纯化的磷酸激活的三磷酸腺苷，fmoL水平蛋白质的混合多肽测序，环境敏感的荧光核苷酸类似物，笼形核苷酸，多肽和脂肪酸，测量胚胎平滑肌收缩能力的技术，以及原位肌球蛋白轻链交换。项目1和核心A将测量肌球蛋白产物释放的动力学，并测试基本轻链和肌球蛋白可变表达组合的假设，以及基本轻链和肌球蛋白重链亚型可变表达组合决定平滑肌肉对镁ADP和交叉桥动力学的可变亲和力的假设。项目2和核心A将确定调节平滑肌肌球蛋白序列的蛋白激酶和磷酸酶。结合项目1的功能方法，项目3将使用这些方法来识别调节血管紧张素诱导的平滑肌肥大的激酶、磷酸酶及其转录靶点，并验证血管紧张素II在血管发育过程中控制血管细胞分化和成熟以及介导成年动物SMC收缩质量的适应性变化的假设。项目2将确定敲除血管紧张素原、血管紧张素AT2或AT1受体和MHOX对SMC在发育过程中投资和/或生长分化/成熟的影响。将在转基因和敲除动物(项目1、2和3)中评估可能的肌球蛋白磷酸激活剂telokin的生理作用。

This multi-disciplinary program will elucidate the signal transduction pathways that regulate vascular smooth muscle development, hypertrophy, gene expression and contraction-processes that are subject to abnormalities contributing to high blood pressure, atherosclerosis, vasospasm, post-angioplasty restenosis and shock. The Projects are mutually interdependent, converge on investigation of a common theme of understanding smooth muscle cell contraction and growth, and require the complementary expertise available in molecular biology (Project 3), biochemistry (Project 2), physiology (Project 1) and biophysics and generation of novel probes (Core A). Newly developed tools and methods will be utilized for identifying and characterizing protein kinases and phosphatases, their upstream and downstream targets, and crossbridge- state kinetics. New reagents and methods include gamma-phosphate- activated ATP for affinity purification of protein kinases, mixed peptide sequencing of fmol level proteins, environmentally sensitive fluorescent nucleotide analogous, caged nucleotides, peptides and fatty acids, techniques for measuring contractility of embryonic smooth muscle, and the exchange of myosin light chains in situ. Project 1 and Core A will measure the kinetics of product release from myosin and test the hypothesis that a combination of variable expression of essential light chain and myosin and test the hypothesis that a combination of variable expression of essential light chain and myosin heavy chain isoforms determines the variable affinity of smooth muscles for MgADP and crossbridge kinetics. Project 2 and Core A will identify protein kinases and phosphatases that regulate smooth muscle myosin sequencing. In conjunction with the functional methods of Project 1, Project 3 will use these methods to identify kinases, phosphatases and their transcriptional targets that regulate angiotensin-induced smooth muscle hypertrophy, also testing the hypothesis that angiotensin-II plays an important role in control of smooth muscle cell differentiation and maturation during vascular development and mediates adaptive changes in contractile mass of SMC in adult animals. The effects of knockout of angiotensinogen, the angiotensin AT2 or AT1 receptors and MHox on SMC investment and/or growth differentiation/maturation during development will be determined in Project 2. The physiological role of telokin, a putative smooth muscle myosin phosphate activator, will be evaluated in transgenic and knockout animals (Projects 1, 2 and 3).