REGULATION OF VASCULAR SMOOTH MUSCLE CONTRACTION

在平滑肌中，肌球蛋白轻链的重要性 已经充分建立了用于力调节的磷酸化。 然而，在这方面， 收缩可以在肌球蛋白水平没有变化的情况下发生 轻链磷酸化，并且在力作用期间达到峰值后， 激活时，肌球蛋白轻链磷酸化水平福尔斯下降， 部队维修。 因此，大量证据表明， 不依赖于肌球蛋白轻链的力调节系统 磷酸化，可能存在。 该补助金的目标是确定 平滑肌力调节的分子机制。 本申请 将测试两个独立的假设。 第一个假设是， 在松弛的平滑肌中存在大量附着的横桥， 这些相连的横桥参与了收缩， 肌球蛋白轻链磷酸化水平无变化。 这一假设的具体目标是：1。 来确定相对 肌动球蛋白ADP跨桥的群体存在于僵硬和放松状态 细胞; 2. 测定肌动球蛋白ADP的力学特性 天桥;及3. 以确定肌动蛋白ADP跨桥是否参与 在肌球蛋白光水平没有变化的收缩中， 链磷酸化 第二个假设是， 由于非交叉桥梁受力结构的发展。 这一假设的具体目标是：1。 来确定十字桥 力激活期间存在的状态;以及2. 部队维修。 测试 这些特定的目标，力量和刚度将在一个完整的 以及用钙或钙离子激活的透化平滑肌细胞， 激动剂 在透化细胞中，僵硬溶液、含有 腺苷三磷酸双磷酸酶，以及具有低ATP浓度，高ADP浓度， 而升高的磷酸盐浓度将被用来增加 肌动球蛋白、肌动球蛋白ADP和肌动球蛋白ADPPi的数量 跨桥国家 分析了刚度与频率的关系， 每个跨桥状态的振荡将使用伪随机 白色噪声和/或肌肉长度的正弦振荡。 然后它将 可以确定跨桥状态，其是：1. 中存在 松弛的平滑肌; 2. 负责宫缩的发生， 肌球蛋白轻链磷酸化水平的变化;和3. 本 在部队启动和维持期间。 在部队维持期间， 数据将区分是否一个群体的附着去磷酸化 （闩锁）横桥发展，整个横桥周期率放缓， 合作机制导致人口发展缓慢， 循环去磷酸化横桥，或非横桥受力 结构形成。 本补助金申请中提出的研究将 为检验肌动球蛋白ADP跨桥的作用提供了直接证据 力调节和非跨桥结构力维持。 这些数据将有助于阐明力激活和力的机制。 上维护

DESCRIPTION: In smooth muscle, the importance of myosin light chain phosphorylation for force regulation is well established. However, contractions can occur in the absence of a change in the level of myosin light chain phosphorylation and, after reaching a peak during force activation, the level of myosin light chain phosphorylation falls during force maintenance. Thus, a large body of evidence suggests that a regulatory system for force, independent of myosin light chain phosphorylation, may exist. The goal of this grant is to determine the molecular mechanism of force regulation in smooth muscle. This application will test two independent hypotheses. The first hypothesis is that a population of attached crossbridges are present in relaxed smooth muscle, and these attached crossbridges participate in contractions which occur in the absence of a change in the level of myosin light chain phosphorylation. Specific aims for this hypothesis are: 1. to determine the relative population of actomyosinADP cross-bridges present in both rigor and relaxed cells; 2. to determine the mechanical characteristics of actomyosinADP crossbridges; and 3. to determine if actomyosinADP crossbridges participate in contractions which occur without a change in the level of myosin light chain phosphorylation. The second hypothesis is that force maintenance is due to the development of non-cross-bridge force bearing structures. Specific aims for this hypothesis are: 1. to determine the cross-bridge states present during force activation; and 2. force maintenance. To test these specific aims, force and stiffness will be determined in single intact and permeabilized smooth muscle cells activated with either calcium or an agonist. In permeabilized cells, rigor solution, rigor solution containing apyrase, and solutions with low ATP concentration, high ADP concentration, and elevated phosphate concentration will be used to increase the relative population of the actomyosin, actomyosinADP, and actomyosinADPPi cross-bridge states. The relationship between stiffness and frequency of oscillation of each cross-bridge state will be determined using pseudorandom white noise and/or sinusoidal oscillation of muscle length. It will then be possible to determine the cross-bridge states which are: 1. present in relaxed smooth muscle; 2. responsible for contractions which occur without a change in the level of myosin light chain phosphorylation; and 3. present during both force activation and maintenance. During force maintenance, the data will differentiate whether a population of attached dephosphorylated (latch) crossbridges develop, the entire cross-bridge cycling rate slows, a cooperative mechanism leads to the development of a population of slowly cycling dephosphorylated crossbridges, or non-cross-bridge force bearing structures are formed. The studies proposed in this grant application will provide direct evidence to test the roles of both actomyosinADP crossbridges in force regulation, and non-cross-bridge structures in force maintenance. The data will aid in elucidating the mechanism of force activation and force maintenance.