REGULATION OF VASCULAR SMOOTH MUSCLE CONTRACTION

描述：在平滑肌中，肌球蛋白轻链的重要性 用于力量调节的磷酸化是很好的确定的。然而， 在肌球蛋白水平没有变化的情况下，可能会发生收缩 轻链磷酸化，在作用力中达到峰值后 激活时，肌球蛋白轻链磷酸化水平在 部队维护。因此，大量证据表明， 独立于肌球蛋白轻链的力调节系统 可能存在磷酸化。这笔赠款的目标是确定 平滑肌力调节的分子机制。此应用程序 将检验两个独立的假设。第一个假设是一个 连接的交叉桥存在于松弛的平滑肌中， 这些连接的交叉桥参与收缩发生在 肌球蛋白轻链磷酸化水平无变化。 这一假说的具体目的是：1.确定 肌动蛋白ADP跨桥的群体呈现僵硬和松弛两种状态 细胞；2.测定肌动蛋白ADP的力学特性。 交叉桥；以及3.确定肌动蛋白ADP交叉桥是否参与 在肌球蛋白光水平没有变化的情况下发生的宫缩 链磷酸化。第二个假设是，武力维持是 由于无跨桥承重结构的发展。 这一假设的具体目的是：1.确定跨桥 在部队激活期间存在的状态；以及2.部队维持。为了测试 这些具体的目标、力和刚度将完整地确定。 钙或血管紧张素转换酶激活的通透性平滑肌细胞 激动剂。在渗透细胞中，冷冻液，冷冻液含有 Apyrase，以及低ATP浓度，高ADP浓度的溶液， 而升高的磷酸盐浓度将用来增加相对 肌动蛋白、肌动蛋白ADP和肌动蛋白ADPPI的群体 跨桥状态。结构的刚度与频率的关系 每个跨桥状态的振荡将使用伪随机来确定 肌肉长度的白噪声和/或正弦振荡。那么它将会是 可以确定以下跨桥状态：1.存在于 松弛的平滑肌；2.负责无收缩的收缩 肌球蛋白轻链磷酸化水平的变化；以及3.存在 在部队激活和维护期间。在部队维护期间， 数据将区分一个附着的种群是否去磷酸化 (闩锁)跨桥发展，整个跨桥循环速度减慢，a 合作机制导致人口发展缓慢 自行车脱磷交叉桥，或非交叉桥承重 结构就形成了。在这项拨款申请中建议的研究将 为测试两种肌动蛋白ADP交叉桥的作用提供直接证据 在受力调整方面，以及在非跨桥结构受力维护方面。 这些数据将有助于阐明力的激活和力的机制 维修。

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.