CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

Catch--平滑肌的机械化学和调节

基本信息

批准号：
2607923
负责人：
MARION Joyce SIEGMAN
金额：
$ 27.14万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-12-01 至 1999-07-31
项目状态：
已结题

项目摘要

In disease states, such as hypertension, the primary defect may be traced to the regulation of the mechanisms for maintenance of tone in smooth muscle. The basic mechanism(s) controlling tone are not well understood. A unique and important characteristic of all smooth muscles is the ability to vary the energy cost of force production and maintenance during the time course of isometric contractions. It has become obvious that there are many similarities among the mechanical and energetic properties of mammalian smooth muscles during the "latch" state and molluscan muscles during "catch," yet the molecular basis of these states and their regulation is not well understood. A unique feature of catch muscle is the operation of a mechanism that appears to control the detachment rate of the crossbridge. Such a mechanism has not yet been identified in mammalian muscle, but would be of great importance as a means of regulating tone and consequently physiological functions, such as blood pressure. The elucidation of such a mechanism in catch thus has the potential to provide important insights into normal and abnormal function of the cardiovascular, pulmonary, digestive, and reproductive systems. The overall goal of this proposal is to define the state of the myosin crossbridge and its kinetics of turnover at rest and during phasic and catch contractions in the anterior byssus retractor muscle (ABRM) of Mytilus edulis, and to identify proteins whose change in phosphorylation state is the molecular basis for the regulation of the transition of the crossbridge into and out of the catch state. The Specific Aims are to determine: (1) in permeabilized ABRM, the nucleotide and/or inorganic phosphate bound to myosin under different mechanical conditions, including the resting state (crossbridges detached or weakly bound to actin), phasic contraction (crossbridges cycling and maintaining force) and the catch state (crossbridges likely to be attached and maintaining force, but cycling very slowly); (2) the rate of nucleotide and/or phosphate turnover on myosin under different mechanical conditions using single turnover techniques in which radiolabeled nucleotides are generated by photolysis of caged compounds, and thereby gain quantitative information concerning the rate of the myosin ATPase cycle under these very different mechanical conditions. We will also determine whether the rapid relaxation from catch initiated by cAMP is associated with a rapid exchange of the products of ATP hydrolysis bound to myosin, in order to directly address the question of whether myosin changes its state in the transition from catch to rest. (3) during the release of catch, which proteins show changes in levels of phosphorylation on a time course that corresponds to the change in state of myosin and/or relaxation following cAMP formation from photolysis of caged cAMP. These experiments will identify proteins which are candidates for the molecular regulator(s) of catch.

在诸如高血压之类的疾病状态下，主要缺陷可以追溯到调节机制以平滑维持音调肌肉。基本机制控制音调尚不清楚。所有平滑肌的独特而重要的特征是能够改变力产生和维护的能源成本在等距收缩期间。它变得明显机械和能量之间有许多相似之处在“闩锁”状态和 “捕获”期间的软体动物肌肉，但这些状态的分子基础而且他们的法规尚未得到很好的理解。捕获的独特功能肌肉是似乎控制着机制的操作十字架的脱离率。这样的机制尚未在哺乳动物肌肉中鉴定出来，但很重要调节语气和因此生理功能的手段，作为血压。因此，阐明这种机制在捕获中提供对正常和异常的重要见解的潜力心血管，肺，消化和生殖的功能系统。该提议的总体目标是定义肌球蛋白Crossbridge及其在休息和阶段期间的动力学并捕获缩回牵引力肌肉（ABRM）的收缩 Mytilus Edulis，并鉴定其磷酸化变化的蛋白质状态是调节过渡的分子基础 Crossbridge进入捕获状态。具体目的是确定：（1）在透化的ABR中，核苷酸和/或无机在不同的机械条件下与肌球蛋白结合的磷酸盐，包括静止状态（Crossbridges分离或弱约束肌动蛋白），阶段收缩（Crossbridges循环和维持力）和捕获状态（越野桥可能附着并维护力量，但骑自行车非常缓慢）；（2）核苷酸和/或的速率在不同的机械条件下，使用放射标记核苷酸的单个周转技术是通过对笼化合物的光解产生，从而获得定量有关这些肌球蛋白ATPase周期速率的信息机械条件非常不同。我们还将确定是否营地发起的捕捞量快速放松与快速相关交换与肌球蛋白结合的ATP水解产品，以便直接解决了肌球蛋白是否改变状态的问题从捕获到休息的过渡。（3）在发行捕获期间，蛋白质在时间过程中显示出磷酸化水平的变化对应于肌球蛋白状态和/或放松的变化。从笼中的光解进行营地形成。这些实验会鉴定蛋白质是分子调节剂的候选蛋白抓住。