PHOSPHORYLATION CALCIUM AND SMOOTH MUSCLE CONTRACTION

尽管近年来取得了重大进展， 平滑肌细胞内调节的正常机制 肌肉是不完全理解的。 长期目标 该项目旨在全面了解 负责平滑肌收缩的机制 应用这些知识来了解发生在 影响平滑肌功能的病理生理状态。 这 该项目是基于假设，两个钙依赖 监管体系并行不悖，一为快速发展 压力的快速循环，磷酸化的横桥， 另一种是压力的缓慢发展和维持， 缓慢循环的非磷酸化锁桥。 具体目标 本申请的目的是：1）确定是否增加 细胞内游离钙（Ca 2+）激活平滑肌 收缩通过两个独立的途径; 2）确定 参与这些Ca 2+依赖性途径的蛋白质系统; 3） 确定激活剂Ca 2+的来源是否影响 支持收缩的具体监管机制;以及 4)确定这两种假定的Ca 2+调节机制 存在于非血管平滑肌中， 相似的比例。 来自猪主动脉的肌动球蛋白和“去污剂 “去皮”和完整的猪颈动脉纤维将是 本次调查的主要模型。 实验将 用MLC激酶的新型特异性抑制剂进行，或 ATP类似物CTP 测量将包括Ca-+和 Mg ~（2+）激活ATP酶和肌动球蛋白的超沉淀， 和应激、ML磷酸化、交联桥循环速率和 附着在完整且具有破坏力的皮肤纤维中。 的蛋白质 参与这第二个Ca 2+依赖系统将被确定 通过结合放射性标记的化合物（Ca、Mg、W-7和 钙调素）转化为蛋白质，然后 通过柱色谱法分离。 活化剂的来源 Ca 2+将被隔离，各种 压力、MLC磷酸化和跨桥上的Ca 2+源 检查循环率。 这些研究的结果将是 与使用牛气管和大鼠肠的研究相比， 平滑肌，以确定Ca 2+依赖的调节， 其他平滑肌 从这获得的信息 申请将提供直接证据证明存在两个 平滑肌中的Ca 2+依赖性调节系统，定位 并表征参与该调节的蛋白质，以及 识别激活剂Ca 2+来源与 平滑肌收缩的机制。 知识 关于平滑肌收缩的调节， 理解改变的作用的必要前提 与病理生理疾病状态相关的平滑肌。

In spite of the major advances that have occurred in recent years, the normal mechanisms in the intracellular regulation of smooth muscle is incompletely understood The. long-term objectives of this project are to obtain a complete understanding of the mechanism(s) responsible for contraction of smooth muscle and to apply this knowledge to understand the changes that occur in pathophysiological states affecting smooth muscle function. This project is based on the hypothesis that two Ca2+ dependent regulatory systems act in parallel, one for the rapid development of stress by rapidly cycling, phosphorylated crossbridges, and the other for the slow development and maintenance of stress by slowly cycling, unphosphorylated latchbridges. The specific aims of this application are to: 1) determine if an increase in intracellular free calcium (Ca2+) activates smooth muscle contraction by two independent pathways; 2) determine the protein system(s) involved in these Ca2+ dependent pathways; 3) determine if the source of the activator Ca2+ influences the specific regulatory mechanism that will support contraction; and 4) determine if these two putative Ca2+ regulatory mechanisms are present in non-vascular smooth muscles in a quantitatively similar ratio. Actomyosin from swine aorta, and "detergent skinned" and intact fibers of swine carotid artery will be the primary models for this investigation. Experiments will be performed with novel specific inhibitors of the MLC kinase or with the ATP analog, CTP. Measurements will include Ca-+ and Mg2+ activation of ATPase and superprecipitation of actomyosin, and stress, ML phosphorylation, crossbridge cycling rate and attachment in intact and detegent skinned fibers. The proteins involved in this second Ca2+ dependent system will be identified by binding of radiolabelled compounds (Ca, Mg, W-7, and calmodulin) to proteins on nitrocellulose membranes and then separated by column chromatography. The sources of activator Ca2+ will be isolated pharmacologically and the effects of various Ca2+ sources on stres, MLC phosphorylation and crossbridge cycling rate examined. The results of these studies will be compared to studies using bovine trachealis and rat intestinal smooth muscle to determine the Ca2+ dependent regulation of other smooth muscles. The information gained from this application will provide direct evidence for the presence of two Ca2+ dependent regulatory systems in smooth muscle, localize and characterize the protein(s) involved in this regulation, and discern the relationship between the source of activator Ca2+ and the mechanism by which smooth muscle contracts. Knowledge concerning the regulation of smooth muscle contraction is a necessary prerequisite for understanding the role of alterations in smooth muscle associated with pathophysiological disease states.