PHOSPHORYLATION CALCIUM AND SMOOTH MUSCLE CONTRACTION

尽管近年来取得了重大进展， Smooth细胞内调节的正常机制 肌肉是不完全理解的。的长期目标 这个项目是为了全面了解 负责平滑肌收缩的机制(S) 应用这些知识来了解在 影响血管平滑肌功能的病理生理状态。这 该项目基于两个钙离子依赖的假设 监管系统并行行动，一个是为了快速发展 通过快速循环，磷酸化的十字桥，以及 其他用于缓慢发展和维持压力，通过 缓慢地骑行，没有磷酸化的锁桥。具体目标 这项申请的目的是：1)确定是否增加 细胞内游离钙(Ca2+)激活血管 通过两条独立的途径进行收缩；2)确定 蛋白质系统(S)参与这些钙依赖途径；3) 确定激活剂钙的来源是否影响 支持收缩的具体监管机制；以及 4)确定这两种可能的钙调节机制 在非血管平滑肌中存在数量上的 相似的比率。猪主动脉中的肌动球蛋白和“洗涤剂” “剥皮”和完整的猪颈动脉纤维将是 这次调查的主要模型。实验将是 使用新的MLC激酶的特异性抑制剂或 用三磷酸腺苷类似物CTP。测量将包括Ca-+和 镁离子激活ATPase和肌动球蛋白的超沉淀， 应激、ML磷酸化、跨桥循环速率和 附着在完整的和肮脏的皮肤纤维上。这些蛋白质 参与这第二个钙依赖系统将被确定 通过结合放射性标记化合物(钙、镁、W-7和 钙调蛋白)转化为硝酸纤维素膜上的蛋白质，然后 用柱层析法分离。激活剂的来源 Ca~(2+)将从药理上分离出来，并具有多种作用 STRES、MLC磷酸化和交叉桥上的钙来源 检查自行车的骑行率。这些研究的结果将是 与使用牛气管和大鼠肠道的研究相比较 确定平滑肌细胞内钙离子依赖的调节 其他光滑的肌肉。从这个过程中获得的信息 申请书将提供两人在场的直接证据 平滑肌中依赖钙离子的调节系统，定位 并对参与这一调控的蛋白质(S)进行了表征，以及 认清激活剂钙的来源与关系 平滑肌肉收缩的机制。知识 关于平滑肌收缩的调节是一个 理解变更在其中的作用的必要前提 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.