REGULATION OF ENERGY TRANSDUCTION IN SMOOTH MUSCLE

平滑肌能量传导的调节

• 批准号: 2226831
• 负责人: THOMAS M BUTLER
• 金额: $ 33万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-06 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226831
• 关键词: adenosine diphosphate; adenosine triphosphate; chemical kinetics; computer simulation; enzyme activity; flash photolysis; high performance liquid chromatography; intermolecular interaction; laboratory rabbit; muscle contraction; muscle metabolism; myosin light chain kinase; myosins; phosphoprotein phosphatase; phosphoproteins; phosphorylation; portal vein; vascular smooth muscle

The overall goal of this proposal is to elucidate the mechanism(s) responsible for regulation of contractile protein interaction and the resulting mechanical output in normally functioning smooth muscle. The mechanism of regulation of smooth muscle is not fully understood, nor is it known how this process is modified in pathological conditions such as hypertension and asthma. Such information on normal function will provide the framework for future studies on the etiology of altered smooth muscle function as well as therapeutic strategies. The specific questions deal with how phosphorylation of the myosin light chain regulates the number of myosin molecules which are activated as well as the cycling rate and force output from an activated myosin crossbridge. The first specific aim is to determine whether there are different pools of activated crossbridges with different cycling rates, and, if so, how myosin light chain phosphorylation affects the distribution and the cycling rate of each pool. How myosin light chain phosphorylation mediates an increase in the cycling rate of unphosphorylated myosin will be determined. Isometric conditions and mechanical perturbations such as shortening and stretching will be studied. This will give insight into how the kinetics of crossbridge cycling are controlled by the mechanical state of the muscle, as well as the effects of smooth muscle's unique regulatory system on this process. Other experiments will specifically test whether the degree of Cooperative activation of unphosphorylated myosin can be modulated by receptor activation, and whether the known activation mechanisms can account for the regulation of number of crossbridges cycling and their cycling rate during calcium-mediated contractions. The main experimental approach 'will be a single turnover protocol to determine the rate at which ADP bound to myosin is replaced with 3H-ADP from splitting of 3H-ATP formed by photolysis of "caged" 3H-ATP. The second aim is to quantitate the interaction of myosin light chain kinase and myosin light chain phosphatase with the crossbridge cycle in smooth muscle. The action of these enzymes on intermediates in the crossbridge cycle may alter the kinetics of the completion of the cycle. A method will be developed to permit measurement of the rate of phosphate turnover in the myosin light chain. This will involve measurement of the rate at which 33P-phosphate from gamma33P-ATP (derived from photolysis of caged gamma33P-ATP) appears in the light chain. The experiments will give a quantitative assessment of the rates of kinase and phosphatase activities compared to the kinetics of the crossbridge cycle. The final specific aim will determine how the ATPase of smooth muscle is controlled by mechanical conditions. Specifically, ATPase measurements will be made during brief shortenings and stretches and will be compared to isometric conditions. The experiments will allow determination of the relationship between ATP utilization by myosin and work output from the muscle, and will provide evidence as to whether the variation in velocity of shortening under different conditions results from an internal loading of fast cycling crossbridges by those with slower cycling rates.

该提案的总体目标是阐明机制 负责调节收缩蛋白相互作用和 在正常运作的平滑肌中导致机械输出。这 平滑肌调节机制尚不完全了解，也不是 它知道如何在病理条件（例如 高血压和哮喘。有关正常功能的此类信息将提供 未来研究平滑肌病因的框架 功能以及治疗策略。具体问题交易 肌球蛋白轻链的磷酸化如何调节数量 激活的肌球蛋白分子以及循环速率和力 从激活的肌球蛋白Crossbridge输出。第一个具体目的是 确定是否有不同的激活的跨桥 不同的循环速率，如果是的，则是肌球蛋白轻链 磷酸化会影响每个磷酸化的分布和循环速率 水池。肌球蛋白轻链磷酸化如何介导 将确定未磷酸化肌球蛋白的循环速率。等距 条件和机械扰动，例如缩短和拉伸 将被研究。这将深入了解如何 Crossbridge骑自行车由肌肉的机械状态控制， 以及平滑肌独特的调节系统对此的影响 过程。其他实验将特别测试是否的程度 未磷酸化肌球蛋白的合作激活可以通过 受体激活以及已知的激活机制是否可以 解释Crossbridges骑自行车及其数量的调节 钙介导的收缩期间的循环速率。主要实验 方法'将是一个单一的失误协议，以确定速率 与肌球蛋白结合的ADP被3H-ATP拆分替换为3H-ADP 由“笼” 3H-ATP的光解形成。第二个目的是定量 肌球蛋白轻链激酶和肌球蛋白轻链的相互作用 磷酸酶与平滑肌中的Crossbridge循环。动作 在Crossbridge循环中中间体上的这些酶可能会改变 周期完成的动力学。将开发一种方法 允许测量肌球蛋白光中磷酸盐离职率 链。这将涉及测量33p-磷酸盐的速率 来自Gamma33p-ATP（源自笼中的光解gamma33p-atp） 在轻链中。实验将对 与动力学相比，激酶和磷酸酶活性的发生率 Crossbridge循环。最终的具体目的将决定 平滑肌的ATPase受到机械条件的控制。 具体而言，将在短暂缩短期间进行ATPase测量 并伸展并将与等距条件进行比较。这 实验将允许确定ATP之间的关系 肌球蛋白的利用和肌肉的工作输出，并将提供 关于缩短速度的变化是否在 不同条件是由于快速循环的内部加载而导致的 骑自行车速率较慢的人的跨桥。