REGULATION OF ENERGY TRANSDUCTION IN SMOOTH MUSCLE

平滑肌能量传导的调节

• 批准号: 2392719
• 负责人: THOMAS M BUTLER
• 金额: $ 35.71万
• 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/2392719
• 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 在正常功能的平滑肌中产生机械输出。这 平滑肌的调节机制尚不完全清楚，也不清楚 它知道这个过程在病理条件下是如何改变的，例如 高血压和哮喘。有关正常功能的此类信息将提供 平滑肌改变病因学的未来研究框架 功能以及治疗策略。具体问题处理 肌球蛋白轻链的磷酸化如何调节 被激活的肌球蛋白分子以及循环速率和力量 来自激活的肌球蛋白横桥的输出。第一个具体目标是 确定是否存在不同的已激活交叉桥池 不同的循环速率，如果是这样，肌球蛋白轻链如何 磷酸化影响每个的分布和循环速率 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。这将有助于深入了解动力学如何 过桥骑行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。其他实验将专门测试是否达到以下程度： 非磷酸化肌球蛋白的协同激活可以通过以下方式调节 受体激活，以及已知的激活机制是否可以 考虑对自行车过桥数量及其影响的规定 钙介导的收缩期间的循环速率。主要实验 方法“将是一个单一的周转协议来确定利率 与肌球蛋白结合的 ADP 被 3H-ATP 裂解后的 3H-ADP 取代 由“笼中的”3H-ATP 光解形成。第二个目标是量化 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横桥循环。的行动 跨桥循环中间体上的这些酶可能会改变 完成循环的动力学。将开发一种方法 允许测量肌球蛋白光下磷酸盐周转率 链。这将涉及测量 33P-磷酸盐的速率 来自 gamma33P-ATP（源自笼中 gamma33P-ATP 的光解）出现 在轻链中。实验将给出定量评估 激酶和磷酸酶活性的速率与动力学相比 跨桥循环。最终的具体目标将决定如何 平滑肌的ATP酶受机械条件控制。 具体来说，ATPase 测量将在短暂的缩短期间进行 和拉伸并将与等长条件进行比较。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 证据表明缩短速度的变化是否在 快速循环的内部负载导致不同的条件 那些骑自行车速度较慢的人过桥。