REGULATION OF ENERGY TRANSDUCTION IN SMOOTH MUSCLE

这项建议的总体目标是阐明机制(S) 负责调节收缩蛋白的相互作用和 导致机械输出产生正常功能的平滑肌肉。这个 对平滑肌的调节机制还不完全清楚，也不是很清楚 它知道这个过程是如何在病理条件下被修改的，比如 高血压和哮喘。这些关于正常功能的信息将提供 未来对血管平滑肌病变病因研究的框架 功能和治疗策略。具体的问题涉及到 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度和作用力 来自激活的肌球蛋白交叉桥的输出。第一个具体目标是 确定是否存在不同的激活交叉桥池 不同的循环速度，如果是这样，肌球蛋白轻链是如何 磷酸化影响每一个的分布和循环速率 游泳池。肌球蛋白轻链磷酸化如何介导细胞内 将测定未磷酸化肌球蛋白的循环速率。等轴测 条件和机械扰动，如缩短和拉伸 将会被研究。这将使我们洞察到， 横桥自行车是由肌肉的机械状态控制的， 以及平滑肌独特的调控系统对此的影响 进程。其他实验将专门测试是否程度 非磷酸化肌球蛋白的协同激活可以通过 受体激活，以及已知的激活机制是否可以 说明了人行横桥骑行数量的调节及其影响因素 钙介导的收缩过程中的循环率。主要的实验 方法将是一个单一的周转协议，以确定速度 与肌球蛋白结合的哪一种ADP被~3H-ATP裂解后的~H-ADP所取代 由笼状的~3H-三磷酸腺苷光解而成。第二个目标是量化 肌球蛋白轻链与肌球蛋白轻链的相互作用 磷酸酶与平滑肌中的交叉桥循环。…的行为 交叉桥循环中中间产物上的这些酶可能会改变 循环完成的动力学。将开发一种方法来 允许测量肌球蛋白光中的磷酸盐周转率 链条。这将包括测量33P-磷酸盐的速率 来自伽马33P-ATP(来源于笼养的伽马33P-ATP的光解)出现 在轻链上。实验将给出一个定量的评估 酶和磷酸酶活性的速率与动力学的比较 跨桥自行车赛。最终的具体目标将决定如何 肌细胞的ATPase受机械条件的控制。 具体来说，ATPase的测量将在简短的缩写期间进行 和拉伸，并将其与等轴测条件进行比较。这个 实验将允许确定三磷酸腺苷之间的关系 肌球蛋白的利用和肌肉的功输出，并将提供 关于缩短速度的变化是否存在于 不同的情况是由快速循环的内部负荷造成的 骑行速度较慢的人过桥。

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.