REGULATION OF ENERGY TRANSDUCTION IN SMOOTH MUSCLE

本提案的总体目标是阐明机制 负责调节收缩蛋白相互作用， 导致正常功能的平滑肌中的机械输出。的 平滑肌的调节机制尚未完全了解， 已知该过程在病理条件下如何改变， 高血压和哮喘。这些关于正常功能的信息将提供 平滑肌改变病因学未来研究的框架 功能以及治疗策略。具体问题涉及 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 从激活的肌球蛋白横桥输出。第一个具体目标是 确定是否有不同的激活的跨桥池， 不同的循环速率，如果是这样，肌球蛋白轻链 磷酸化影响每个细胞的分布和循环速率， 池肌球蛋白轻链磷酸化是如何介导肌球蛋白轻链磷酸化增加的？ 测定未磷酸化肌球蛋白的循环速率。等距 条件和机械扰动，如缩短和拉伸 将被研究。这将使我们深入了解 过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节系统对此的影响 过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 说明自行车过桥数量的规定及其 钙介导的收缩期间的循环速率。主要实验 方法“将是一个单一的营业额协议，以确定率在 其中与肌球蛋白结合的ADP被3 H-ATP裂解产生的3 H-ADP取代 由“笼状”3 H-ATP的光解形成。第二个目标是量化 肌球蛋白轻链激酶与肌球蛋白轻链的相互作用 磷酸酶与平滑肌中的过桥循环。的作用 这些酶在桥环的中间体上可以改变 完成循环的动力学。将开发一种方法， 允许测量肌球蛋白光中磷酸盐周转率 链这将涉及测量33 P-磷酸盐 从γ 33 P-ATP（来自笼状γ 33 P-ATP的光解）中出现 在轻链中。这些实验将定量评估 激酶和磷酸酶活性的速率与 十字桥周期最终的具体目标将决定如何 平滑肌的ATP酶受机械条件的控制。 具体而言，将在短暂缩短期间进行ATP酶测量 并将其与等长条件进行比较。的 实验将允许确定ATP之间的关系 肌球蛋白的利用和肌肉的功输出，并将提供 证据表明，是否在缩短速度的变化下， 不同的条件是由快速循环的内部负荷引起的 人行横桥的人骑自行车的速度较慢。

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.