PHOSPHORYLATION CALCIUM AND SMOOTH MUSCLE CONTRACTION

20000道尔顿肌球蛋白轻链的磷酸化 钙-钙调蛋白-MLC激酶复合体是最被广泛接受的机制 假设是为了启动平滑肌的收缩。然而，它一直是 表明在没有高水平的MLC的情况下，压力可以保持 磷酸化。这种压力维持状态被称为“闩锁”。 并以低水平的MLC磷酸化和低水平的 缩短了速度。这表明压力是由 脱磷，慢行或不骑行过桥。尽管 闩锁的概念目前正在被接受，有几个主要问题 对于这种状态的形成机制仍未得到回答。这 应用程序将解决这些问题。具体来说：1)是MLC 磷酸化是形成闩锁状态的先决条件，如下所述 Chatterjee和Murphy(Science 211：464,1983)或者闩锁可以由 直接机制？；以及2)如果MLC被磷酸化，则去磷酸化 是形成闩锁的必要条件吗？高渗透性洗涤剂 猪颈动脉的皮肤纤维将被用来回答这些问题 问题。蒙皮纤维会暴露在镁离子中，建议为MLC 磷酸化不依赖于收缩，或在存在钙离子的情况下 钙调素抑制剂。MLC磷酸化、等渗性的测定 缩短速度(表示平均跨桥骑行率)，以及 硬度和最大拉伸阻力(表示 在这些情况下)将决定是否缓慢地 可以在没有MLC的情况下形成循环或非循环连接的闩锁桥 磷酸化。MLC还将被不可逆地硫代磷酸化(MLC 磷酸酶抗性)和[Ca~(2+)]的变化。的参数 将测量磷酸化、速度和硬度，以确定是否 在恒定水平的MLC磷酸化过程中，可以形成闩锁桥。 这些研究将增加关于钙离子调节的重要信息。 血管平滑肌。这一初步建议将开始一个长期的 关于收缩机械的基本生理学的计划 血管平滑肌与该组织中发生的改变 高血压。如果可以在没有先前MLC情况下形成锁存状态 磷酸化导致应力和/或硬度的增加，然后 钙离子“泄漏”到肌细胞的增加在 高血压可能会激活这种状态。这将导致增加 血管阻力和动脉压升高。这位利亚森会 有助于了解高血压发生的变化。

Phosphorylation of the 20000 dalton myosin light chain (MLC) by a Ca-calmodulin-MLC kinase complex is the most widely accepted mechanism hypothesized to initiate contraction in smooth muscle. However it has been shown that stress can be maintained without high levels of MLC phosphorylation. This state of stress maintenace has been termed "latch" and is characterized by low levels of MLC phosphorylation and low shortening velocities. This suggests stress is maintained by dephosphorylated, slowly or non-cycling cross bridges. Although the concept of latch is currently gaining acceptance, several major questions remain unanswered about the mechanism of formation of this state. This application will address these questions. Specifically: 1) is MLC phosphorylation a prerequisite for latch state formation as suggested by Chatterjee and Murphy (Science 211:464,1983) or can latch be formed by a direct mechanism?; and 2) if the MLC phosphorylated, is dephosphorylation necessary for the formation of latch? The hyperpermeable, detergent skinned fiber of the swine carotid artery will be used to answer these questions. Skinned fibers will be exposed to Mg2+, suggested to be a MLC phosphorylation independent contraction, or to Ca2+ in the presence of calmodulin inhibiters. Measurement of MLC phosphorylation, isotonic shortening velocity (indicative of average cross bridge cycling rate), and stiffness and peak resistance to stretch (indicative of average number of attached cross bridges) during these conditions will determine if slowly cycling or non-cycling attached latch bridges can be formed without MLC phosphorylation. The MLC will also be irreversibly thiophosphorylated (MLC phosphatase resistant) and the [Ca2+] varied. The parameters of phosphorylation, velocity, and stiffness will be measured to determine if latch bridges can be formed during constant level of MLC phosphorylation. These studies will add significant information about the Ca2+ regulation of vascular smooth muscle. This initial proposal will begin a long term program to relate the basic physiology of the contractile machinery in vascular smooth muscle to alterations that occur in this tissue in hypertension. If a latch state can be formed without prior MLC phosphorylation resulting in an increase in stress and/or stiffness, then the increase Ca2+ "leak" into the muscle cell demonstrated during hypertension may activate this state. This would result in an increase in vascular resistance and an increase in arterial pressure. This liason will aid in the understanding of the changes that occur in hypertension.