NITRIC OXIDE REGULATION OF SMOOTH MUSCLE TONE

描述：(改编自申请人的摘要)一氧化氮(NO) 以及产生NO的化合物(例如硝化甘油)起作用 围手术期越来越重要的治疗作用 (例如，在治疗急性肺性和系统性高血压方面)。 NO对血管平滑肌的作用机制尚不完全清楚 明白了。最近的证据表明，NO不能松弛平滑肌 只有通过降低细胞内钙离子浓度([Ca~(2+)]i)， 重点放在大多数以前的工作上，还通过减少力量的量 对给定的[Ca~(2+)]i产生(即“肌丝Ca~(2+)敏感性”)。 我们的初步数据表明有两类机制 对于这些对肌丝钙敏感性的影响：1)机制 NO诱导的鸟苷环3‘，5’-单磷酸增加 (CGMP)和2)独立于cGMP的机制。这样做的目的是 建议探索专门与cGMP有关的新机制- NO依赖(AIM A)和非依赖cGMP(AIM B)对血管内皮细胞的影响 肌丝对钙离子的敏感性。对钙离子的抑制作用- 钙调素对收缩蛋白的激活及其抑制作用 膜受体连接的第二信使系统调节 CGMP依赖和非cGMP依赖的肌丝钙敏感性 我们将研究NO的影响。这些研究将由 使用一种独特的通透性平滑肌制剂，其中 细胞内NO和cGMP的浓度可以单独控制 在[Ca~(2+)]恒定的条件下，I.在该制剂中 调节肌丝钙敏感性的信使系统仍然存在 完好无损。初步研究支持cGMP-cGMP- NO对肌丝钙敏感性的依赖作用是介导的 完全通过抑制正常情况下的第二信使系统 调节肌丝对钙的敏感性，而cGMP- NO的独立作用是通过抑制这两类物质来实现的 细胞过程。肌球蛋白轻链的生化测定 磷酸化，肌球蛋白轻链酶，肌球蛋白ATPase，和 蛋白激酶C的活性将确定具体的机制 参与了这些行动。对平滑肌力学的测量将 深入了解肌动蛋白-肌球蛋白交叉桥动力学的影响。这些 实验将揭示产生力量的肌动蛋白比例是否- 肌球蛋白交叉桥或每个交叉桥的平均作用力减少。一个 对产生这些效应的复杂机制的理解可能 提供对其他组织中NO作用机制的洞察 在平滑肌中发现的细胞内信号传导系统 在其他类型的细胞中是常见的。此外，对这些问题的解释 机制可以为未来的发展提供战略建议 治疗用的平滑肌松弛药。

DESCRIPTION: (Adapted from the applicant's abstract) Nitric oxide (NO) and compounds that generate NO (e.g., nitroglycerin) play an increasingly important therapeutic role in the perioperative period (e.g., in the treatment of acute pulmonary and systemic hypertension). The mechanisms of action of NO on smooth muscle are not fully understood. Recent evidence suggests that NO relaxes smooth muscle not only by reducing cytosolic calcium (Ca2+) concentration ([Ca2+]i), the focus of most previous work, but also by reducing the amount of force produced for a given [Ca2+]i (i.e., "myofilament Ca2+ sensitivity"). Our preliminary data suggest two categories of mechanisms responsible for these effects on myofilament Ca2+ sensitivity: 1) mechanisms mediated by NO-induced increases in guanosine cyclic 3',5'-monophosphate (cGMP), and 2) mechanisms that are independent of cGMP. The goal of this proposal is to explore novel mechanisms specifically related to cGMP- dependent (AIM A) and cGMP-independent (AIM B) effects of NO on myofilament Ca2+ sensitivity in smooth muscle. Both inhibition of Ca2+- calmodulin activation of the contractile proteins and inhibition of membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity by cGMP-dependent and cGMP-independent effects of NO will be studied. These studies will be facilitated by the use of a unique permeabilized smooth muscle preparation in which intracellular concentrations of NO and cGMP can be separately manipulated under conditions of constant [Ca2+]i. In this preparation, second messenger systems that regulate myofilament Ca2+ sensitivity remain intact. Preliminary studies support the overall hypothesis that cGMP- dependent effects of NO on myofilament Ca2+ sensitivity are mediated exclusively by inhibition of the second messenger systems that normally function to regulate myofilament Ca2+ sensitivity, whereas cGMP- independent effects of NO are mediated by inhibition of both categories of cellular processes. Biochemical measurements of myosin light chain phosphorylation, and myosin light chain inase, myosin ATPase, and protein kinase C activities will identify the specific mechanisms involved in these actions. Measurements of smooth muscle mechanics will provide insight into effects on actin-myosin crossbridge kinetics. These experiments will reveal whether the fraction of force-generating actin- myosin crossbridges or the mean force per crossbridge is reduced. An understanding of the complex mechanisms producing these effects may provide insights into mechanisms of NO actions in other tissues, as many of the intracellular signal transduction systems found in smooth muscle are common to other cell types. Additionally, elucidation of these mechanisms may suggest strategies for the future development of therapeutic smooth muscle relaxants.