NITRIC OXIDE REGULATION OF SMOOTH MUSCLE TONE

描述：（改编自申请人摘要）一氧化氮（NO） 以及产生NO的化合物（例如，硝酸甘油）发挥作用， 在围手术期的治疗作用日益重要 (e.g.,治疗急性肺动脉和全身性高血压）。 NO对平滑肌的作用机制尚不完全清楚 明白 最近的证据表明，NO松弛平滑肌， 仅通过降低胞浆钙浓度（[Ca 2 +]i）， 以往工作的重点，而且还减少了力量的数额 对于给定的[Ca 2 +]i（即，“肌丝Ca 2+敏感性”）。 我们的初步数据表明，两类机制负责 对于这些对肌丝Ca 2+敏感性的影响：1）机制 由NO诱导的鸟苷环3 '，5'-单磷酸增加介导 （cGMP），和2）独立于cGMP的机制。 这个目标 该计划旨在探索与cGMP相关的新机制- 依赖性（AIM A）和cGMP非依赖性（AIM B）NO对 平滑肌肌丝Ca ~（2+）敏感性。 抑制Ca 2 +- 钙调素激活收缩蛋白和抑制 膜受体连接的第二信使系统， cGMP依赖性和cGMP非依赖性肌丝Ca 2+敏感性 将研究NO的影响。 这些研究将由 一种独特的透化平滑肌制剂的用途， NO和cGMP的细胞内浓度可以分别控制 在[Ca ~（2+）]i恒定的条件下， 在这篇文章中，第二 调节肌丝Ca 2+敏感性的信使系统仍然存在， 完整 初步研究支持cGMP- NO对肌丝Ca ~（2+）敏感性的依赖性作用是介导的 仅仅是通过抑制第二信使系统， 功能调节肌丝Ca 2+敏感性，而cGMP- NO的独立作用是通过抑制这两种类型来介导的 细胞过程。 肌球蛋白轻链的生化测量 磷酸化，和肌球蛋白轻链酶，肌球蛋白ATP酶，和 蛋白激酶C活性将确定具体机制 参与这些行动。 平滑肌力学的测量将 提供对肌动蛋白-肌球蛋白交联动力学影响的深入了解。 这些 实验将揭示产生力的肌动蛋白的部分， 肌球蛋白横桥或每个横桥的平均力降低。 一个 了解产生这些效应的复杂机制， 提供深入了解NO在其他组织中的作用机制，因为许多 平滑肌细胞内的信号传导系统 与其他细胞类型相同。 此外，阐明这些 这些机制可能为未来的发展提出战略建议， 治疗性平滑肌松弛剂。

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.