CELL CA2+ AND CONTRACTION IN VASCULAR SMOOTH MUSCLE

我们计划评估一些监管机制，以管理 血管平滑肌收缩。蛋白的磷酸化 2万道尔顿肌球蛋白轻链允许快速过桥 循环(缩短速度)，因此应力发展迅速。 另一种形式的跨桥交互，称为“闩锁”，允许 附着式十字桥，保持压力，大大减少 跨桥自行车和三磷酸腺苷消耗。这两种形式的 交叉桥的相互作用依赖于(钙)i。我们有 测量完整组织中肌球蛋白磷酸化的技术 并对交叉桥的相互作用进行生物物理量化。 这些测量值与细胞内的估计值的相关性 (CA2)是本项目的研究目标。 细胞内游离钙的估计涉及到加载 细胞内光蛋白Aequorin与光子测量 释放作为aequorin结合钙。我们已经报告了一起 光估计(钙)i与肌球蛋白磷酸化的相关性 在K去极化猪颈动脉中膜中。这种相关性支持 Krebs和Beavo接受钙的第四个标准-- 刺激肌球蛋白磷酸化具有生理意义。 (Ca~(2+))对压力维持的敏感性高于对 肌球蛋白磷酸化或缩短速度。 我们计划(1)通过以下方式进一步测试aequorin方法的有效性 Aequorin同时测定单细胞内(Ca~(2+))_i 还有荧光染料。(2)检验(钙)的普适性- 肌球蛋白磷酸化与(钙)-应激关系。这 包括测量(钙)、肌球蛋白磷酸化、缩短 几种激动剂收缩过程中的速度和应力 在环核苷酸依赖的血管扩张过程中。(3)测试 机械扰动的影响，这可能会调解 肌源性反应、(钙)i和(钙)敏感性 收缩装置。

We plan to evaluate some of the regulatory mechanisms governing contraction in vascular smooth muscle. Phosphorylation of the 20,000 dalton light chain of myosin permits rapid crossbridge cycling (shortening velocity) and thus rapid stress development. Another form of crossbridge interaction, termed "latch," allows attached crossbridges to maintain stress with greatly reduced crossbridge cycling and ATP consumption. Both forms of crossbridge interactions are dependent on (Ca2+)i. We have techniques to measure myosin phosphorylation in intact tissues and to biophysically quantitate crossbridge interactions. Correlation of these measurements with estimates of intracellular (Ca2+) in smooth muscle is the goal of this project. Estimation of intracellular free Ca2+ involves loading the photoprotein aequorin intracellularly and measuring the photon emission as aequorin binds Ca2+. We already have reported a correlation of light estimated (Ca2+)i with myosin phosphorylation in K+ depolarized swine carotid media. This correlation supports the fourth criterion of Krebs and Beavo for acceptance of Ca2+- stimulated myosin phosphorylation as physiologically significant. The (Ca2+) sensitivity for stress maintenance was higher than for myosin phosphorylation or shortening velocity. We plan to (1) further test the validity of the aequorin method by simultaneously measuring (Ca2+)i in single cells using aequorin and the fluorescent dyes. (2) Test the universality of the (Ca2+)- myosin phosphorylation and (Ca2+)-stress relationships. This involves measuring (Ca2+), myosin phosphorylation, shortening velocity, and stress during contraction with several agonists and during cyclic nucleotide-dependent vasodilation. (3) Test the effect of mechanical perturbations, which could mediate myogenic responses, on (Ca2+)i and the (Ca2+) sensitivity of the contractile apparatus.