CELL CA2+ AND CONTRACTION IN VASCULAR SMOOTH MUSCLE

我们计划评估一些监管机制， 血管平滑肌收缩。 的磷酸化 肌球蛋白的20，000道尔顿轻链允许快速过桥 循环（缩短速度），从而快速应力发展。 另一种形式的跨桥交互，称为“闩锁”， 附加的横桥，以保持应力大大减少 过桥自行车和ATP消耗。 两种形式的 跨桥相互作用依赖于（Ca 2+）i。 我们有 测量完整组织中肌球蛋白磷酸化的技术 并对桥间相互作用进行生物药理学定量。 这些测量值与细胞内 (Ca2+）在平滑肌中的作用是本项目的目标。 细胞内游离Ca 2+的估计包括加载 细胞内发光蛋白水母发光蛋白和测量光子 发光，因为水母发光蛋白结合Ca 2+。 我们已经报道了一个 光估计的（Ca 2+）i与肌球蛋白磷酸化的相关性 在K+去极化猪颈动脉中膜。 这种关联支持 Krebs和Beavo的第四个接受Ca 2+的标准 刺激肌球蛋白磷酸化的生理意义。 维持应激的钙敏感性高于维持应激的钙敏感性。 肌球蛋白磷酸化或缩短速度。 我们计划（1）进一步测试水母发光蛋白方法的有效性， 用水母发光蛋白同时测定单细胞内（Ca ~（2+））_i 和荧光染料。 (2)测试（Ca 2+）-的普适性 肌球蛋白磷酸化和（Ca 2+）-应激的关系。 这 包括测量（Ca 2+），肌球蛋白磷酸化，缩短 速度和应力在收缩与几个激动剂和 在环核苷酸依赖性血管舒张期间。 (3)测试 机械扰动的影响， 肌源性反应，对（Ca 2+）i和（Ca 2+）敏感性的影响。 收缩器

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.