PHOSPHORYLATION, CALCIUM, AND SMOOTH MUSCLE CONTRACTION

尽管依赖于钙离子的肌球蛋白轻链(MLC)磷酸化是一种 血管平滑肌收缩启动的重要一步， 我们已经证明，部队也可以在 缺乏MLC的磷酸化。在本资助期间，我们 测试了两个依赖于钙离子的调节系统在 与控制平滑肌收缩平行：一个人负责 磷酸化交叉桥的力量的快速发展和第二 通过非磷酸化控制力量的缓慢发展或维持 十字桥。我们的结果表明，平滑肌受 两个钙依赖的过程，并认为这些系统作用于 平行的。此外，我们的数据表明，第二钙离子依赖于 过程中可能涉及到细丝蛋白钙调蛋白。这项建议是 基于钙离子和磷酸化依赖的假设 钙调蛋白的去抑制允许固有水平的肌动蛋白激活 肌球蛋白ATPase活性的表达；此表达导致 发展。与这一过程共存和同时存在的是钙离子 依赖的MLC磷酸化显著增加肌球蛋白ATPase 活动和部队发展的速度。我们还将测试两个 另一种假说：1.钙调蛋白去抑制和MLC磷酸化 2.MLC的磷酸化-去磷酸化的速率 是收缩的主要调节器。为了验证我们的假设，猪 颈动脉将被研究为分离的肌球蛋白，Triton X-100 洗涤剂的皮肤纤维和完整的组织。有了这些型号，我们将 测定钙离子对生化指标的依赖性(ATPase活性， 超沉淀和蛋白质磷酸化水平)和机械 (跨桥循环速率和附着量以及力)参数 收缩。具体目标是：1.确定是否发生逆转 抑制钙调蛋白可通过以下方式启动平滑肌收缩： 在没有MLC的情况下，是否可以通过去抑制钙调蛋白来产生作用力 通过MLC的磷酸化能不能产生FORCE？ 钙调蛋白去抑制？；是否存在固有的肌动蛋白激活水平？ 平滑肌肌球蛋白ATPase活性？会增加钙离子和 钙调蛋白抑制钙调蛋白并允许这种活性的表达？ MLC的激酶和/或磷酸酶活性是否受钙离子的调节？ 确定钙离子依赖能否逆转钙调蛋白抑制 我们会问：钙调蛋白在钙离子中是否被磷酸化？ 依赖的，MLC磷酸化的非依赖性收缩？ 钙离子/钙调蛋白依赖的蛋白激酶II与钙调蛋白的关系 磷酸化？3.确定通过以下方式调节的程度 钙调蛋白在血管顺应性生理反应中的作用 我们将研究肌肉：钙调蛋白去抑制是否发生在对 所有刺激模式？；是否需要钙调蛋白磷酸化 在完整的组织中解除抑制？哪一种激酶起作用？

Although Ca2+ dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of contraction in vascular smooth muscle, we have demonstrated that force can also be developed and maintained in the absence of MLC phosphorylation. During the current funded period, we tested the hypothesis that two Ca2+ dependent regulatory systems act in parallel to control smooth muscle contraction: one is responsible for the rapid development of force by phosphorylated crossbridges and the second controls the slow development or maintenance of force by unphosphorylated crossbridges. Our results demonstrated that smooth muscle is regulated by two Ca2+ dependent processes and suggested that these systems act in parallel. Moreover, our data indicate that the second Ca2+ dependent process may involve the thin filament protein caldesmon. This proposal is based on the hypothesis that Ca2+ and phosphorylation dependent disinhibition of caldesmon allows an intrinsic level of actin-activated myosin ATPase activity to be expressed; this expression results in force development. Coexisting and simultaneous with this process is Ca2+ dependent MLC phosphorylation which significantly increases myosin ATPase activity and the rate of force development. We will also test two alternate hypotheses: 1. caldesmon disinhibition and MLC phosphorylation occur in series; and 2. the rate of MLC phosphorylation-dephosphorylation is the primary regulator of contraction. To test our hypothesis, swine carotid arteries will be studied as isolated actomyosin, Triton X-100 detergent skinned fibers and intact tissues. With these models we will determine the Ca2+ dependence of biochemical (ATPase activity, superprecipitation and protein phosphorylation levels) and mechanical (crossbridge cycling rates and attachment, and force) parameters of contraction. The specific aims are: 1. To determine if reversal of caldesmon inhibition will initiate smooth muscle contraction by asking: Can force be developed by caldesmon disinhibition without MLC phosphorylation?; Can force be developed by MLC phosphorylation without caldesmon disinhibition?; Is there an inherent level of actin-activated myosin ATPase activity in smooth muscle?; Will increases in Ca2+ and calmodulin disinhibit caldesmon and allow expression of this activity?; Are MLC kinase and/or phosphatase activities modulated by Ca2+? 2. To determine if caldesmon inhibition can be reversed by Ca2+ dependent phosphorylation we will ask: Is caldesmon phosphorylated during a Ca2+ dependent, MLC phosphorylation independent contraction?; Is MAP kinase or Ca2+/calmodulin dependent protein kinase II responsible for caldesmon phosphorylation? 3. To determine the extent to which regulation by caldesmon plays a role in the physiological response of vascular smooth muscle we will examine: Does caldesmon disinhibition occur in response to all modes of stimulation?; Is caldesmon phosphorylation required for disinhibition in intact tissue?; and Which kinase is responsible?