PHOSPHORYLATION, CALCIUM, AND SMOOTH MUSCLE CONTRACTION

虽然钙离子依赖性肌球蛋白轻链（MLC）磷酸化是一个重要的机制。 血管平滑肌收缩起始的重要步骤， 我们已经证明，武力也可以发展和维持， 没有MLC磷酸化。在目前的融资期内，我们 测试了两个Ca 2+依赖性调节系统在 平行控制平滑肌收缩：一个是负责 通过磷酸化的横桥和第二个 控制缓慢发展或维持部队的非磷酸化 天桥我们的研究结果表明，平滑肌是由 两个Ca 2+依赖的过程，并建议这些系统的作用， 并联此外，我们的数据表明，第二个Ca 2+依赖 这一过程可能涉及细丝蛋白质钙调素。这项建议是 基于Ca 2+和磷酸化依赖于 钙调素的去抑制允许肌动蛋白激活的内在水平 肌球蛋白ATP酶活性表达;这种表达导致力 发展与此过程同时共存的是Ca 2 + 依赖MLC磷酸化，显著增加肌球蛋白ATP酶 活动和力量发展的速度。我们还将测试两个 替代假设：1.钙调素去抑制和MLC磷酸化 连续发生; 2. MLC磷酸化-去磷酸化速率 是收缩的主要调节器。为了验证我们的假设，猪 颈动脉将作为分离的肌动球蛋白Triton X-100进行研究 洗涤剂皮肤纤维和完整的组织。有了这些模型， 测定生化（ATP酶活性， 超沉淀和蛋白磷酸化水平）和机械 （横桥循环速率和附着力）参数 收缩。具体目标是：1.以确定是否逆转 钙调蛋白抑制将通过以下方式启动平滑肌收缩： 无MLC时，钙调素去抑制能产生力吗 磷酸化？力可以通过MLC磷酸化而产生， 钙调素去抑制？是否有一种内在的肌动蛋白激活水平 平滑肌肌球蛋白ATP酶活性;将增加Ca 2+和 钙调素解除钙调素的抑制并允许这种活性的表达？ MLC激酶和/或磷酸酶活性是否受Ca 2+调节？2.到 确定钙调素抑制是否可以通过Ca 2+依赖性逆转 磷酸化我们会问：钙调蛋白在Ca 2 + 依赖性、MLC磷酸化非依赖性收缩？是MAP激酶或 Ca 2 +/钙调蛋白依赖性蛋白激酶II负责钙调素 磷酸化？3.为了确定监管的程度， 钙调素在血管平滑肌的生理反应中起作用 我们将检查肌肉：钙调素去抑制是否发生在对 所有的刺激方式？钙调蛋白磷酸化是 完整组织中的去抑制作用？哪种激酶负责？

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?