PHOSPHORYLATION, CALCIUM, AND SMOOTH MUSCLE CONTRACTION

人们普遍认为，[Ca~(2+)]的增加通过 肌球蛋白轻链(MLC)激酶的激活和磷酸化 MLC。我们已经证明了第二条平行的、独立的途径 通过检验解除抑制的假设来存在调节的存在 通过磷酸化的钙调蛋白允许固有激活的肌球蛋白 与肌动蛋白相互作用并产生力量的桥梁。为了测试这一点 假设，我们将首先使用反义寡核苷酸 一种产生钙离子缺乏的血管组织的方法。有了这个 Caldesmon“敲除”组织，我们可以更精确地确定 这种蛋白在收缩功能中的作用。然后我们将确定哪一个 内源性激酶与钙调蛋白的生理学关系 磷酸化。我们将区分p38、p42和p44成员 催化钙调蛋白的丝裂原活化蛋白激酶家族 磷酸化。我们还将测试备选假设 钙调蛋白不参与平滑肌的调节 收缩本身，但涉及结构的完整性 组织。这些假设将使用完整和Triton X-100进行测试 剥了皮的猪颈动脉条。我们将测定钙离子 肌动蛋白激活的肌球蛋白ATPase活性、作用力、交叉桥、 循环率和附着性、激酶活性、蛋白质含量和 含有内源性钙调蛋白的对照试条中的蛋白质磷酸化， 在缺乏钙离子的条带中，以及在缺乏钙离子的条带中 外源性钙离子被重新引入。以下是具体目标 在这项建议中将追求：1.使用反义抑制h- 培养的猪颈动脉条合成钙调蛋白 动脉；2.确定钙调蛋白在颈动脉条中的作用 对照和钙调蛋白缺乏的组织；3.确定哪种特异性 MAP激酶家族中的激酶是钙调蛋白激酶；以及 确定导致特异体激活的上游途径(S) 鉴定出了激活酶。我们知道存在着不同于 其中MLC的磷酸化起着简单的钙依赖开关的作用。什么 这些途径是存在的，它们是如何受到监管的，目前尚不清楚。卡尔德斯蒙 十多年来一直处于这场争议的中心。这个 成功完成这些具体目标应该提供一个合理的 问题的明确答案：钙调蛋白是否与收缩有关？ 监管。

It is widely accepted that an increase in [Ca2+] initiates contraction by activation of myosin light chain (MLC) kinase and phosphorylation of the MLC. We have demonstrated that a second parallel, independent pathway exists for the regulation by testing the hypothesis that disinhibition of caldesmon by phosphorylation allow inherently activated myosin crossbridges to interact with actin and produce force. To test this hypothesis, we will first utilize an antisense oligodeoxynucleotide approach to produce a caldesmon-deficient vascular tissue. With this caldesmon "knock-out" tissue, we can determine with greater precision, the role of this protein in contractile function. We will then determine which endogenous kinases is physiologically relevant for caldesmon phosphorylation. We will distinguish among p38, p42, and p44 members of the mitogen-activated protein kinase family for catalyzing caldesmon phosphorylation. We will also test the alternate the alternate hypothesis that caldesmon is not involved in the regulation of smooth muscle contraction per se, but involved in the structural integrity of the tissue. These hypothesis will be tested using intact and Triton X-100 skinned strips of swine carotid artery. We will determine the Ca2+ dependence of actin-activated myosin ATPase activity, force, crossbridge, cycling rate and attachment, kinase activities, protein content, and protein phosphorylation in control strips containing endogenous caldesmon, in caldesmon-deficient strips, and in caldesmon-deficient strips to which exogenous caldesmon has been re-introduced. The following specific aims will be pursued in this proposal: 1. To use antisense inhibition of h- caldesmon synthesis in cultured smooth muscle strips from porcine carotid arteries; 2. To determine the roles of caldesmon in carotid strips using control and caldesmon-deficient tissues; 3. To determine which specific kinase within the MAP kinase family is the caldesmon kinase; and 4. To determine the upstream pathway(s) leading to activation of the specific kinases identified. We know that pathways exist other than the one in which MLC phosphorylation acts as a simple Ca2+-dependent switch. What these pathways are and how they are regulated is still unknown. Caldesmon has been at the center of this controversy for over a decade. The successful completion of these specific aims should provide a reasonably definitive answer to the question: Is caldesmon involved in contractile regulation.