PHOSPHORYLATION, CALCIUM, AND SMOOTH MUSCLE CONTRACTION

人们普遍认为，[Ca 2 +]的增加通过以下方式启动收缩： 肌球蛋白轻链（MLC）激酶的激活和肌球蛋白轻链（MLC）的磷酸化 MLC。我们已经证明了第二条平行的独立途径 存在的监管通过测试的假设，解除抑制的 钙调素通过磷酸化使固有的激活肌球蛋白 与肌动蛋白相互作用并产生力的桥梁。为了验证这一 假设，我们将首先利用反义寡脱氧核苷酸 方法来产生钙调素缺乏的血管组织。与此 caldesmon“敲除”组织，我们可以更精确地确定， 这种蛋白质在收缩功能中的作用。然后我们将决定 内源性激酶与钙调素生理相关 磷酸化我们将区分p38，p42和p44成员。 催化钙调素的丝裂原活化蛋白激酶家族 磷酸化我们还将检验备用假设 钙调素不参与平滑肌的调节 收缩本身，但涉及的结构完整性， 组织.这些假设将使用完整和Triton X-100进行检验 猪颈动脉的剥皮条。我们将确定Ca 2 + 依赖肌动蛋白激活的肌球蛋白ATP酶活性，力，交联， 循环速率和附着，激酶活性，蛋白质含量，和 含有内源性钙调素的对照条中的蛋白磷酸化， 在钙调素缺乏的条带中，在钙调素缺乏的条带中， 外源性钙调素被重新引入。以下具体目标 将在本提案中进行：1。使用反义抑制h- 培养的猪颈动脉平滑肌条中钙调素的合成 动脉; 2.为了确定caldesmon在颈动脉条中的作用， 对照和钙调蛋白缺陷组织; 3.为了确定哪些特定的 MAP激酶家族内的激酶是钙调蛋白激酶;和4.到 确定导致特异性抗体激活的上游途径， 已鉴定的激酶。我们知道，除了大脑中的通道， 其中MLC磷酸化充当简单的Ca 2+依赖性开关。什么 这些途径是什么以及它们是如何被调节的仍然是未知的。卡尔德斯蒙 十多年来一直是这场争论的焦点的 这些具体目标的成功实现， 这个问题的明确答案：caldesmon是否参与收缩 调控

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.