MAPK IN THE CONTRACTILE PHENOTYPE OF SMOOTH MUSCLE

描述：（研究者摘要）概述的工作目标 本申请的目的是进一步理解信令 血管平滑肌中的一个系统， 收缩性 特别是，建议研究调查 丝裂原活化蛋白激酶（MAPK）和钙调蛋白在 血管平滑肌收缩。 钙调素的高分子量形式（h-钙调素）是一种有效的， 平滑肌特异性肌动球蛋白ATP酶活性抑制剂， 体外，结合肌动蛋白和肌球蛋白。 h-Caldesmon被磷酸化 在静止的猪颈动脉中， 在药理学刺激下磷酸化增加 表明这一过程可能是收缩的调节过程。 h- 钙调素在蛋白质的羧基末端的两个位点上被磷酸化， 这些位点被丝裂原活化蛋白激酶修饰 （MAPK），在体外。 有待检验的假设是， MAPK是平滑肌的收缩表型， H-钙调蛋白的磷酸化和心肌收缩功能的改变 功能 对此有两个亚假设： 钙调素通过MAPK 1：改变肌动球蛋白动力学，无论是直接或通过 与另一种蛋白质的相互作用，2）允许结构 细胞内的变化是长期改变所必需的， 应力 为了研究这一假设，以下具体 目标将是： 1.为了测量MAPK活性，h-钙调蛋白磷酸化和对 猪颈动脉的药理学刺激。 激动剂将 包括神经体液剂和生长因子。 2.为了检测MAPK活性和h-caldesmon磷酸化水平， 颈动脉受到拉伸。 MAPK活性与h-钙调蛋白 将测量磷酸化水平作为机械负荷的函数 应用于肌肉。 3.确定h-钙调蛋白磷酸化的生化效应 通过MAPK对a）肌动蛋白和肌球蛋白与h-钙调蛋白的结合，B）肌动蛋白 c）肌动球蛋白ATP酶活性。 为此目的进行的研究 将研究h-钙调素的直接生化作用 磷酸化对收缩器官的影响。 MAPK在培养的细胞生长和增殖中起作用;然而， 血管平滑肌收缩表型中的细胞不 正常进行细胞分裂。 这些研究是独一无二的， 研究MAPK参与平滑肌收缩， 这个组织的主要功能。

DESCRIPTION: (Investigator's abstract) The goal of the work outlined in this application is to further understanding of the signalling systems in vascular smooth muscle that result in an alteration of contractility. In particular, studies are proposed to investigate the roles of mitogen-activated protein kinase (MAPK) and caldesmon in vascular smooth muscle contraction. The high molecular weight form of caldesmon (h-caldesmon) is a potent, smooth muscle specific, inhibitor of actomyosin ATPase activity, in vitro, that binds both actin and myosin. h-Caldesmon is phosphorylated in resting porcine carotid arteries and the stoichiometry of phosphorylation increases in response to pharmacological stimulation suggesting that this process may be regulatory for contraction. h- Caldesmon is phosphorylated on two sites in the carboxyl-terminus of the protein; these sites are modified by mitogen-activated protein kinase (MAPK), in vitro. The hypothesis to be tested is that the activation of MAPK is the contractile phenotype of smooth muscle results in the phosphorylation of h-caldesmon and an alteration of contractile function. Two sub-hypotheses to this are that the phosphorylation of h- caldesmon by MAPK 1:alters actomyosin kinetics, either directly or via an interaction with another protein, and 2) allows for structural changes within the cell that are required for prolonged alterations of stress. In order to investigate this hypothesis, the following specific aims will be pursued: 1. To measure MAPK activity, h-caldesmon phosphorylation and force upon pharmacological stimulation of porcine carotid arteries. Agonists will include neurohumoral agents and growth factors. 2. To measure MAPK activity and h-caldesmon phosphorylation in porcine carotid arteries subjected to stretch. MAPK activity and h-caldesmon phosphorylation levels will be measured as a function of mechanical load applied to the muscle. 3. To determine the biochemical effects of h-caldesmon phosphorylation by MAPK on a)actin and myosin binding to h-caldesmon, b) actin polymerization, and c) actomyosin ATPase activity. Studies in this aim will investigate the direct biochemical effects of h-caldesmon phosphorylation on the contractile apparatus. MAPK plays a role in cultured cell growth and proliferation; however, cells in the contractile phenotype of vascular smooth muscle do not normally undergo cell division. These studies are unique in investigating involvement of MAPK in smooth muscle contractility, the primary function of this tissue.