MAPK IN THE CONTRACTILE PHENOTYPE OF SMOOTH MUSCLE

描述：(调查人员摘要)勾勒出的工作目标 在此应用中是对信令的进一步理解 血管平滑肌中的系统，导致血管 伸缩性。特别是，建议进行研究，以调查 丝裂原活化蛋白激酶(MAPK)和钙调蛋白在血管紧张素转换酶中的作用 血管平滑肌收缩。 高分子形式的钙调蛋白(h-钙调蛋白)是一种有效的， 肌动球蛋白ATPase活性的肌动蛋白特异性抑制物 在体外，它可以同时结合肌动蛋白和肌球蛋白。H-Caldesmon是磷酸化的 在静息状态下猪颈总动脉的化学计量比 对药物刺激的反应使磷酸化增加 这表明，这一过程可能会对经济收缩起到监管作用。H- Caldesmon在羧基末端的两个位置上被磷酸化 蛋白质；这些位点被有丝分裂原激活的蛋白激酶修饰 (MAPK)，在体外。需要检验的假设是，激活 MAPK是平滑肌收缩的表型，导致 H-钙调蛋白的磷酸化与收缩功能的改变 功能。关于这一点的两个次级假设是，h-的磷酸化 Caldesmon by MAPK 1：直接或通过改变肌动球蛋白动力学 与另一种蛋白质的相互作用，以及2)允许结构 长期更改所需的单元格内的更改 压力。为了调查这一假设，以下是具体的 我们将致力于以下目标： 1.测定MAPK活性、h-caldesmon磷酸化及对 药物对猪颈动脉的刺激作用。激动剂将 包括神经体液制剂和生长因子。 2.测定猪MAPK活性和h-钙调蛋白磷酸化水平 颈动脉受到拉伸的影响。MAPK活性与h-caldesmon 磷酸化水平将作为机械负荷的函数进行测量 贴在肌肉上。 3.测定h-钙调蛋白磷酸化的生化效应 通过a)肌动蛋白和肌球蛋白与h-caldesmon结合的MAPK，b)肌动蛋白 C)肌动球蛋白ATPase活性。这一目标的研究 将研究h-caldesmon的直接生物化学效应 收缩装置上的磷酸化。 MAPK在培养细胞的生长和增殖中发挥作用；然而， 血管平滑肌收缩表型的细胞不 正常情况下会经历细胞分裂。这些研究是独一无二的 研究MAPK在平滑肌收缩中的作用 这种组织的主要功能。

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.