Regulation of Vascular Smooth Muscle Contraction

描述（由申请人提供）：光滑的收缩特性 肌肉大致分为阶段性（快）和紧张性（慢）。相位平滑 肌肉的特征在于相对快速的力量激活率， 舒张和Vmax，而紧张性平滑肌的特点是缓慢的 力激活率、力松弛率和Vmax。 调节平滑肌收缩特性的机制是 未知其他人和我们已经证明了这种多样性的机制 在收缩性能之间的相位和紧张性平滑肌在于 水平的收缩丝，和差异的动力学， 肌动球蛋白ATP酶（AMATP）。这项拨款的总体目标是调查 AMAATPase动力学差异的分子机制， 紧张性和阶段性平滑肌。我们的假设是 AMATPase的动力学是由于肌球蛋白的剪接变体表达 重链（MHC）和17-kDa肌球蛋白轻链（MLC 17）。具体目标 为了检验这一假设，需要确定MHC和MLC 17的剪接变体是否 是平滑肌AMAATPase动力学的分子决定因素 （具体目标1-3）。此外，我们将确定非肌肉肌球蛋白是否 负责平滑肌中的力维持（具体目标4）。测试 具体目标1-3，我们将强制表达两种剪接变体同种型 MHC和MLC 17在培养的胚胎主动脉和砂囊平滑肌细胞中的表达。 我们将过度表达内源性和替代性MHC亚型， 培养的主动脉和肌胃平滑肌细胞和组织条中的MLC 17。 在强制表达单个收缩蛋白后，我们将确定 无机磷和MgADP的升高对稳态力的影响 单个培养的平滑肌细胞以及平滑肌中的硬度 剥离并将结果与未转染的 对照这些实验将阐明一个表达的影响， 单个收缩蛋白，在隔离，对AMAATPase的动力学， 平滑肌此外，为了确定非肌肉肌球蛋白是否参与 力维持的分子机制（具体目标4），我们将 确定转基因小鼠系中力维持的性质， 缺乏非肌肉肌球蛋白IIB。这些研究的结果应阐明 决定平滑肌AMAATPase动力学的机制，以及 为将来研究平滑肌收缩性 会因疾病状态而改变

DESCRIPTION (provided by the applicant): The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by relatively rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slower rates of force activation, force relaxation and Vmax However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. Others and we have demonstrated that the mechanism for this diversity in contractile properties between phasic and tonic smooth muscle lies at the level of the contractile filaments, and differences in the kinetics of the actomyosin ATPase (AMATPase). The overall goal of this grant is to investigate the molecular mechanism for the differences in the kinetics of the AMATPase in tonic and phasic smooth muscle. Our hypothesis is that differences in the kinetics of the AMATPase are due to splice variant expression of the myosin heavy chain (MHC) and the 17-kDa myosin light chain (MLC17). The Specific Aims to test this hypothesis are to determine if splice variants of MHC and MLC17 are molecular determinants for the kinetics of the AMATPase of smooth muscle (Specific Aims 1-3). In addition, we will determine if non-muscle myosin is responsible for force maintenance in smooth muscle (Specific Aim 4). To test Specific Aims 1-3, we will force the expression of both splice variant isoforms of MHC and MLC17 in cultured embryonic aortic and gizzard smooth muscle cells. We will over-express both the endogenous and the alternative isoform of MHC and MLC17 in cultured aortic and gizzard smooth muscle cells and tissue strips. After forcing the expression of a single contractile protein, we will determine the effect of elevations of inorganic Pi and MgADP on steady state force and stiffness of single cultured smooth muscle cells, as well as in smooth muscle strips and compare the results to those obtained in the non-transfected controls. These experiments will elucidate the effects of the expression of a single contractile protein, in isolation, on the kinetics of the AMATPase of smooth muscle. In addition, to determine if non-muscle myosin participates in the molecular mechanism for force maintenance (Specific Aim 4), we will determine the properties of force maintenance in a transgenic mouse line, which lacks non-muscle myosin IIB. The results of these studies should elucidate the mechanism that determines the kinetics of the AMATPase of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.