Regulation of Vascular Smooth Muscle Contraction

描述(申请人提供)：Smooth的收缩特性 肌肉大致分为相位性(快)和强直性(慢)。阶段平滑 肌肉的特点是力量激活的速度相对较快，力量 松弛和Vmax，而紧张性平滑肌的特点是速度较慢 然而，分子的力激活、力松弛和Vmax的速率 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 时相和紧张性平滑肌之间收缩特性的差异在于 收缩细丝的水平，以及运动的差异 肌动球蛋白ATPase(AMATPase)。这笔赠款的总体目标是调查 细胞内AMATPase动力学差异的分子机制 强直性和相性平滑肌。我们的假设是， 肌球蛋白剪接变异表达对AMATPase动力学的影响 重链(MHC)和17 kDa肌球蛋白轻链(MLC17)。具体目标 检验这一假设的目的是确定MHC和MLC17的剪接变体 是平滑肌AMATPase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 负责肌力的维持(具体目标4)。为了测试 具体目标1-3，我们将强制表达两种剪接变体异构体 MHC和MLC17在培养的胚胎主动脉和肌胃平滑肌细胞中的表达。 我们将过度表达MHC的内源性和替代异构体，并 MLC17在培养的主动脉和肌肌细胞及组织条中表达。 在强制表达单个收缩蛋白后，我们将确定 无机PI和MgADP的升高对稳态力和 单个培养的平滑肌细胞的硬度以及平滑肌细胞的硬度 剥离结果并与未转染组的结果进行比较 控制。这些实验将阐明一种基因表达的影响 分离的单个收缩蛋白对大鼠心肌细胞AMATPase动力学的影响 平滑的肌肉。此外，为了确定非肌肉肌球蛋白是否参与 力量维持的分子机制(特定目标4)，我们将 确定转基因小鼠品系的力量维持特性， 缺乏非肌肉肌球蛋白IIB。这些研究的结果应该能阐明 决定平滑肌AMATPase动力学的机制，以及 为进一步研究平滑肌的收缩能力奠定了基础 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.