REGULATION OF SMOOTH MUSCLE CONTRACTILE PROPERTIES

平滑肌的收缩特性大致分为阶段性（快）和紧张性（慢）。 相性平滑肌的特征在于快速的力激活、力松弛和Vmax，而紧张性平滑肌的特征在于缓慢的力激活、力松弛和Vmax。 然而，调节平滑肌收缩特性的分子机制尚不清楚。 这项资助的总体目标是确定平滑肌收缩特性的分子机制，并阐明快速和缓慢收缩特性的机制。 我们的假设是，收缩蛋白的剪接变体亚型决定平滑肌的收缩特性。检验这一假设的具体目的是确定MLC17（特异性目的1）、MHC（特异性目的2）和MLC磷酸酶（特异性目的3）的剪接变体是否是平滑肌收缩特性的分子决定因素。 我们还将确定MLC磷酸酶的剪接变体同种型是否影响激动剂诱导的力增强的幅度或敏感性（具体目标3）。 为了测试这些特定目的，我们将在培养的胚胎主动脉和肌胃平滑肌细胞中强制表达MLC17、MHC和MLC磷酸酶的两种剪接变体同种型。 我们将确定在培养的主动脉和肌胃平滑肌细胞中正常存在或不表达的同种型的表达对培养的平滑肌细胞的机械性能的影响。 迫使单个收缩蛋白表达后，我们将确定单个培养的平滑肌细胞的最大力、力激活和力松弛速率、Vmax和MLC20磷酸化，并将结果与未转染的结果进行比较对照。 这些实验将阐明一个单一的收缩蛋白的表达，在隔离，对培养的平滑肌细胞的机械性能的影响。 这些研究的结果应该阐明的机制，决定平滑肌的收缩特性，并形成一个基础，为未来的调查如何平滑肌收缩性改变疾病状态。

The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by a rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slow rates of force activation, force relaxation and Vmax. However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. The overall goal of this grant is to determine the molecular mechanism for the contractile properties of smooth muscle and to elucidate the mechanism for fast and slow contractile properties. Our hypothesis is that splice variant isoforms of contractile proteins determine the contractile properties of smooth muscle. The specific aims to test this hypothesis are to determine if splice variants of MLC17 (Specific Aim 1), MHC (Specific Aim 2) and MLC phosphatase (Specific Aim 3) are molecular determinants of the contractile properties of smooth muscle. We will also determine if splice variants isoforms of MLC phosphatase effect either the magnitude or sensitivity of agonist induced force enhancement (Specific Aim 3). To test these Specific Aims we will force the expression of both splice variant isoforms of MLC17, MHC and MLC phosphatase in cultured embryonic aortic and gizzard smooth muscle cells. We will determine the effects of the expression of the isoform normally present or not expressed in the cultured aortic and gizzard smooth muscle cells on the mechanical properties of cultured smooth muscle cells. After forcing the expression of a single contractile protein, we will determine the maximum force, the rates of force activation and force relaxation, Vmax, and MLC20 phosphorylation of single cultured smooth muscle cells 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 mechanical properties of cultured smooth muscle cells. The results of these studies should elucidate the mechanism that determines the contractile properties of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.