MYOSIN ISOFORMS & CALCIUM REGULATION OF ACTOMYOSIN ATPASE IN DETRUSOR

肌球蛋白异构体

• 批准号: 6105779
• 负责人: SAMUEL K. CHACKO
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-18 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105779
• 关键词: RNA splicing; RNase protection assay; actins; animal tissue; calcium ion; gene expression; human genetic material tag; human tissue; hypertrophy; immunofluorescence technique; in situ hybridization; muscle contraction; myosins; northern blottings; nuclear runoff assay; phosphorylation; polymerase chain reaction; protein isoforms; protein localization; smooth muscle; urinary bladder; urinary bladder disorder; urination disorder; western blottings

We propose to use smooth muscle tissue from the rabbit model for outlet obstruction and human biopsy samples from patients with outlet obstruction (from Core B) to elucidate the cellular and molecular mechanisms underlying the changes in contractility associated with bladder smooth muscle hypertrophy and remodeling in outlet obstruction and it's reversal. Our preliminary data indicate that the expression of myosin isoforms, regulation of actomyosin ATPase, and the organization of myofilaments in different stages of bladder remodeling are important factors that affect the ability of smooth muscle to generate the active force need to empty the bladder. Based on these data, we hypothesize that altered contractility of the bladder smooth muscle associated with outlet obstruction is due to either changes in the composition of myosin isoforms, organization or myosin into the contractile apparatus. and/or the Ca2+ regulation of actomyosin. We will test this hypothesis using the rabbit model for partial outlet obstruction and human detrusor muscle from patients undergoing surgery for outlet obstruction. Specially, our experiments will address the splicing of the myosin mRNA at the 3' end (SM1 and SM2) or the 5' end (insert near the ATP-binding region on the head of the myosin molecules), different in smooth muscles from decompensated bladder walls as compared to normal and compensated bladders (during outlet obstruction and after reversal)? Is there a difference in the expression of these myosin isoforms at transcriptional and translational levels in bladder wall smooth muscle at different stages of compensation and decompensation? (3) What effect does exchanging LC17 isoforms have on the function and the regulation of myosin isolated from normal and hypertrophied smooth muscle? (4) Is the smooth muscle myosin isolated from the detrusor of decompensated bladder functionally different as compared to normal and compensated bladders? (5) Is the expression of myosin light chain kinase (MLCK), which plays a role in the regulation of actomyosin ATPase and force generation, altered during decompensation as compared with the normal and compensation stage? (6) What is the effect of addition of MLCK on the myosin light chain phosphorylation and force in chemically skinned smooth muscle fiber preparations made from normal and compensated bladder (during outlet obstruction and after the reversal of outlet obstruction) detrusor smooth muscles? Alterations in the proteins that form the contractile apparatus will be correlated with changes in the regulation of the entry of Ca2+ into the cytosol (Project 1) and velocity of force generation and regulation of cross-bridge cycling (Project 3). Together, the data from experiments outlined in the Urology Research Center proposal will identify the molecular mechanisms that are responsible for the contractile dysfunction in outlet obstruction.

我们建议使用来自兔子模型的平滑肌组织作为出口 梗阻和来自出口梗阻患者的人体活检样本 （来自核心 B）阐明细胞和分子机制 与膀胱平滑相关的收缩力变化的基础 出口阻塞时的肌肉肥大和重塑及其逆转。 我们的初步数据表明，肌球蛋白亚型的表达， 肌动球蛋白 ATP 酶的调节和肌丝的组织 膀胱重塑的不同阶段是影响膀胱重塑的重要因素 平滑肌产生需要排空的主动力的能力 膀胱。根据这些数据，我们假设改变了 与出口相关的膀胱平滑肌的收缩性 阻塞是由于肌球蛋白成分的变化 同种型、组织或肌球蛋白进入收缩装置。和/或 肌动球蛋白的 Ca2+ 调节。我们将使用以下方法检验这一假设 兔部分出口梗阻模型和人逼尿肌模型 因出口阻塞而接受手术的患者。特别是，我们的 实验将解决肌球蛋白 mRNA 在 3' 端的剪接问题 （SM1 和 SM2）或 5' 端（插入靠近 ATP 结合区） 肌球蛋白分子的头部），与平滑肌不同 失代偿膀胱壁与正常膀胱和代偿膀胱相比 （出口阻塞期间和反转后）？有什么区别吗 这些肌球蛋白亚型在转录和 不同阶段膀胱壁平滑肌的翻译水平 代偿和失代偿？ (3) 更换LC17有什么影响 亚型对分离的肌球蛋白的功能和调节有影响 正常平滑肌和肥大平滑肌？ (4) 是平滑肌肌球蛋白 与失代偿膀胱的逼尿肌分离功能不同 与正常膀胱和代偿膀胱相比？ (5) 的表达式为 肌球蛋白轻链激酶 (MLCK)，在调节 肌动球蛋白 ATP 酶和力的产生，在失代偿期间发生改变 与正常阶段和补偿阶段相比？ （6）有什么作用 添加 MLCK 对肌球蛋白轻链磷酸化和力的影响 化学剥皮平滑肌纤维制剂，由正常和 代偿性膀胱（在出口梗阻期间和逆转后 出口梗阻）逼尿肌平滑肌？蛋白质的改变 形成收缩装置的变化将与 调节 Ca2+ 进入细胞质（项目 1）和速度 过桥骑行的力产生和调节（项目 3）。 泌尿学研究中概述的实验数据一起 中心提案将确定以下分子机制： 造成出口梗阻的收缩功能障碍。