MYOSIN ISOFORMS & CALCIUM REGULATION OF ACTOMYOSIN ATPASE IN DETRUSOR

我们建议使用兔模型的平滑肌组织作为出口 出口梗阻患者的梗阻和人体活检样本 (from核心B）阐明细胞和分子机制 与膀胱平滑肌相关的收缩性变化的基础 出口梗阻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 平滑肌产生排空所需主动力的能力 膀胱基于这些数据，我们假设 与出口相关的膀胱平滑肌收缩性 阻塞是由于肌球蛋白组成的变化 同种型、组织或肌球蛋白进入收缩装置。和/或 肌动球蛋白的Ca ~（2+）调节。我们将使用 兔膀胱出口部分梗阻模型和人逼尿肌 因出口梗阻接受手术的患者。特别是，我们的 实验将解决肌球蛋白mRNA在3'端的剪接 (SM1和SM 2）或5'端（插入到靠近 肌球蛋白分子的头部），在平滑肌中不同于 与正常和代偿膀胱相比，失代偿膀胱壁 （出口阻塞期间和反转后）？是否有差异 这些肌球蛋白同种型在转录水平的表达， 在不同阶段的膀胱壁平滑肌中的翻译水平 代偿和失代偿(3)交换LC 17有什么影响 同种型对肌球蛋白的功能和调节的影响 正常和肥大的平滑肌(4)平滑肌肌球蛋白 从失代偿膀胱的逼尿肌分离功能不同 与正常和补偿性膀胱相比？(5)表达了 肌球蛋白轻链激酶（MLCK），在调节 肌动球蛋白ATP酶和力的产生，在失代偿期间改变， 与正常和补偿阶段相比？(6)效果如何 MLCK对肌球蛋白轻链磷酸化的作用和对肌球蛋白轻链磷酸化的影响 化学去皮的平滑肌纤维制品， 补偿膀胱（在出口阻塞期间和 出口梗阻）逼尿肌平滑肌？蛋白质的改变 形成收缩器官的细胞将与 调节Ca 2+进入胞质溶胶（项目1）和速度 力的产生和跨桥循环的调节（项目3）。 总之，泌尿学研究中概述的实验数据 中心的建议将确定分子机制， 导致出口梗阻时的收缩功能障碍。

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.