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标本 (摘自核心B)以阐明细胞和分子机制 与膀胱顺畅相关的收缩能力变化的潜在原因 出口梗阻的肌肉肥大和重构及其逆转。 我们的初步数据表明，肌球蛋白亚型的表达， 肌动球蛋白ATPase的调节和肌丝的组织 不同阶段的膀胱重塑是影响膀胱重塑的重要因素。 平滑肌产生主动力量的能力需要排空 膀胱。基于这些数据，我们假设改变了 与出口相关的膀胱平滑肌的收缩特性 梗阻是由于肌球蛋白组成的改变 肌球蛋白的异构体、组织或肌球蛋白进入收缩装置。和/或 肌动球蛋白的钙调节。我们将使用 兔出口部分梗阻模型及人逼尿肌的实验研究 因出口梗阻而接受手术的患者。特别是我们的 实验将解决肌球蛋白mRNA在3‘端的剪接 (SM1和SM2)或5‘端(插入到 肌球蛋白分子的头部)，在平滑肌肉中不同于 失代偿膀胱壁与正常和代偿性膀胱的比较 (在出口阻塞期间和反转后)？在这方面有区别吗？ 这些肌球蛋白亚型在转录和转录水平上的表达 慢性前列腺炎患者不同时期膀胱壁平滑肌的转录水平 补偿和失代偿？(3)交换LC17有什么影响 肌球蛋白亚型对肌球蛋白功能和调节的影响 正常和肥大的平滑肌？(4)平滑肌肌球蛋白 从失代偿膀胱逼尿肌分离出来的功能不同 与正常和补偿的膀胱相比？(5)是 肌球蛋白轻链激酶(MLCK)在细胞周期调控中的作用 肌动球蛋白ATPase和力的产生，在失代偿过程中改变为 与正常和补偿阶段相比？(6)有什么影响 加入MLCK对肌球蛋白轻链磷酸化和肌球蛋白轻链作用力的影响 化学去皮的平滑肌纤维制剂，由正常和 代偿性膀胱(出口梗阻期间和逆转后 出口梗阻)逼尿肌平滑？蛋白质的变化 形成收缩装置的信号将与 钙离子进入胞浆的调节(方案1)和速率 关于跨桥自行车的力量产生和调节(项目3)。 总而言之，《泌尿学研究》概述的实验数据 中心的提案将确定以下分子机制 负责出口梗阻的收缩功能障碍。

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.