Excitation contraction coupling in bladder smooth muscle

描述(由申请人提供)：良性前列腺增生症(BPH)的患病率随着男性年龄的增长而增加。虽然BPH没有生命危险，但这些症状越来越多地干扰生活质量，下尿路症状如尿频、膀胱排空不全、尿流中断或无力以及尿漏等。膀胱的一个主要组成部分，负责许多方面的排尿行为是膀胱平滑肌。不幸的是，我们对正常膀胱的平滑肌调节的了解还不完全，这阻碍了对疾病状态下发生的变化的研究。因此，这项建议的重点是通过对膀胱平滑肌兴奋-收缩偶联的详细机制的理解来填补这些空白。这一提议将检验的全球假设是，刺激膀胱平滑肌产生导致收缩的信号通路的激活，以及改变给定刺激产生的最终力水平的调节通路。这些途径主要包括肌球蛋白轻链(MLC)激酶、MLC磷酸酶、活化的和结构性活性的Rho激酶以及蛋白激酶C(PKC)。这项提议将测试的特定假设是，膀胱平滑肌含有一种结构性活性的拉伸依赖的Rho激酶，它为未受刺激的膀胱壁的肌肉细胞设定基调。对膀胱平滑肌的刺激然后启动一系列事件，涉及钙依赖的MLC磷酸化、激活的Rho激酶和PKC，这些事件通过线性和串扰通路微调通过作用于MLC激酶和磷酸酶而产生的力的水平。事实是，经过细胞培养的平滑肌表型调节为非肌肉表型，这阻碍了对平滑肌的研究。为了避免这个问题，我们开发了一种新的器官培养的膀胱平滑肌组织，它保持了活性和收缩表型，并允许引入siRNA。这项提议的第一个目标将是继续开发这种器官培养制剂，并展示siRNA敲除的效用。第二个目标将检验这样的假设，即蛋白激酶C是调节膀胱平滑肌收缩的信号的组成部分，并且蛋白激酶C的特定亚型参与了这一信号的特定步骤。我们还将检验蛋白激酶C影响Rho激酶活性的假设。第三个目的是研究Rho激酶在调节膀胱平滑肌收缩中的作用。我们将验证这样一种假设，即一个结构性活跃的、依赖拉伸的Rho激酶设定了膀胱平滑肌的基调，并且第二个Rho激酶参与了MLC磷酸酶活性的调节。这些研究将使我们能够开发一个详细的膀胱平滑肌调节的机制模型，我们将使用该模型来构建旨在了解BPH等疾病状态下发生的调节变化的研究。

DESCRIPTION (provided by applicant): The prevalence of benign prostatic hyperplasia (BPH) increases as men age. Although BPH is not life threatening, the symptoms increasingly interfere with the quality of life, with lower urinary tract symptoms such as frequent urination, the sensation of incomplete bladder emptying, interrupted orweak urinary stream, and urinary leakage. One major component of the urinary bladder that is responsible for many aspects of voiding behavior is bladder smooth muscle. Unfortunately our understanding of the regulation of smooth muscle in normal bladder is incomplete which has hampered investigations into the changes that occur during diseased states. Therefore the focus of this proposal is to fill these gaps by developing a detailed mechanisitic understanding of excitation-contraction coupling of bladder smooth muscle. The global hypothesis that this proposal will test is that stimulation of bladder smooth muscle produces activation of signaling pathways leading to contraction as well as modulatory pathways that alter the final level of force produced for a given stimulus. These pathways include primarily the myosin light chain (MLC) kinase, the MLC phosphatase, activated and constitutively active Rho kinases, and protein kinase C (PKC). The specific hypothesis that this proposal will test is that bladder smooth muscle contains a constitutively active stretch dependent Rho kinase that sets the tone on the muscle cells in the unstimulated bladder wall. Stimulation of the bladder smooth muscle then initiates a series of events involving calcium dependent MLC phosphorylation, activated Rho kinase, and PKC which by linear and cross-talk pathways fine tune the levels of force produced via actions on the MLC kinase and phosphatase. The fact that smooth muscles subjected to cell culture phenotypically modulate into a non-muscle phenotype has hampered smooth muscle research. In order to circumvent this problem, we have developed a novel organ-cultured bladder smooth muscle tissue that maintains viability and the contractile phenotype and allows introduction of siRNA. The first aim of this proposal will be to continue the development of this organ-cultured preparation and demonstrate the utility of siRNA knock-down. The second aim will test the hypothesis that protein kinase C is an integral part of the signaling in the regulation of bladder smooth muscle contraction and that specific isoforms of protein kinase C are involved in specific steps in this signaling. We will also test the hypothesis that protein kinase C affects the activity of Rho kinase. The third aim will examine the role of Rho kinase in the regulation of bladder smooth muscle contraction. We will test the hypothesis that a constitutively active, stretch-dependent Rho kinase sets the basal tone in bladder smooth muscle and that a second Rho kinase is involved in the regulation of MLC phosphatase activity. These studies will allow us to develop a detailed, mechanistic model of bladder smooth muscle regulation which we will use to construct studies aimed at understanding the changes in regulation that occur in disease states such as BPH.