Excitation contraction coupling in bladder smooth muscle

描述（由申请人提供）：良性前列腺增生（BPH）的患病率随着男性年龄的增长而增加。虽然前列腺增生不会危及生命，但其症状越来越多地影响生活质量，下尿路症状包括尿频、膀胱排空不完全的感觉、尿流中断或微弱以及尿漏。膀胱的一个主要组成部分是膀胱平滑肌，它负责许多方面的排尿行为。不幸的是，我们对正常膀胱平滑肌调节的理解是不完整的，这阻碍了对病变状态下发生的变化的调查。因此，本研究的重点是通过对膀胱平滑肌兴奋-收缩耦合的详细机制理解来填补这些空白。该提案将验证的整体假设是，刺激膀胱平滑肌会激活导致收缩的信号通路，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、活化和构成活性的Rho激酶和蛋白激酶C （PKC）。该提案将验证的具体假设是，膀胱平滑肌含有一种构成性活性拉伸依赖的Rho激酶，该激酶在未受刺激的膀胱壁的肌肉细胞上设置基调。刺激膀胱平滑肌引发一系列事件，包括钙依赖的MLC磷酸化、激活的Rho激酶和PKC，这些事件通过线性和串音途径微调MLC激酶和磷酸酶作用产生的力的水平。平滑肌受细胞培养表型调节为非肌肉表型的事实阻碍了平滑肌的研究。为了避免这个问题，我们开发了一种新的器官培养膀胱平滑肌组织，它保持活力和收缩表型，并允许引入siRNA。本提案的第一个目标将是继续发展这种器官培养的制剂，并证明siRNA敲除的实用性。第二个目的将验证蛋白激酶C是膀胱平滑肌收缩调控信号传导的一个组成部分的假设，并且蛋白激酶C的特定同工型参与了该信号传导的特定步骤。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激酶在膀胱平滑肌收缩调节中的作用。我们将验证这样的假设，即一个构成性活性的、依赖拉伸的Rho激酶设定膀胱平滑肌的基底张力，第二个Rho激酶参与MLC磷酸酶活性的调节。这些研究将使我们能够建立一个详细的、机制的膀胱平滑肌调节模型，我们将用它来构建旨在理解在前列腺增生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.