Detrusor Overactivity and Bladder Smooth Muscle Dysfunction

描述（由申请人提供）： 逼尿肌过度活动（DO）是一种使人衰弱的下尿路疾病，对男性和女性的生活质量都有深远的影响。尽管其相当普遍，膀胱过度活动症的病理生理学仍不清楚，反映了控制排尿的机制的不完全掌握，特别是关于膀胱平滑肌的调节。我们以前建立了膀胱平滑肌小窝，专门的膜结构域，调节各种信号分子，调节收缩反应的几种生理刺激，并成为显着改变的动物模型的DO。此外，小窝的耗竭导致类似于在患有DO的动物中观察到的模式的表型。这些动物模型表现出过度的膀胱自发活动和受损的小窝介导的嘌呤能信号调节。基于这些发现，我们将研究嘌呤能系统的一个新的组成部分，成为突出的小窝失调。我们建议，ATP释放膀胱平滑肌细胞通过连接蛋白半通道和自分泌/旁分泌激活嘌呤能受体的调节小窝，此外，小窝介导的调节这一过程的损失增加ATP释放，促进嘌呤能受体激活，导致逼尿肌过度活动。在具体的目标1，我们将确定是否嘌呤介导的膀胱功能的调制是由小窝在BSM调节，使用体外功能测定和蛋白质相互作用的评估。具体目标2将使用基于细胞的测定和成像方法来确定BSM释放ATP的机制。在具体目标3中，我们将证明连接子介导的ATP释放是由小窝调节的。这种可能性将使用形态学，功能测定，基因/蛋白质表达和共聚焦显微镜来解决。最后，使用分子干预，我们将证明，逼尿肌过度活动可以改善部分通过重新建立小窝介导的调节连接子依赖的ATP释放和嘌呤能受体信号。通过恢复小窝蛋白表达来恢复异常表型将通过全面的体内和体外功能评价来评估，并得到细胞和分子研究的支持。新提出的连接子依赖性嘌呤能信号转导受小窝调节的范例挑战了目前膀胱平滑肌功能的概念。该项目致力于通过阐明新的调节途径来推进我们目前对BSM功能的理解，还包括膀胱功能障碍的病理生理学。因此，这些发现可能最终为设计更好的排尿障碍靶向药物治疗提供基础。

DESCRIPTION (provided by applicant): Detrusor overactivity (DO) is a debilitating disorder of the lower urinary tract that has a profoun impact on quality of life in both men and women. Despite its considerable prevalence, the pathophysiology of the overactive bladder is still unclear, reflecting an incomplete grasp of the mechanisms controlling micturition, particularly regarding the regulation of bladder smooth muscle. We previously established that bladder smooth muscle caveolae, specialized membrane domains that regulate a variety of signalling molecules, modulate contractile responses to several physiologic stimuli and become significantly altered in animal models of DO. Furthermore, depletion of caveolae results in a phenotype resembling the pattern observed in animals with DO. These animal models exhibit exaggerated bladder spontaneous activity and impaired caveolae-mediated regulation of purinergic signaling. Based on these findings, we will investigate a novel component of the purinergic system that becomes prominent with caveolae dysregulation. We propose that ATP release by bladder smooth muscle cells through connexin hemichannels and its autocrine/paracrine activation of purinergic receptors are regulated by caveolae; moreover, loss of caveolae-mediated regulation of this process augments ATP release and facilitates purinergic receptor activation, leading to detrusor overactivity. In specifc aim 1, we will determine whether purinergically-mediated modulation of bladder function is regulated by caveolae in BSM, using in vitro functional assays and assessment of protein interactions. Specific aim 2 will identify the mechanism by which ATP is released by BSM, using cell-based assays and imaging approaches. In Specific Aim 3, we will demonstrate that connexon-mediated ATP release is regulated by caveolae. This possibility will be addressed using morphology, functional assays, gene/protein expression and confocal microscopy. Finally, using molecular interventions, we will demonstrate that detrusor overactivity can be ameliorated in part by re-establishing caveolae-mediated regulation of connexon-dependent ATP release and purinergic receptor signaling. Reversal of the abnormal phenotype by restoring caveolin expression will be assessed by comprehensive in vivo and in vitro functional evaluation, supported by cellular and molecular studies. The proposed novel paradigm of connexon- dependent purinergic signalling regulated by caveolae challenges current concepts of bladder smooth muscle function. This project endeavours to advance our current understanding not only of BSM function by elucidating new pathways of regulation, but also the pathophysiology of bladder dysfunction. Consequently, these findings may ultimately provide the foundation for the design of better targeted pharmacotherapy for micturition disorders.