Detrusor Overactivity and Bladder Smooth Muscle Dysfunction

描述(由申请人提供)： 逼尿肌过度活动(DO)是一种衰弱的下尿路疾病，对男性和女性的生活质量都有严重影响。尽管其发病率相当高，但膀胱过度活动的病理生理学仍不清楚，反映了对排尿控制机制的不完全了解，特别是关于膀胱平滑肌的调节。我们先前已经证实，膀胱平滑肌小窝是一种特殊的膜域，调节各种信号分子，调节对几种生理刺激的收缩反应，并在DO动物模型中发生显著变化。此外，小窝的耗尽导致表型类似于在DO动物身上观察到的模式。这些动物模型表现出夸大的膀胱自发活动和小窝介导的嘌呤能信号调节受损。基于这些发现，我们将研究嘌呤能系统中的一种新成分，该成分在小窝失调时变得突出。我们认为，膀胱平滑肌细胞通过缝隙连接蛋白半微管释放ATP及其对嘌呤能受体的自分泌/旁分泌激活受小窝的调节；此外，小窝的丧失对这一过程的调节增加了ATP的释放，并促进了嘌呤能受体的激活，导致逼尿肌过度活动。在特定的目标1中，我们将使用体外功能分析和蛋白质相互作用的评估来确定在BSM中，嘌呤介导的膀胱功能调节是否受到小凹的调节。特定目标2将使用基于细胞的分析和成像方法来确定BSM释放ATP的机制。在特定的目标3中，我们将证明连接蛋白介导的ATP释放是由小凹调节的。这种可能性将通过形态、功能分析、基因/蛋白质表达和共聚焦显微镜来解决。最后，利用分子干预，我们将证明，通过重新建立连接蛋白依赖的ATP释放和嘌呤能受体信号的小窝介导的调节，可以部分改善逼尿肌过度活动。通过恢复小窝蛋白表达来逆转异常表型将通过全面的体内和体外功能评估进行评估，并辅之以细胞和分子研究。新提出的连接蛋白依赖的嘌呤能信号转导由小凹调节的新范式挑战了目前关于膀胱平滑肌功能的概念。这个项目不仅致力于通过阐明新的调节途径来促进我们对BSM功能的理解，而且还致力于促进膀胱功能障碍的病理生理学研究。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.