Detrusor Overactivity and Bladder Smooth Muscle Dysfunction

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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.