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.