Regulation of bladder smooth muscle contraction and a new organ culture model to study bladder smooth muscle biology

It is well accepted that phosphorylation of the 20 kDa regulatory myosin light chain (MLC) catalyzed by the MLC kinase and dephosphorylation catalyzed by the MLC phosphatase plays a primary role in the regulation of smooth muscle contraction and relaxation. Inhibition of MLC phosphatase causes a net increase in MLC phosphorylation levels. The goal of this dissertation was to clarify the signaling pathways in intact bladder smooth muscle, especially the roles of protein kinase C (PKC) and Rho kinase (ROCK) and their downstream effectors in regulating MLC phosphatase activity and force during the phasic and sustained phases of bladder smooth muscle agonist stimulated contraction and stretch-induced basal tone. To achieve these goals, the studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of PKCpotentiated PP1 inhibitory protein of 17 kDa (Thr38-CPI-17) and myosin phosphatase targeting subunit (Thr696/Thr85O-MYPT1) were measured at different time points during carbachol and phorbol dibutyrate stimulation and at different degrees of stretch using site specific antibodies. By removing Ca2+ during stretch, the significance of Ca2+ signaling in the stretch-induced regulation was also studied in this dissertation.;Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In addition ROCK phosphorylates both Thr850MYPT1 and CPI-17, partly through cross-talk with the PKC pathway. Secondly, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state. Lastly, our results show that stretch-induced force generation in bladder smooth muscle is regulated by an increase in MLC phosphorylation through a stretch-induced influx of extracellular Ca2+ and an inhibition of MLC phosphatase activity.;This dissertation also describes the development and characterization of a novel bladder smooth muscle organ culture model. The viability of this model was tested by its contractility and smooth muscle specific protein expression following 9 days of culture. Our results suggest that this novel organ culture model maintains the contractile phenotype of smooth muscle for up to 9 days. Therefore this organ culture model provides a new and useful method to study bladder smooth muscle biology with preserved contractility and smooth muscle phenotype.