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

Tanchun Wang

doi:10.17918/00008197

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.

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

Files and links (1)

Abstract

Metrics

Details

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

Files and links (1)

Abstract

Metrics

Details

Drexel University Social media