The overall aim will be to investigate the mechanism of protein dephosphorylation involved in the regulation of smooth muscle contraction. Details of how myosin light chain kinase phosphorylates myosin light chains and brings about activation of smooth muscle contraction have been well characterized. Also, cAMP-dependent protein kinase is known to both phosphorylate and inactivate myosin light chain kinase, as well as to cause relaxation of smooth muscle. Specific phosphatases which dephosphorylate myosin light chains or myosin light chain kinase thus would be expected to have opposite effects on tension generation - relaxation in the case of the former, and activation in the case of the later. However, we have previously shown that smooth muscle phosphatase IV actually dephosphorylates myosin light chains and increases tension at submaximal Ca2+. This research is designed to test the ability of different phosphatases to dephosphorylate myosin light chains and myosin light chain kinase in skinned smooth muscle cells and to analyse their effect on the physiological variables, contraction and relaxation to determine which phosphatase is most likely to be operating in vivo. Inhibitors to type 1 phosphatase will be used to determine which types of phosphatase are involved in the dephosphorylation of myosin light chains or myosin light chain kinase. These inhibitors are known to inhibit phosphatase activity in vitro and will be used to inhibit type 1 phosphatase activity in skinned smooth muscle cells. Their effect on protein phosphorylation and tension will be characterized. A more cautious approach of using antibodies which are known to inhibit phosphatase activity """"""""in vitro"""""""" will be used to further characterize the phosphatase regulation. Results from these studies should give us a better understanding of the role phosphatases play in the regulation of contraction and relaxation in smooth muscle and important information regarding the type of phosphatases involved. This information is crucial for a complete understanding of the mechanisms involved in the regulation of smooth muscle contraction in vivo.
