Understanding the physiological mechanism of the regulation of smooth muscle contraction and relaxation is essential for determining the mechanism responsible for pathological disorders of airway disease. It is well accepted that the initiation of smooth muscle contraction is regulated by Ca2+-dependent phosphorylation of myosin by myosin light chain kinase. However, the mechanism of the subsequent tension maintenance state, called the latch state, is not well understood. Several hypotheses have been proposed to account for the latch state. Caldesmon has been proposed to be responsible for the latch mechanism because it inhibits actomyosin ATPase activity although the mechanism by which caldesmon inhibits the ATPase is not understood. Caldesmon is the major calmodulin binding protein in smooth muscle and also binds to actin, myosin and tropomyosin. Caldesmon function is also modified by phosphorylation. We recently identified that the protein kinase responsible for the phosphorylation is Ca2+ calmodulin dependent protein kinase II (CaM PKII). Although this class of kinase has been identified from other tissues, CaM PKII has not been purified from smooth muscle and the role of this kinase on the regulation of smooth muscle contraction is not known. Recently, a troponin T like protein, called calponin, was isolated from smooth muscle and it was found that this protein inhibits actomyosin like caldesmon does. The proposed projects study the role of caldesmon, calponin and CaM PKII on the regulation of smooth muscle contraction to clarify the complex mechanism of regulation of smooth muscle. Furthermore, the proposed projects study the alteration of the properties of contractile proteins as well as regulatory proteins in pathological airway smooth muscle. The experiments will be directed: 1) To study the regulatory mechanism of caldesmon function; A) Identification of phosphorylation sites which affect caldesmon function. B) To study the functional sites of caldesmon using monoclonal antibodies. C) To study the effects of caldesmon on myosin filament formation and myosin conformation. 2) To study the role of calponin on the contractile apparatus. 3) To study the subcellular localization of CaM PKII isozymes as well as caldesmon. 4) To study the structure and function of smooth muscle CaM PKII. 5) To study the alteration of smooth muscle contractile apparatus and its regulatory system in the pathological airway smooth muscle. The proposed studies will use techniques of protein biochemistry, immunochemistry, microscopy and molecular cloning.
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