A major feature of acute lung injury is pulmonary edema resulting from an increase in permeability of the microvasculature of the lung. The fluid accumulation is widely believed to result from active opening of junctions between endothelial cells. The primary HYPOTHESIS that serves as a foundation for the proposed work states that exposure to inflammatory mediators activates the endothelial cell actomyosin based contractile system. This hypothesis dictates that phosphorylation of myosin II regulatory light chains catalyzed by myosin light chain kinase in parallel with the assembly of the filamentous cytoskeleton (actin, associated myosin II) is essential for activation of endothelial cell contraction. The functional correlate of the activated actomyosin based contractile system is development of endothelial cell isometric tension. This hypothesis will be tested utilizing intact and saponin permeabilized preparations to gain a detailed understanding of the cascade of events leading to opening of gaps between endothelial cells of the vasculature as it occurs in situ. We will correlate the development and regulation of endothelial cell isometric tension with changes in the cytoskeletal proteins actin, myosin IIA, myosin IIB and myosin light chain kinase. The proposed studies will: 1) characterize the role of phosphorylation of myosin II regulatory light chains in basal and agonist stimulated isometric contraction; 2) establish the contributions of site specific phosphorylations i.e. monophosphorylation (Serl9) versus diphosphorylation (Serl9/Thrl8) of myosin II regulatory light chains in the development and maintenance of endothelial cell contraction; 3) characterize endothelial cell myosin II heavy chain isoforms and 4) biochemically and physiologically characterize a novel high molecular weight myosin light chain kinase recently identified in endothelial cells. Results from these studies will provide useful new information about the cellular and biochemical mechanisms regulating endothelial cell contraction and increased vascular permeability.
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