Loss of the semi-selective endothelial cell barrier increases vascular permeability, produces life-threatening pulmonary edema, and is a cardinal feature of inflammatory lung injury. The regulatory mechanisms underlying endothelial cell barrier dysfunction, however, are poorly understood. Our studies support our original working model that barrier dysfunction evoked by the multifunctional serine protease, thrombin, results from endothelial cell contraction, gap formation, and increased paracellular fluid and proteins transport. Our data indicate that thrombin-induced endothelial contractile events are critically dependent upon activation of a novel, high molecular weight, Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). We have recently cloned this unique non-muscle MLCK isoform and hypothesize that this enzyme provides the molecular machinery for force generation and endothelial cell barrier dysfunction. In SA#1 we will utilize standard molecular biological techniques to express and purify endothelial cell MLCK in order to more closely examine its enzymatic characteristics. Our findings indicate thrombin-mediated MLCK activation, MLCK phosphorylation and barrier function are under key regulation by cAMP-dependent protein kinase A and protein kinase C, with possibly contributory regulation by Ca2+ and calmodulin availability) are poorly understood, SA#2 will explore the role of MLCK phosphorylation/dephosphorylation in the regulation of MLCK activities. Finally, both the endothelial cell and smooth muscle MLCK gene contain specific domains whose functions is likely involved in contractile protein binding and possibly myosin filament stabilization. Based upon our preliminary characterization of a functional complex of MLCK and MLCK- binding proteins, SA#3 will identify relevant contractile and signaling effectors which bind MLCK, and determine both the sites and functional consequence of MLCK binding. These studies will define the biochemical basis of endothelial cell actomyosin activation and elucidate the role of contractile proteins in control of barrier function. This information may mead to novel therapies designed to preserve the endothelial cell barrier, reduced alveolar flooding, and limit the patient morbidity characteristic of acute lung injury syndromes.
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