and Specific Aims.) A key vascular endothelial cell (EC) function is the regulation of exchanges across the capillary wall between circulating blood and the interstitial fluid. EC barrier dysfunction is an important hallmark of inflammation and results in tissue edema and organ dysfunction and is central development of life-threatening pulmonary edema, vascular ischemia and atherosclerosis. Barrier dysfunction induced by the multifunctional serine protease, thrombin, involves contraction-induced intercellular gap formation linked to modulation of actin polymerization and myosin phosphorylation. Thrombin-induced EC contractile events were dependent upon G protein-coupled phosphoinositide metabolism resulting in increased [Ca2+i] and protein kinase C (PKC) activation. Direct PKC activation by phorbol esters produced gap formation and EC barrier dysfunction, whereas agents which increased cAMP were found to protect EC against thrombin-induced gap formation and barrier dysfunction. The mechanisms by which both PKC and cAMP-dependent protein kinase A (PKA) regulate actomyosin interactions are unknown. Preliminary data indicate both PKC and PKA modulate myosin light chain kinase activity and that EC MLCK has specific biochemical and regulatory features which indicate it is distinct from MLCKs derived from other tissues. The application proposes to examine early, intermediate and terminal activation sequences involved in the phosphorylation of myosin light chains (MLC20) and the actin binding protein, caldesmon77, resulting in productive actomyosin interaction, cellular contraction, gap formation, and loss of EC barrier function.
The Specific Aims are: 1) to investigate potential mechanisms of cAMP-dependent protein kinase A (PKA)-mediated barrier protection; 2) to characterize protein kinase C-mediated regulation of actomyosin interactions; 3) to examine the role of Ca2+/calmodulin/phosphocalmodulin in regulation of MLCK activity; and 4) to clone, sequence, and purify myosin light chain kinase (MLCK) from vascular EC. Because preservation of a cellular barrier to plasma proteins is vital, these studies which define the biochemical basis of EC actomyosin activation may further enhance understanding of the role of contractile proteins in control of EC barrier function.
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