Loss of endothelial barrier function is important in the development of Adult Respiratory Distress Syndrome. The transcellular transport of albumin and other macromolecules via endothelial caveolae is a factor regulating endothelial barrier function. Using pulmonary microvascular endothelial cells, we have identified specific interactions between caveolin-1 and Src-family tyrosine kinases that may regulate albumin uptake and its transport via caveolae. The primary objective of the proposed investigation is to address the roles of caveolin-1 and Src in endocytosis and regulation of endothelial barrier function. Caveolin-1 and Src knockout mice will be used to investigate (1) the function of caveolin-1 in internalization of caveolae and transendothelial transport in pulmonary microvascular endothelial cells and (2) the role of dynamin in mediating fission of caveolae from the membrane and transcellular albumin permeability in endothelial cells. Our hypothesis is that Src-dependent phosphorylation of caveolin-1 and dynamin is essential for formation of caveolae and their scission from the membrane. We will measure vessel wall albumin permeability in the mouse lung preparation from caveolin-1 knockout mice. Mutant caveolin-1, Src, and dynamin cDNA constructs will be used in cell culture experiments to examine the role of Src-dependent interactions in regulating caveolin-1 and dynamin functions. Cholera toxin subunit B and albumin endocytosis and transcellular transport will be determined by electron microscopy, confocal microscopy, and measurement of iodinated tracers in cultured cells. Molecular-genetic and biochemical strategies will be used to elucidate the domain-specific and phosphorylation-dependent interactions between caveolin-1, Src, and dynamin. Thus, these studies will elucidate the Src-dependent signaling mechanisms that regulate endocytosis and transport across the pulmonary vascular endothelial barrier.
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