Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability

The actomyosin complex is the major cytoskeletal component that controls ce ll contraction. In this study, we investigated the effects of actomyosin in teraction on endothelial barrier function and gap formation. Activated myos in light chain kinase (MLCK) protein was transferred into coronary venular endothelial cell (CVEC) monolayers. Uptake of the activated protein resulte d in a significant shift in myosin light chain (MLC) from an unphosphorylat ed to a diphosphorylated form. In addition, MLCK induced a hyperpermeabilit y response of the monolayer as measured by albumin transendothelial flux. M icroscopic examination of MLCK-treated CVECs revealed widespread gap format ion in the monolayer, loss of peripheral beta-catenin, and increases in act in stress fibers. Inhibition of all of the above responses by a specific ML CK inhibitor suggests they are the direct result of exogenously added MLCK. These data suggest that activation of MLCK in CVECs causes phosphorylation of MLC and contraction of CVECs, resulting in gap formation and concomitan t increases in permeability. This study uses a novel technique to measure t he effects of an activated kinase on both its substrate and cellular morpho logy and function through direct transference into endothelial cells.