HYDROGEN PEROXIDE-INDUCED CYTOSKELETAL REARRANGEMENT IN CULTURED PULMONARY ENDOTHELIAL-CELLS

Although the signaling pathways leading to hydrogen peroxide (H2O2)-in duced endothelial monolayer permeability remain ambiguous, cytoskeleta l proteins are known to be essential for maintaining endothelial integ rity and regulating solute flux through the monolayer. We have recentl y demonstrated that thrombin-induced actin reorganization in bovine pu lmonary artery endothelial cells (BPAEC) requires activation of both m yosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a lime- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP , but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 an d calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to th e cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2? transiently attenuated PKC activity but s lightly increased the phosphorylation of the prominent PKC substrate a nd actin-binding protein, myristoylated alanine-rich C kinase substrat e (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to b elow basal levels by 30 min. On the other hand, H2O2 induced a time-an d dose-dependent phosphorylation of myosin light chains which was elim inated by both MLCK and PKC inhibitors. These data suggest that MLCK c ontributes to H2O2-induced myosin light chain phosphorylation and acti n rearrangement and that PKC may play a permissive role. Neither of th ese enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton. (C) 1998 Wiley-Liss, Inc.