Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca2+-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca2+/calmodulin- dependent protein kinase II (CaM kinase II) activities in a Ca2+- and time-dependent manner in bovine pulmon ary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increase s in monolayer permeability to albumin and decreases in transendothelial el ectrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin- induced myosin light chain phosphory lation was unaffected. Furthermore, a cell-permeable N-myristoylated synthe tic filamin peptide (containing the COOH-terminal CaM kinase II phosphoryla tion site) attenuated both thrombin-induced filamin phosphorylation and dec reases in TER. Together, these studies indicate that CaM kinase II activati on and filamin phosphorylation may participate in thrombin-induced cytoskel etal reorganization and endothelial barrier dysfunction.