Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II.
Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca(2+)-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca(2+)- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical 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 phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.
Borbiev
T
T
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Verin
A D
AD
Shi
S
S
Liu
F
F
Garcia
J G
JG
D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D003285:Contractile Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D064448:Filamins; D008840:Microfilament Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011651:Pulmonary Artery; D013347:Subcellular Fractions / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003285:Contractile Proteins; D004791:Enzyme Inhibitors; D064448:Filamins; D008840:Microfilament Proteins; D018994:Myosin Light Chains; D013449:Sulfonamides; D013917:Thrombin; C072105:KN 93; D000096926:Benzenesulfonamides