FILAMIN TRANSLOCATION IS AN EARLY ENDOTHELIAL-CELL INFLAMMATORY RESPONSE TO BRADYKININ - REGULATION BY CALCIUM, PROTEIN-KINASES, AND PROTEIN PHOSPHATASES

Endothelial cell (EC) cytoskeletal proteins are one of the earliest pr imary targets of second messenger cascades generated in response to in flammatory agonists. Actin binding proteins, by modulating actin gelat ion-solation state and membrane-cytoskeleton interactions, in part reg ulate cell motility and cell-cell apposition. This in turn can also mo dulate interendothelial junctional diameter and permeability. Nonmuscl e filamin (ABP-280), a dimeric actin-crosslinking protein, promotes or thogonal branching of F-actin and links micro filaments to membrane gl ycoproteins. In the present study, immunoblot analysis demonstrates th at filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-con fluency, Both bradykinin and ionomycin cause filamin redistribution fr om the peripheral cell border to the cytosol of confluent EC. Forskoli n, an activator of adenylate cyclase, blocks filamin translocation. Br adykinin activation of EC is not accompanied by significant proteolyti c cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of cal cium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP- dependent protein kinase causes translocation of filamin in unstimulat ed cells. Calyculin A, an inhibitor of protein phosphatases, also caus es translocation of filamin in the absence of an inflammatory agent. M L-7, an inhibitor of myosin light chain kinase and phorbol myristate a cetate, an activator of protein kinase C, do not cause filamin movemen t into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocatio n is initiated by the calcium/calmodulin-dependent protein kinase wher eas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeab ility by increasing cytoplasmic calcium, which disassembles the microf ilament dense peripheral band by releasing filamin from F-actin. (C) 1 996 Wiley-Liss, Inc.