| Abstrakt: |
Moy, Alan B., Ken Blackwell, Ning Wang, Kari Haxhinasto, Mary K. Kasiske, James Bodmer, Gina Reyes, and Anthony English. Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics. Am J Physiol Lung Cell Mol Physiol 287: L153-LI67, 2004. First published March 5, 2004; 10.1152/ajplung.00292.2003.--The mechanisms of phorbol ester- and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibu- tyrate (PDBU) mediated slow force velocity and less force than thrombin. Taxol did not attenuate PDBU-mediated tension, while it reversed nocodazole-mediated tension. PDBU-mediated tension was not affected by acrylamide; PDBU increased cell stiffness and produced greater declines in transendothelial resistance (TER) than acrylamide. Thus PDBU caused a net increase in tension and did not unload microtubule or intermediate filaments. Microfilament remod- eling, determined on the basis of immunocytochemistry and actin solubility, lacked the sensitivity and specificity to predict actin- dependent mechanical properties. Thrombin increased myosin light chain (MLC) kinase site-specific MLC phosphorylation, according to peptide map analysis, whereas PDBU did not increase PKC-specific MLC phosphorylation. The initial PDBU-mediated tension development temporally correlated with PDBU-mediated decline in TER and increased low-molecular-weight caldesmon (/-CAD) phosphorylation. PDBU-mediated tension development and decreases in TER were associated with a temporal loss of endothelial cell-matrix adhesion, based on a numerical model of TER. Although, on the basis of immunocytochemistry, thrombin-mediated tension was associated with actin insolubility, actin reorganization, and gap formation, these changes did not predict thrombin-mediated gap formation, based on TER and time-lapse differential interference contrast microscopy. These data suggest that PDBU may disrupt endothelial barrier function through loss of cell-matrix adhesion through /-CaD-dependent actin contraction. phorbol ester dibutyrate; porcine pulmonary artery endothelial cells; myosin light chain; low-molecular-weight caldesmon [ABSTRACT FROM AUTHOR] |
