Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics.
The mechanisms of phorbol ester- and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibutyrate (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 remodeling, 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 (l-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 l-CaD-dependent actin contraction.
Moy
Alan B
AB
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, 52242, USA. alan-moy@uiowa.edu
Blackwell
Ken
K
Wang
Ning
N
Haxhinasto
Kari
K
Kasiske
Mary K
MK
Bodmer
James
J
Reyes
Gina
G
English
Anthony
A
D000199:Actins / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D007382:Intermediate Filaments / Q000187:drug effects / Q000502:physiology; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D008970:Molecular Weight; D018994:Myosin Light Chains / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011651:Pulmonary Artery / Q000378:metabolism*; D013552:Swine; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014655:Vascular Resistance / Q000187:drug effects; D014661:Vasoconstriction / Q000187:drug effects
D000199:Actins; D002148:Calmodulin-Binding Proteins; D018994:Myosin Light Chains; D015240:Phorbol 12,13-Dibutyrate; D013917:Thrombin