Metabolites of the phospholipase D pathway regulate H2O2-induced filamin redistribution in endothelial cells.
Hypoxia/reoxygenation injury to cultured endothelial cells results in cytoskeletal rearrangement and second messenger activation related to increased monolayer junctional permeability. Cytoskeletal rearrangement by reactive oxygen species may be related to specific activation of the phospholipase D (PLD) pathway. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) or metabolites of the PLD pathway for 1-60 min. Changes in cAMP levels, Ca2+ levels, PIP2 production, filamin distribution, and intercellular gap formation are then quantitated. H2O2-induced filamin translocation from the membrane to the cytosol occurs after 1-min H2O2 treatment, while intercellular gap formation significantly increases after 15 min. H2O2 and phosphatidic acid exposure rapidly decrease intracellular cAMP levels, while increasing PIP2 levels in a Ca2+-independent manner. H2O2-induced cAMP decreases are prevented by inhibiting phospholipase D. H2O2-induced cytoskeletal changes are prevented by inhibiting phospholipase D, phosphatidylinositol-4-phosphate kinase, phosphoinositide turnover, or by adding a synthetic peptide that binds PIP2. These data indicate that metabolites produced downstream of H2O2-induced PLD activation may mediate filamin redistribution and F-actin rearrangement.
Hastie
L E
LE
Microvascular Research Laboratory, Biological Sciences and Photonics Center, Boston University, Massachusetts 02215, USA.
Patton
W F
WF
Hechtman
H B
HB
Shepro
D
D
D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000032:analysis / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D064448:Filamins; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism*; D010539:Permeability; D010712:Phosphatidic Acids / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014471:Umbilical Veins
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D010712:Phosphatidic Acids; D018129:Phosphatidylinositol Phosphates; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D010739:Phospholipase D; D002118:Calcium