Oxidant-sensitive protein phosphorylation in endothelial cells.
Reactive oxygen is an important regulator of vascular cell biology; however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal transduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H2O2 to examine oxidant-sensitive changes in phosphorylation state. Addition of H2O2 increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intracellular free Ca2+ in endothelial cells did not change following addition of 100 microM H2O2, nor did the ability of the cells to respond to bradykinin. H2O2-induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H-8, staurosporin, or calphostin c). Two-dimensional analysis of phosphoproteins from homogenates of 32P-labeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H2O2. Simultaneous addition of 10 eta M PMA and 50 microM H2O2 decreased the oxidant-stimulated phosphorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H2O2-sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase.
Barchowsky
A
A
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755.
Williams
M E
ME
Benz
C C
CC
Chepenik
K P
KP
D000704:Analysis of Variance; D000818:Animals; D001011:Aorta; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D016877:Oxidants; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; C049925:fura-2-am; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2