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 mu M 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 P-32-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 mu M 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.