OXIDANT-SENSITIVE AND PHOSPHORYLATION-DEPENDENT ACTIVATION OF NF-KAPPA-B AND AP-1 IN ENDOTHELIAL-CELLS

Relatively low concentrations of reactive oxygen cause reversible alte rations of endothelial cell signal transduction and gene transcription . The hypothesis that low levels of oxidant stress activate retention of trans-acting proteins in the nucleus was investigated by determinin g time and dose requirements for oxidant-stimulated nuclear protein bi nding to consensus DNA sequences for nuclear factor (NF)-kappa B or ac tivator protein 1 (AP-1). Nuclear proteins were extracted from low pas sage porcine aortic endothelial cells 15 min to 24 h after addition of increasing concentrations of H2O2 Electrophoretic mobility shift assa ys demonstrated that protein binding to NF-kappa B and AP-1 sequences increases over 1-2 h after stress relative to time-matched controls an d resolves by 24 h. The selective protein kinase C inhibitor, calphost in C, prevents similar to 30% of this increase. Inhibition of tyrosine kinase activity by herbimycin A (5 mu M) completely inhibits the resp onse to H2O2. Exposure of intact cells to H2O2 increases substrate pho sphorylation in pp60(src) immunoprecipitates. The activity of pp60(src ) in immunoprecipitates from control cells or of recombinant pp60(src) increases after in vitro addition of H2O2. H2O2-stimulated pp60(src) activity is reduced by pretreatment of the enzyme preparation with N-a cetylcysteine. These data indicate that oxidants increase nuclear leve ls of trans-acting factors in endothelial cells and that these increas es require oxidant-sensitive changes in both tyrosine and serine/threo nine phosphorylations.