Oxidant-sensitive and phosphorylation-dependent activation of nf-kb and ap-1 in endothelial cells. Barchowsky, Aaron, Sara R. Munro, Salvatore J. Morana, Matthew P Vincenti, and Melinda Treadwell. Departments of Pharmacology and Toxicology and of Biochemistry, Dartmouth Medical School, Hanover, NH 03755
APStracts 2:0117L, 1995.
Relatively low concentrations of reactive oxygen cause reversible alterations 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 determining time and dose requirements for oxidant -stimulated nuclear protein binding to consensus DNA sequences for NF -kB or AP-1. Nuclear proteins were extracted from low passage porcine aortic endothelial cells 15 minutes to 24 hours after addition of increasing concentrations of H2O2. Electrophoretic mobility shift assays demonstrated that protein binding to NF-kB and AP-1 sequences increases over 1-2 hours following stress relative to time matched controls and resolves by 24 hours. The selective protein kinase c inhibitor, calphostin c, prevents approximately 30 % of this increase. Inhibition of tyrosine kinase activity by herbimycin A (5 [mu]M) completely inhibits the response to H2O2. Exposure of intact cells to H2O2 increases substrate phosphorylation in pp60src immunoprecipitates. The activity of pp60src in immunoprecipitates from control cells or of recombinant pp60src increases following in vitro addition of H2O2. H2O2-stimulated pp60src activity is reduced by pre-treatment of the enzyme preparation with N-acetylcysteine. These data indicate that oxidants increase nuclear levels of trans -acting factors in endothelial cells and that these increases require oxidant-sensitive changes in both tyrosine and serine/threonine phosphorylations. 

Received 20 May 1994; accepted in final form 22 June 1995.
APS Manuscript Number L147-4.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 18 July 1995.