Hydrogen peroxide induces nitric oxide and proteosome activity in endothelial cells: A bell-shaped signaling response

We investigated nitric oxide ((NO)-N-.)-mediated proteosomal activation in bovine aortic endothelial cells (BAEC) treated with varying fluxes of hydrogen peroxide (H2O2) generated from glucose/glucose oxidase (Glu/GO). Results revealed a bell-shaped (NO)-N-. signaling response in BAEC treated with Glu/GO (2-20 mU/ml). GO treatment (2 mU/ml) enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and (NO)-N-. release in BAEC. With increasing GO concentrations, phospho eNOS and (NO)-N-. levels decreased. Bell-shaped responses in proteasomal function and (NO)-N-. induction were observed in BAEC treated with varying levels of GO (2-10 mU/ml). Proteosomal activation induced in GO-treated BAEC was inhibited by N-omega-nitro-L-arginine-methyl ester pretreatment, suggesting that (NO)-N-. mediates proteasomal activation. Intracellular (NO)-N-. induced by H2O2 was detected by isolating the 4,5-diaminoflourescein (DAF-2)/(NO)-N-./O-2-derived green fluorescent product using the high-performance liquid chromatography-fluorescence technique, a more rigorous and quantitative methodology for detecting the DAF-2/(NO)-N-./O-2 reaction product. Finally, the relationships between H2O2 flux, proteasomal activation/inactivation, endothelial cell survival, and apoptosis are discussed. (c) 2007 Elsevier Inc. All rights reserved.