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

We investigated nitric oxide (*NO)-mediated proteosomal activation in bovine aortic endothelial cells (BAEC) treated with varying fluxes of hydrogen peroxide (H(2)O(2)) generated from glucose/glucose oxidase (Glu/GO). Results revealed a bell-shaped *NO 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 release in BAEC. With increasing GO concentrations, phospho eNOS and *NO levels decreased. Bell-shaped responses in proteasomal function and *NO 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 mediates proteasomal activation. Intracellular *NO induced by H(2)O(2) was detected by isolating the 4,5-diaminoflourescein (DAF-2)/*NO/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/O(2) reaction product. Finally, the relationships between H(2)O(2) flux, proteasomal activation/inactivation, endothelial cell survival, and apoptosis are discussed.