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.
Thomas
Simmy
S
Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Kotamraju
Srigiridhar
S
Zielonka
Jacek
J
Harder
David R
DR
Kalyanaraman
B
B
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D004790:Enzyme Induction; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D046988:Proteasome Endopeptidase Complex / Q000096:biosynthesis* / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D046988:Proteasome Endopeptidase Complex