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Heatshock protein 90 regulates endothelial nitric oxide synthase generation of nitric oxide and superoxide anion



Heatshock protein 90 regulates endothelial nitric oxide synthase generation of nitric oxide and superoxide anion



FASEB Journal 15(5): A781



The balance of NO and O2- plays a critical role in vascular biology. The association of Hsp90 with eNOS in endothelial cells (EC) increases NO generation. The effects of Hsp90 on EC O2- generation however, remain unknown. As eNOS can generate both NO and O2- we hypothesized that Hsp90 may also modulate eNOS production of O2-. To test this hypothesis bovine coronary EC (BCEC) were pretreated with geldanamycin (GA, 10 ug/mL, 30 min) and stimulated with A23187 (5 uM). BCEC generation of NO and O2- generation were quantified by a fluorescent assay for nitrite and ferricytochrome c assay for O2-, respectively. cNOS protein-protein interactions were examined by immunoprecipitation and immunoblotting. GA significantly decreased A23187-stimulated nitrite production, an index of NO. In contrast to its inhibitory effects on NO GA significantly increased A23187-stimulated O2- generation. More importantly, this increase in A23187-stimulated O2- generation in GA-treated BCEC was inhibited by L-NAME (1 mM) suggesting that inhibiting Hsp90 activity increases O2- generation by eNOS. A23187-stimulation increases the association of Hsp90 and calmodulin (CaM) with eNOS apprx2-fold and apprx5-fold, respectively. Although GA inhibits Hsp90 activity it does not prevent Hsp90 or CaM from associating with eNOS. Pretreating BCEC with GA increases the association of Hsp90 and CaM with eNOS apprx2.0-fold and apprx6-fold, respectively. A23187-stimulation of GA-treated BCEC cultures further increases the association of Hsp90 and CaM with eNOS. These data indicate that activation of eNOS in BCEC cultures with intact Hsp90 activity increases NO production while activation of eNOS in BCEC cultures where Hsp90 activity is inhibited increases eNOS-dependent O2- production. These observations indicate that not only is Hsp90 association and conformation change with eNOS critical for NO production but also for limiting eNOS-dependent O2- generation.

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