Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase

The balance of nitric oxide (·NO) and superoxide anion (O ₂^.- plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ·NO As eNOS is capable of generating both ·NO and O ₂^.- we hypothesized that Hsp90 might also mediate eNOS-dependent O₂^.- production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 μg/ml; 17.8 μM) and then stimulated with the calcium ionophore, A23187 (5 μM). GA significantly decreased A23187-stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased A23187-stimulated eNOS-dependent O₂^.- production (p < 0.001, n = 8). A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ·NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O₂^.- production.