Nitrooleate Mediates Nitric Oxide Synthase Activation in Endothelial Cells

Nitrated lipids such as nitrooleate (OLA-NO₂) can act as endogenous peroxisome proliferator-activated receptor gamma (PPARγ) ligands to exert vascular protective effects. However, the molecular mechanisms regarding nitric oxide (NO) production and its regulation are not fully defined in the vasculature. Here, we show that OLA-NO₂ increased endothelial NO release by modulating activation of endothelial nitric oxide synthase (eNOS) in endothelial cells. Treatment with OLA-NO₂ (3 μM) increased NO release in a time-dependent manner. OLA-NO₂ decreased protein expression of eNOS and caveolin-1 (Cav-1) but increased heat shock protein 90 (Hsp90) expression. Immunoprecipitation analysis confirmed that OLA-NO₂ replaced eNOS/Cav-1 with eNOS/Hsp90 interaction, resulting in increasing eNOS activity. OLA-NO₂ also induced eNOS phosphorylation at Ser633 and Ser1177 and eNOS dephosphorylation at Ser113 and Thr495. In addition, OLA-NO₂ induced phosphorylation of Akt and extracellular signal-regulated protein kinase (ERK1/2), which might contribute to eNOS activation. Collectively, these results substantiate a new functional role for nitrated fatty acid, demonstrating that OLA-NO₂ exerts vascular protective effects by increasing NO bioavailability through eNOS phosphorylation/dephosphorylation and interaction with associated proteins such as Hsp90 and Cav-1.