Ceramide‐induced disruption of endothelial nitric oxide synthase dimerization in bovine aortic endothelial cells (BAECs) is not secondary to peroxynitrite formation

Abstract

We have used pharmacological and genetic approaches in mice, arteries, and/or BAECs to show that ceramide impairs arterial function by decreasing eNOS activity in a tissue autonomous manner. eNOS dimerization is required for optimal eNOS enzymatic activity. Peroxynitrite uncouples eNOS by disrupting protein dimer formation.We hypothesized that ceramide‐induced superoxide anion (O2•−) generation disrupts eNOS dimer formation as a result of increased peroxynitrite accumulation. BAECs were treated for 3 h with: vehicle (V); 500 μM palmitate (P); or P + the ceramide synthesis inhibitor myriocin (M, 10 μM). Relative to V, P increased: ceramide accumulation (HPLC) by 1.6±0.1‐fold (n=6); reactive oxygen species (ROS) generation (DCFDA fluorescence) by 3.2±0.2‐fold (n=31); and O2• − production (ESR) by 1.27±0.08‐fold (n=11, all p<0.05). These changes were blunted (p<0.05) in BAECs treated with P + M. Three estimates of nitrotyrosine formation [i.e., immunoblotting (n=14–18), ELISA (n=16), and immunostaining (n=12)] showed no evidence for P‐evoked peroxynitrite accumulation. Despite the lack of P‐evoked peroxynitrite formation, insulin (n=10) and VEGF (n=10)‐mediated increases (p<0.05) in p‐eNOS at S1177 and S617, and the eNOS monomer : dimer ratio were prevented (p<0.05) by P, but were restored by co‐incubation of P + M. These findings indicate that ceramide‐evoked disruption of agonist‐mediated eNOS phosphorylation and dimerization occurs via a peroxynitrite – independent mechanism. NIHR15HL091493, ADA7‐08‐RA‐164