Premium
Ceramide‐induced disruption of endothelial nitric oxide synthase dimerization in bovine aortic endothelial cells (BAECs) is not secondary to peroxynitrite formation
Author(s) -
Wilson Lloyd,
Zhang Quanjiang,
Arrant Colton,
Pettey Dix,
Kowalski Chris,
McCamey Dane,
Boehme Christoph,
Baker William,
Soorappan Rajasekaran N,
Abel E Dale,
Symons J David
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.978.16
Subject(s) - peroxynitrite , enos , ceramide , chemistry , superoxide , nitric oxide , reactive oxygen species , peroxynitrous acid , biochemistry , medicine , endocrinology , nitric oxide synthase , enzyme , apoptosis , biology , organic chemistry
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 (O 2 • − ) 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 O 2 • − 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