Premium
The interplay between eNOS palmitoylation and phosphorylation: the triacsin C effect (1075.9)
Author(s) -
Blakeman Nina,
Weis Margaret
Publication year - 2014
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.28.1_supplement.1075.9
Subject(s) - enos , palmitoylation , phosphorylation , serine , chemistry , thioredoxin , nitric oxide , biochemistry , medicine , nitric oxide synthase , oxidative stress , cysteine , enzyme , organic chemistry
Decreased endothelial‐derived (NO) is central to the development of vascular disease. Earlier, we showed that triacsin C (TriC), a long chain fatty acyl‐CoA synthetase inhibitor, increased NO in cultured endothelial cells, and decreased endothelial nitric oxide synthase (eNOS) palmitoylation. Inhibiting eNOS palmitoylation suggests a decrease in NO production; however, eNOS regulation is complex. This leads us to hypothesize that the interplay between eNOS palmitoylation and phosphorylation regulates a phosphoryl switch increasing NO synthesis. After incubating bEnd.3 cells with TriC for 24 hours, we measured the phosphorylation status of key regulatory residues of eNOS. The mean relative density of each site (p‐eNOS/total eNOS) is summarized:Vehicle N=3 TriC N=3p‐serine 1177 0.85 ± 0.07 0.39 ± 0.03 p< 0.0001p‐serine 635 0.12 ± 0.02 0.10 ± 0.02 nsp‐serine 617 0.04 ± 0.01 0.05 ± 0.01 nsp‐threonine 495 0.60 ± 0.06 0.70 ± 0.09 nsp‐serine 116 0.17 ± 0.03 0.07 ± 0.01 p<0.0157Phosphorylation can mediate eNOS uncoupling and superoxide generation. To address this concern, we evaluated TriC’s effect to alter lipid peroxidation or induce the thioredoxin/thioredoxin reductase (Trx/TrxR) system. TriC significantly decreased thiobarbituric acid (TBA)‐malondialdehyde (MDA) adduct formation. Although not an indication of their activity level, no significant change to Trx/TrxR protein expression was seen.Treatment Trx TrxRVehicle 0.05 ± 0.01 0.05 ± 0.01TriC 0.06 ± 0.01 0.06 ± 0.02Treatment MDA Concentration (µM)Vehicle 100.0 ± 6.1TriC 50.9 ± 4.7 ********=p<0.0001; n=9 These results support our hypothesis that TriC regulates an eNOS phosphoryl switch increasing NO synthesis, without increasing oxidative stress.