Abstract 3633: Flow-induced Dilation is Mediated by Akt-dependent Activation of eNOS-derived H2O2 in Mouse Cerebral Arteries
Introduction. Endothelial nitric oxide synthase (eNOS) produces superoxide leading to H2O2-dependent dilations to acetylcholine (ACh) in isolated mouse cerebral and human coronary arteries. Akt increases eNOS activity responsible for flow-mediated dilation (FMD). It is unknown, however, if endogenous H2O2 mediates FMD.
Hypothesis. Akt-dependent activation of eNOS leads to the production of functionally relevant levels of H2O2 responsible for FMD.
Methods. Cerebral arteries (100 to 150 μm in diameter) were isolated from 12±2 week-old C57Bl/6 male mice. FMD (0 to 10 μl/min, 2-mu;l step-increase at constant internal pressure of 60 mm Hg; shear stress = 0 to 50 dyn/cm2) were induced in vessels pre-constricted with phenylephrine (30 μM) at a pO2 value of 150 mm Hg using a gas mixture of 12% O2, 5% CO2, 83% N2. Simultaneously to diameter acquisition, H2O2 or NO production was detected by the fluorescent dyes CMH2CFDA or DAF-2, respectively. Results are expressed as mean±SEM of n = 6 to 8 mice per experiment.
Results. FMD (at 10 μl/min, 25±3% of maximal diameter) were reduced (P<0.05) by endothelial removal (6±1%) or eNOS inhibition with N-nitro-L-arginine (L-NNA; 11±1%), but not by the specific NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (PTIO; 24±3%). Likewise, FMD was not associated with a rise in NO-associated fluorescence, while addition of PEG-catalase and silver diethyldithio-carbamate (DETC; superoxide dismutase inhibitor) reduced (P<0.05) FMD to 10±2% and 15±1%, respectively, suggestive of the implication of H2O2 in FMD. Accordingly, a rise in H2O2-associated fluorescence (+133±19 a.u.) was observed simultaneously to FMD, and was reduced by L-NNA, PEG-catalase and DETC (+55±10, +64±4 and +50±10 a.u., respectively; P<0.05). Furthermore, specific inhibition of Akt by triciribine (1 μM) prevented FMD together with H2O2-associated rise in fluorescence (3±1% and +23±4 a.u., respectively, P<0.05). Triciribine, however, neither limited ACh-induced dilation nor the rise in H2O2-associated fluorescence.
Conclusion. In C57Bl/6 mouse cerebral arteries, Akt-dependent activation of eNOS-derived H2O2 accounts for flow-mediated dilation. This suggests a physiological role for eNOS-derived free radicals.