flow-induced dilation is mediated by akt-dependent activation of endothelial nitric oxide synthase-derived hydrogen peroxide in mouse cerebral arteries

Abstract

Background and Purpose-: Endothelial nitric oxide synthase produces superoxide under physiological conditions leading to hydrogen peroxide (H2O2)-dependent dilations to acetylcholine in isolated mouse cerebral arteries. The purpose of this study was to investigate whether H2O2 was involved in flow-mediated dilation (FMD). Methods-: Cerebral arteries were isolated from 12±2-week-old C57Bl/6 male mice. FMD (0 to 10 μL/min, 2-μL step increase at constant internal pressure) was induced in vessels preconstricted with phenylephrine (30 μmol/L). Simultaneously to diameter acquisition, H2O2 or nitric oxide production was detected by the fluorescent dyes CMH2CFDA or 4,5-diaminofluorescein diacetate, respectively. Results are expressed as mean±SEM of 6 to 8 mice. Results-: FMD (at 10 μL/min, 25±3% of maximal diameter) was prevented (P<0.05) by endothelium removal (6±1%) or endothelial nitric oxide synthase inhibition with N-nitro-l-arginine (11±1%) but not by the specific nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (24±3%). Addition of PEG-catalase and silver diethyl dithio-carbamate (superoxide dismutase inhibitor) reduced (P<0.05) FMD to 10±2% and 15±1%, respectively. Simultaneously to FMD, H2O2-associated rise in fluorescence (+133±19 a.u.) was prevented by N-nitro-l-arginine, PEG-catalase, and silver diethyl dithio-carbamate (+55±10, +64±4, and +50±10 a.u., respectively; P<0.05). Inhibition of FMD by PEG-catalase was fully restored by the addition of tetrahydrobiopterin, a cofactor of endothelial nitric oxide synthase (23±3%); this functional reversal in dilation was associated with the simultaneous increase in nitric oxide-associated fluorescence (+418±58 a.u., P<0.05), which was prevented by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (+93±26 a.u.). Akt inhibition with triciribine prevented FMD and H2O2-associated rise in fluorescence (3±1% and +23±4% a.u., respectively; P<0.05), but not acetylcholine-induced dilation. Conclusion-: In healthy C57Bl/6 mouse cerebral arteries, Akt-dependent activation of endothelial nitric oxide synthase-derived H2O2 mediates flow-dependent dilation. © 2009 American Heart Association, Inc.