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(Stroke. 2009;40:1827.)
© 2009 American Heart Association, Inc.

Original Contributions

Flow-Induced Dilation Is Mediated by Akt-Dependent Activation of Endothelial Nitric Oxide Synthase-Derived Hydrogen Peroxide in Mouse Cerebral Arteries

From the Université de Montréal, Department of Surgery, Institut de Cardiologie de Montréal, Centre de recherche, Montréal, Québec, Canada.

Correspondence to Eric Thorin, PhD, Institut de Cardiologie de Montréal, centre de recherche, 5000, rue Bélanger, Montréal, Québec, H1T 1C8, Canada. E-mail eric.thorin{at}umontreal.ca

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.

Key Words: endothelium • nitric oxide • oxygen radicals • resistance arteries

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