Flow-induced dilation is mediated by Akt-dependent activation of endothelial nitric oxide synthase-derived hydrogen peroxide in mouse cerebral arteries. Stroke 40: 1827–1833

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 122-week-old C57Bl/6 male mice. FMD (0 to 10 L/min, 2-L step increase at constant internal pressure) was induced in vessels preconstricted with phenylephrine (30 mol/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 meanSEM of 6 to 8 mice. Results—FMD (at 10 L/min, 253 % of maximal diameter) was prevented (P0.05) by endothelium removal (61%) or endothelial nitric oxide synthase inhibition with N-nitro-L-arginine (111%) but not by the specific nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (243%). Addition of PEG-catalase and silver diethyl dithio-carbamate (superoxide dismutase inhibitor) reduced (P0.05) FMD to 102 % and 151%, respectively. Simultaneously to FMD, H2O2-associated rise in fluorescence (13319 a.u.) was prevented by N-nitro-L-arginine, PEG-catalase, and silver diethyl dithio-carbamate (5510, 644, and 5010 a.u., respectively; P0.05). Inhibition of FMD by PEG-catalase was fully restored by the addition of tetrahydrobiopterin, a cofactor of endothelial nitric oxide synthase (233%); this functional reversal in dilation was associated with the simultaneous increase in nitri