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

BACKGROUND AND PURPOSE Endothelial nitric oxide synthase produces superoxide under physiological conditions leading to hydrogen peroxide (H(2)O(2)) -dependent dilations to acetylcholine in isolated mouse cerebral arteries. The purpose of this study was to investigate whether H(2)O(2) 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 microL/min, 2-microL step increase at constant internal pressure) was induced in vessels preconstricted with phenylephrine (30 micromol/L). Simultaneously to diameter acquisition, H(2)O(2) or nitric oxide production was detected by the fluorescent dyes CMH(2)CFDA or 4,5-diaminofluorescein diacetate, respectively. Results are expressed as mean+/-SEM of 6 to 8 mice. RESULTS FMD (at 10 microL/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, H(2)O(2)-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 H(2)O(2)-associated rise in fluorescence (3+/-1% and +23+/-4% a.u., respectively; P<0.05), but not acetylcholine-induced dilation. CONCLUSIONS In healthy C57Bl/6 mouse cerebral arteries, Akt-dependent activation of endothelial nitric oxide synthase-derived H(2)O(2) mediates flow-dependent dilation.