Flow-induced dilation is mediated by Akt-dependent activation of endothelial nitric oxide synthase-derived hydrogen peroxide in mouse cerebral arteries.
OBJECTIVE
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.
Drouin
Annick
A
Institut de Cardiologie de Montréal, Centre de Recherche, Montréal, Québec, Canada.
Thorin
Eric
E
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D002536:Cerebral Arteries / Q000378:metabolism*; D002560:Cerebrovascular Circulation / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D051058:Oncogene Protein v-akt / Q000502:physiology*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014664:Vasodilation / Q000502:physiology*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D001708:Biopterins; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D002374:Catalase; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D051058:Oncogene Protein v-akt; C003402:sapropterin; D000109:Acetylcholine