Endothelin Mediates Superoxide Production and Vasoconstriction through Activation of NADPH Oxidase and Uncoupled Nitric-Oxide Synthase in the Rat Aorta

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Abstract
Experiments were designed to test the hypothesis that elevated levels of endothelin 1 (ET-1) in the vasculature activate NADPH oxidase and/or uncoupled nitric-oxide synthase (NOS), resulting in production, and mediate increased constriction. Rat aortic rings were incubated with ET-1 or vehicle in the presence and absence of superoxide dismutase (SOD), ebselen (glutathione peroxidase mimetic), apocynin (NADPH oxidase inhibitor), l-NAME (Nω-nitro-l-arginine methyl ester) (NOS inhibitor), tetrahydrobiopterin (BH4) (NOS cofactor), or selective ETA and ETB receptor antagonists (BQ-123 [cyclo(d-Asp-Pro-d-Val-Leu-d-Trp)] and A-192621 [[2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid]], respectively). production was monitored by oxidized dihydroethidine staining and/or lucigenin chemiluminescence. ET-1 significantly increased production compared with vehicle. SOD, ebselen, and apocynin inhibited the ET-1-induced increase in in intact and endothelium-denuded aorta. l-NAME and BH4 inhibited the ET-1-induced increase in in intact tissue, whereas these two compounds had no effect on ET-1-induced in endothelium-denuded aorta. Preincubation with BQ-123 or A-192621, individually, had no effect on ET-1-induced ; however combining both antagonists inhibited the ET-1-stimulated increase in . Rat aortic rings were incubated with ET-1 or vehicle in the presence or absence of sepiapterin (BH4 synthesis substrate) or apocynin and mounted on wire myographs to determine isometric force generation in response to increasing KCl concentrations. ET-1 increased the contractile response to KCl compared with vehicle. Treatment with either sepiapterin or apocynin attenuated the ET-1-mediated increase with no effect of sepiapterin or apocynin alone. These data support the hypothesis that ET-1 increases vascular tone, in part, through ETA/ETB receptor activation of production from NADPH oxidase and NOS uncoupling.