Endothelin mediates superoxide production and vasoconstriction through activation of NADPH oxidase and uncoupled nitric-oxide synthase in the rat aorta

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 O₂^-. 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 (N^ω-nitro-L-arginine methyl ester) (NOS inhibitor), tetrahydrobiopterin (BH₄) (NOS cofactor), or selective ET_A and ET_B 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). O₂^-. production was monitored by oxidized dihydroethidine staining and/or lucigenin chemiluminescence. ET-1 significantly increased O₂^-. production compared with vehicle. SOD, ebselen, and apocynin inhibited the ET-1-induced increase in O₂ ^-. in intact and endothelium-denuded aorta. L-NAME and BH₄ inhibited the ET-1-induced increase in O₂^-. in intact tissue, whereas these two compounds had no effect on ET-1-induced O ₂^-. in endotheliumdenuded aorta. Preincubation with BQ-123 or A-192621, individually, had no effect on ET-1-induced O₂ ^-.; however combining both antagonists inhibited the ET-1-stimulated increase in O₂^-.. 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 ET_A/ET_B receptor activation of O₂^-. production from NADPH oxidase and NOS uncoupling.