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 (Nomega-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). 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 O2-* in endothelium-denuded 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 ETA/ETB receptor activation of O2-* production from NADPH oxidase and NOS uncoupling.
Loomis
E Dabbs
ED
Vascular Biology Center, Medical College of Georgia, CB-3213, 1459 Laney Walker Blvd., Augusta, GA 30912, USA.
Sullivan
Jennifer C
JC
Osmond
David A
DA
Pollock
David M
DM
Pollock
Jennifer S
JS
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D001393:Azoles / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054833:Isoindoles; D049449:Luminescence; D008163:Luminescent Measurements; D008297:Male; D019255:NADPH Oxidases / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D016566:Organoselenium Compounds / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D014661:Vasoconstriction / Q000187:drug effects*
C120508:A 192621; D000098:Acetophenones; D001393:Azoles; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D054833:Isoindoles; D016566:Organoselenium Compounds; D011759:Pyrrolidines; D013481:Superoxides; D001708:Biopterins; C042986:ebselen; C056165:acetovanillone; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D019331:NG-Nitroarginine Methyl Ester