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 O 2. 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 -nitro-L-arginine methyl ester) (NOS inhibitor), tetrahydrobiopterin (BH 4 ) (NOS cofactor), or selective ET A and. production was monitored by oxidized dihydroethidine staining and/or lucigenin chemiluminescence. ET-1 significantly increased O 2 .production compared with vehicle. SOD, ebselen, and apocynin inhibited the ET-1-induced increase in O 2 . in intact and endothelium-denuded aorta. L-NAME and BH 4 inhibited the ET-1-induced increase in O 2 . 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 O 2 . ; however combining both antagonists inhibited the ET-1-stimulated increase in O 2 . .Rat aortic rings were incubated with ET-1 or vehicle in the presence or absence of sepiapterin (BH 4 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 O 2 . production from NADPH oxidase and NOS uncoupling.

Abstract-Experiments were conducted to test the hypothesis that hypertension produced by chronic ET-1 infusion is mediated by NADPH oxidase-dependent superoxide production. Mean arterial pressure (MAP) was continuously monitored in male Sprague Dawley rats by telemetry. After baseline measurements, rats were placed on a high-salt diet (8% NaCl) and osmotic minipumps were implanted to infuse ET-1 (5 pmol/kg per minute intravenous) for 12 days. Control rats were maintained on the high-salt diet only. Separate groups of rats were also infused with ET-1 and given the superoxide dismutase mimetic, tempol (1 mmol/L), or the NADPH oxidase inhibitor, apocynin (1.5 mmol/L), in the drinking water. Infusion of ET-1 significantly increased MAP when compared with baseline values (132Ϯ3 versus 114Ϯ2 mm Hg, PϽ0.05). Neither tempol nor apocynin treatment had any effect on the increase in MAP produced by ET-1 when compared with baseline values (127Ϯ5 versus 113Ϯ2 and 130Ϯ3 versus 115Ϯ2 mm Hg, respectively). Plasma 8-isoprostane, an indicator of oxidative stress, was significantly increased in ET-1-infused rats compared with rats on a high-salt diet alone (128Ϯ33 versus 51Ϯ5 pg/mL; PϽ0.05). Both tempol and apocynin treatment significantly attenuated the ET-1-induced increase in plasma 8-isoprostane (72Ϯ10 and 61Ϯ6 pg/mL, respectively). Similarly, ET-1 infusion also significantly increased aortic superoxide production (chemiluminescence and dihydroethidium staining techniques), which was prevented by both tempol and apocynin. These data provide evidence that chronic ET-1 infusion increases vascular NADPH oxidase-dependent superoxide production but does not account for chronic ET-1-induced hypertension. Key Words: endothelin Ⅲ oxidative stress Ⅲ sodium E ndothelin-1 (ET-1) is a potent vasoconstrictor peptide that can produce vasoconstriction primarily through ET A receptor activation. 1 Salt-dependent models of hypertension such as DOCA-salt rats 2,3 and chronic angiotensin II-infused rats 4 display a high level of ET-1 mRNA and peptide expression. Furthermore, ET A receptor antagonists attenuated the increase in blood pressure in these models, suggesting an important role of ET-1 in the pathogenesis of salt-dependent hypertension. 2,5,6 Additionally, ET-1 is believed to contribute to hypertension in the black population that is known to be predominantly salt-sensitive. 7,8 Considerable recent attention has been paid to the possibility that ET-induced vasoconstriction may be dependent, at least in part, on the production of superoxide anion. 9 -11 In cultured pulmonary artery smooth muscle cells, Wedgwood et al have shown that exposure to ET-1 significantly increases superoxide production, and this effect can be blocked by pre-incubation with the ET A receptor antagonist PD-156707. 11 Additionally, exogenously applied ET-1 can stimulate superoxide anion formation in rat aortic rings. 9,12 In conscious rats, Sedeek et al observed that 9 days of ET-1 infusion significantly increased blood pressure that was completely abolished when ...

We hypothesize that endothelin-A receptor stimulation contributes to the elevated blood pressure and superoxide production in endothelin-B receptor-deficient rats on a high salt diet. Experiments were conducted on homozygous endothelin-B-deficient (sl/sl) and wild-type rats (wt) fed a high salt diet (8% NaCl) for 3 weeks. Separate groups were given normal drinking water or water containing the endothelin-A receptor antagonist, ABT-627 (5 mg/kg per day; n = 8-9 in all groups). On a normal salt diet, (sl/sl) rats had a significantly elevated systolic blood pressure compared with wt (138 +/- 3 vs 117 +/- 4 mmHg, respectively; P < 0.05). High salt diet caused a significant increase in systolic blood pressure in (sl/sl) rats compared with wt (158 +/- 2 vs 138 +/- 3 mmHg, respectively; P < 0.05). Endothelin-A receptor blockade decreased systolic blood pressure in (sl/sl) rats on high salt (125 +/- 5 mmHg; P < 0.05 vs without antagonist) without affecting the systolic blood pressure in wt (119 +/- 4 mmHg). Aortic superoxide production (lucigenin chemiluminescence) and plasma 8-isoprostane were elevated in sl/sl rats and were significantly reduced by endothelin-A receptor blockade in sl/sl, but not in wt rats. These findings suggest that endothelin-1, through the endothelin-A receptor, contributes to salt-induced hypertension and vascular superoxide production in endothelin-B-deficient rats.

Epidemiological evidence suggests that advancing age affects the cardiovascular system of men and women differently. The purpose of this study was to determine whether the effects of aging on nitric oxide synthase (NOS), oxidative stress, and vascular function are different in males and females. Mesenteric arteries from young (3 mo) and old (24 mo) male and female Fischer 344/Brown Norway rats were studied. Western blot analysis and NOS activity were performed on the homogenized mesenteric arterial bed separated into cytosolic and membrane-associated fractions. Plasma 8-isoprostane measurements assessed oxidative stress. Vascular reactivity was determined by using a wire myograph in the absence and presence of a NOS inhibitor, N(omega)-nitro-l-arginine, to examine endothelial function and basal and stimulated nitric oxide release. In additional arteries, reactivity was performed in the presence of polyethylene glycol-SOD to assess the impact of superoxide on vascular function. Among females, aging was associated with a decline in membrane-associated NOS activity and membrane-associated NOS III protein expression. Advancing age in males was associated with increased cytosolic NOS III protein expression. Among both males and females, advancing age resulted in increased oxidative stress. Vascular function was maintained with age in arteries from both males and females, and there was no difference in either basal or stimulated nitric oxide release with age. Despite sex-specific effects of advancing age on the NOS system and increases in markers of oxidative stress, vascular function is maintained in mesenteric arteries from aged Fischer 344/Brown Norway rats. These data suggest that age-related alterations in the resistance vasculature are complex and likely involve multiple compensating vasoactive pathways.