NADPH Oxidase–Derived Superoxide Augments Endothelin-1–Induced Venoconstriction in Mineralocorticoid Hypertension
Deoxycorticosterone acetate (DOCA)–salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ETA receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ETB receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ETA receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10−9 mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1–induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1–treated vena cavas of normal rats. Finally, in vivo ETA receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1–induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.
Accumulating evidence indicates that increased arterial superoxide (O2−) contributes to hypertension development in both animals and humans by inactivating nitric oxide (NO) and impairing arterial endothelium-dependent relaxation.1–3 An increase in venous O2− might also inactivate NO and alter venous functions (eg, impaired relaxation or augmented constriction). However, the role of venous O2− production in hypertension and its effect on venoconstriction are unknown. Because hypertension involves multifactorial hemodynamic alterations, venoconstriction augmented by O2− might contribute to increased blood pressure by enhancing cardiac output and shifting blood from the veins to arteries.4–6 Consistent with this notion, endothelin-1 (ET-1) stimulates venous constriction, resulting in significant changes in blood volume distribution, cardiac output, and blood pressure.5,6
The factors controlling venoconstriction are complex and include both neural and humoral mechanisms. The increased sympathetic nerve activity results in augmented venoconstriction, which predominates in the development of spontaneous hypertension.4 Various humoral factors, such as angiotensin II (Ang II) and ET-1, also induce venoconstriction.7 Ang II causes impaired arterial NO-mediated relaxation by increasing O2− in Ang II–induced hypertensive rats.8 We and others have recently reported that arterial O2− levels are also elevated in deoxycorticosterone acetate (DOCA)–salt hypertension,9–14 a model known for its suppressed plasma renin level.15 Studies from our laboratory and others have also shown that both ET-1 and NADPH oxidase activities are increased in arteries of DOCA-salt hypertension models,11,13,16 resulting in NADPH oxidase activation and O2− formation by way of ETA receptors.13 In contrast, ETB receptors have been shown to mediate the protective effect against vascular and renal injuries in DOCA-salt hypertension.17 On the other hand, our most recent studies have demonstrated that venous O2− levels are also increased in DOCA-salt hypertension,18 and ET-1 and its receptors play important roles in maintaining venous tone in this model.19,20 However, the effect of increased venous O2− on ET-1–induced venoconstriction remains unknown. Based on the aforementioned findings, the present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and O2− via its ETA receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Our results demonstrate, for the first time, that (1) ET-1 increases O2− levels through its ETA receptors in both ET-1–treated vena cavas of normal rats and vena cavas of DOCA-salt rats, (2) O2− augmented ET-1–induced venoconstriction, and (3) enhanced venous NADPH oxidase activity plays a key role in increased venous O2− levels in response to ET-1 in this model.
DOCA-Salt Hypertensive Rats and In Vivo Pharmacologic Intervention
DOCA-salt hypertension was created in adult, male Sprague-Dawley rats as previously described.12–14 In brief, rats (250 to 275 g, Charles River, Portage, Mich) underwent uninephrectomy (flank incision, left side), and a silicone rubber DOCA implant (200 mg/kg) was placed subcutaneously between the shoulder blades. Sham rats were also uninephrectomized but received no implant. DOCA-salt rats received 1.0% NaCl and 0.2% KCl in water to drink, and sham rats received tap water. All animals were fed standard rat chow and had ad libitum access to both food and drinking solution. Hypertension develops gradually in this model, with arterial pressure rising gradually but steadily over a 4-week period. During the third week, some DOCA-salt rats received ABT-627 (Abbott Laboratories), a selective ETA receptor antagonist, 2 mg/kg body weight per day in their drinking water, for 2 weeks.13 Blood pressure was measured by noninvasive, tail-cuff methods in conscious but restrained rats. The vessels used were collected between weeks 4 and 6 after DOCA implantation. All animal procedures were in accordance with the institutional guidelines of Michigan State University.
Venous O2− Measurements
Venous O2− was quantified by lucigenin chemiluminescence, as previously described.13,14 Isolated vena cava segments (4 mm long) were assayed for O2− levels, which were expressed as nanomoles per minute per milligram tissue. In addition, in situ detection of O2− was performed by confocal microscopy with use of the oxidative fluorescent dye dihydroethidium (DHE, Sigma), as described previously.12–14 DHE is freely permeable to cell membranes and fluoresces red when oxidized to ethidium bromide by O2−. Veins from DOCA-salt or sham rats were imaged for DHE fluorescence with the aid of a Zeiss 210 confocal microscope with a 590-nm long-pass filter.
To determine the direct effects of ET-1 and ET receptors on O2− production, vein segments of normal rats were incubated in Eagle’s minimum essential medium (Fisher) at 37°C with the ETB receptor agonist sarafotoxin s6c (S6c, 10−7 mol/L, 4 hours; Sigma) or ET-1 (10−11 to 10−9 mol/L, 4 hours) and preincubated with or without the selective ETA antagonist ABT-627 (3×10−8 mol/L, 1 hour). To determine the effects of ET-1, tempol, flavoprotein, NADPH oxidase, xanthine oxidase, and nitric oxide synthase (NOS) on O2− production, vein segments of normal, DOCA-salt, or sham rats were incubated at 37°C with or without ET-1 (10−9 mol/L, 10 minutes) or preincubated with the superoxide dismutase mimetic tempol (10−3 mol/L, 30 minutes; Sigma), ABT-627 (3×10−8 mol/L, 1 hour), diphenylene iodonium (DPI, 10−4 mol/L, 30 minutes; Sigma), apocynin (10−4 mol/L, 1 hour; Calbiochem), allopurinol (10−6 mol/L, 1 hour; Sigma), or Nω-l-arginine methyl ester (l-NAME, 10−4mol/L, 1 hour; Sigma), respectively. All concentrations used were based on our preliminary experiments and published studies.10,13,20
ELISA Enzyme Immunoassay for ET-1
The ET-1 levels of vena cava tissue and rat plasma were determined as described previously.13 In brief, blood from vena cavas of DOCA-salt and sham rats was collected with EDTA as an anticoagulant, and plasma was obtained by centrifuging at 1000g. The cleaned and weighed veins from sham or DOCA-salt rats were frozen in liquid N2, homogenized for 1 minute in 1 mol/L acetic acid (1 mL/50 mg tissue) containing 1.5×10−5 mol/L pepstatin (Sigma), and immediately boiled for 10 minutes. After being chilled, the homogenate was centrifuged at 20 000g for 30 minutes at 4°C, and the supernatant was stored at −80°C until use. The supernatant and plasma were subjected to enzyme immunoassay for ET-1 with a commercial ELISA kit (R&D Systems). Tissue ET-1 levels were expressed as picograms per gram tissue weight, and plasma ET-1 levels were expressed in moles per liter.
NADPH Oxidase Assay
Vena cavas of sham and DOCA-salt rats were homogenized in lysis buffer (10−1 mol/L K2HPO4, 10−3 mol/L phenylmethylsulfonyl fluoride, and 0.2% Triton X-100). The homogenates were centrifuged at 12 000g at 4°C for 30 minutes and then subjected to protein assay (Bio-Rad). NADPH oxidase activities were measured by lucigenin chemiluminescence assay (5×10−6 mol/L lucigenin, Sigma) in the presence of its substrate NADPH (10−4 mol/L, Sigma) as previously described.11 No enzymatic activity could be detected in the absence of NADPH. Reactions were initiated by addition of 10 to 20 μL tissue homogenates containing 25 to 50 μg extracted protein. The enzyme activity was expressed as nanomoles per minute per milligram protein.
Isolated vena cava ring segments (4 mm long) were placed in physiologic salt solution consisting of (in mmol/L) NaCl, 130; KCl, 4.7; KH2PO4, 1.18; MgSO4 · 7H2O, 1.17; CaCl2 · 2H2O, 1.6; NaHCO3, 14.9; dextrose, 5.5; and Na2EDTA, 0.03. Vessels were cleaned of fat and connective tissue, left with an intact endothelium, mounted on stainless steel hooks, and placed on stainless steel holders in tissue baths (30 mL) for isometric-tension recordings with Grass polygraphs and transducers (Astro-Med) or PowerLab for the Macintosh (ADInstruments).20 Vessels were placed under 1 g optimal resting tension.20 Vessels from sham and DOCA-salt rats were placed in the same bath, thus controlling for experimental variations. Tissue baths were filled with warmed, aerated (95% O2, 5% CO2) physiologic salt solution. Vessels were challenged with a maximal contraction to norepinephrine (10−5 mol/L). Functional integrity of the endothelial cells was evaluated by testing relaxation to acetylcholine (10−6 mol/L) in strips contracted with the adrenergic agonist norepinephrine (10−8 to 10 −7 mol/L). Cumulative concentration-response curves to agonists were generated. ET-1 contracts tissues slowly, so tissues were exposed to each concentration of ET-1 for a minimum of 5 minutes before a higher concentration of the agonist was added. In some experiments, the selective ETA receptor antagonist ABT-627 (3×10−8 mol/L), the superoxide dismutase mimetic tempol (10−3 mol/L), or vehicle was incubated with the vessels for 1 hour before addition of ET-1. Venoconstrictions are represented by percentages of maximal contraction to norepinephrine at 10−5 mol/L.
Data are expressed as mean±SEM. Repeated-measures ANOVA was used for comparison of multiple values obtained from the same subject, whereas factorial ANOVA was used for comparing data obtained from 2 independent samples of subjects. The Bonferroni procedure was used to control type I error. A value of P<0.05 was considered significant.
Effect of ET-1 on O2− Levels and Venoconstriction in Vena Cavas of Normal Rats
After 4 hours of incubation, ET-1 concentration-dependently increased O2− levels and venoconstriction in vena cavas of normal rats. Pretreatment with ABT-627 (3×10−8 mol/L), a selective ETA receptor antagonist, completely reversed the effect of ET-1 on O2− production. However, the selective ETB receptor agonist S6c had no effect on O2− production (Figure 1A). ET-1 also induced venoconstriction that was concentration dependent, which was significantly inhibited by ABT-627, as shown by the rightward shift of the dose-response curve (Figure 1B).
In Vivo Blockade of ETA Receptors on Blood Pressure and Increased Venous O2− Levels in DOCA-Salt Rats
There was a significant increase in average systolic blood pressure (170±3 vs 115±2 mm Hg; n=24, *P<0.01) and venous O2− levels (Figure 2) in DOCA-salts rats compared with sham-operated controls. In vivo blockade of ETA receptors for 2 weeks with ABT-627 (in drinking water) significantly lowered blood pressure in DOCA-salt rats (175±3 vs 149±6 mm Hg; n=5, P<0.05), with a concomitant decrease in venous O2− levels in the same group of DOCA-salt rats (Figure 2).
Venous ET-1, NADPH Oxidase, and O2− Levels in DOCA-Salt Rats
There was no significant difference in ET-1 levels in vena cavas between sham and DOCA-salt rats (0.88±0.28 vs 0.80±0.18 pg/g tissue). Similarly, the plasma ET-1 levels were not significantly different between sham and DOCA-salt rats (5.02±0.26 vs 5.74±1.18×10−13 mol/L, n=5–8, P>0.05).
Although venous ET-1 levels were not different between sham and DOVA-salt rats, the endogenous NADPH oxidase activity of vena cavas was significantly increased in DOCA-salt rats compared with that of sham rats, which was reduced by the NADPH oxidase inhibitor apocynin (10−4 mol/L; Figure 3A). As a result, short-term treatment of vena cavas with ET-1 (10−9 mol/L, 10 minutes) further increased O2− levels in DOCA-salt rats but not in sham rats in vitro, an effect that was abolished by both ABT-627 and the superoxide dismutase mimetic tempol (Figure 3B).
Role of O2− on ET-1–Induced Venoconstriction in DOCA-Salt Rats
Consistent with the aforementioned biochemical data, tempol (10−3 mol/L) significantly reduced ET-1–induced venoconstriction in vena cavas of DOCA-salt rats (Figure 4A). In contrast, such an inhibitory effect was not observed in sham rats (Figure 4B). There was no difference in the logarithm of the median effective concentration (logEC50, −8.25±0.03 vs −8.29±0.06, P>0.05) or maximum response (553.2±68.5 vs 482.4±58.5, P>0.05) induced by ET-1 between sham and DOCA-salt rats in either the absence or presence of tempol (−8.19±0.01 vs −8.30±0.00 in logEC50; 503.1±57.7 vs 367.3±21.2 in maximum response).
Effect of NADPH Oxidase, NOS, and Xanthine Oxidase on Venous O2− Levels
Both DPI (10 −4 mol/L), a flavoprotein inhibitor, and apocynin (10−4 mol/L), an NADPH oxidase inhibitor, abolished increases in O2− levels in ET-1–treated (10−9 mol/L) vena cavas of normal rats (Figure 5A) and the vena cavas of DOCA-salt rats (Figure 5B).
In contrast, the xanthine oxidase inhibitor allopurinol (10−6 mol/L) and the NOS inhibitor l-NAME (10−4 mol/L) had no such effects on vena cavas of either ET-1–treated normal rats (data not shown) or of DOCA-salt rats (Figure 5B).
In Situ Detection of Venous O2−
Compared with untreated vessels of normal rats (Figure 6A), incubation with the O2−-sensitive dye DHE resulted in a marked increase in ethidium bromide fluorescence (ie, red color) throughout the vessel wall of ET-1–treated vena cavas of normal rats (Figure 6B) or vena cavas of DOCA-salt rats (Figure 6C). The O2− fluorescence intensity was markedly reduced by DPI in vena cavas of DOCA-salt rats (Figure 6D) compared with the untreated vena cavas (Figure 6C).
The results of the present study demonstrate, for the first time, that (1) ET-1 increases venous O2− levels via its ETA receptor, resulting in augmented ET-1–induced venoconstriction in vena cavas of DOCA-salt but not sham rats and (2) enhanced venous NADPH oxidase activity is a major source of venous O2− production in this model.
It has been reported that increased arterial O2− contributes to vasoconstriction by inactivating NO in both animals and humans with cardiovascular diseases, including hypertension.1–3,21–23 Arterial O2− levels are markedly increased in DOCA-salt hypertension, a model with low plasma renin but high arterial ET-1 levels.9–14 However, it was not clear whether ET-1 also elevates venous O2− levels in this model. Furthermore, the effect of O2− on venoconstriction in DOCA-salt hypertension has not been reported. In the present study, our findings showed that (1) ET-1 concentration-dependently stimulated O2− production in vitro in vena cavas of normal rats; (2) NADPH oxidase and O2− levels were elevated in vena cavas of DOCA-salt rats compared with the sham rats; and (3) tempol, a superoxide dismutase mimetic, significantly inhibited ET-1–induced venoconstriction in DOCA-salt but not in sham rats. Together, these data suggest that ET-1 increases venous O2−, which promotes ET-1–induced venoconstriction in DOCA-salt hypertension.
The increased venous O2− levels in DOCA-salt rats might augment ET-1–induced venoconstriction by removing the venodilator effects of NO, because tempol shifted the ET-1–induced venoconstriction curve rightward. In addition, O2− might act as a direct venoconstrictor, consistent with previous demonstrations of its vasoconstrictor properties.21,22 In contrast, baseline O2− levels in sham rats were apparently too low to be scavenged by tempol, which did not shift the ET-1–induced venoconstriction curve rightward in vessels from sham rats. The rationale of using ET-1 at a concentration of 10−9 mol/L to stimulate O2− was based on our experimental observations that venous O2− levels produced by 10−9 mol/L ET-1 in normal rats have already exceeded those found in DOCA-salt rats. The time period required for exogenous ET-1 treatment to induce venoconstriction was between 5 and 10 minutes in the organ chamber, as we determined in the present study. On the other hand, a 4-hour incubation with ET-1 was required to increase O2− in vena cavas of normal rats. Hence, it might not be possible for exogenous ET-1 treatment to produce enough extra O2− in vena cavas of sham rats in such a short period. Indeed, we found that in vena cavas of DOCA-salt but not of sham rats, exogenous ET-1 treatment for 10 minutes produced extra O2−, which was suppressed by both ABT-627 and tempol. The latter result is correlated with our finding in the present study that NADPH oxidase activity was significantly higher in vena cavas of DOCA-salt rats compared with those of sham rats. It might further explain why tempol did not significantly affect the ET-1–induced venoconstriction in vena cavas of sham rats and why O2− potentiated the ET-1–induced vasoconstriction in DOCA-salt rats. In contrast to the finding from our previous study, which was that arterial ET-1 levels are significantly increased in DOCA-salt rats,12 venous and plasma ET-1 levels were not different between DOCA-salt and sham rats. Although there was no apparent increase in venous ET-1 content, our findings of significantly elevated venous NADPH oxidase activity in DOCA-salt rats might account for the increased O2− levels observed in vena cavas in response to the same amount of circulatory ET-1 stimulation. In agreement with this possibility, our data showed that short-term ET-1 treatment for 10 minutes further increased O2− levels in vena cavas of DOCA-salt rats only but not of sham rats.
Because ET-1–induced alterations in venous tone have been shown to result in significant changes in blood volume distribution, cardiac output, and blood pressure,6,7 examination of the effect of O2− on ET-1/ET-1 receptor–mediated venoconstriction might provide a basic understanding of its potential contribution to blood pressure regulation in DOCA-salt hypertension. Our data showed that O2− formation is independent of ETB receptor activation, and ETA receptors mediate both O2− formation and venoconstriction induced by ET-1 in the vena cava. In vivo administration of the selective ETA antagonist ABT-627 significantly decreased blood pressure in DOCA-salt rats, with a concomitant reduction in venous O2− levels. These findings are in agreement with published data that ET-1 produces O2− via its ETA receptors in rebound pulmonary hypertension23 and that the O2− scavenger tempol reduces blood pressure in Ang II–induced hypertensive rats.2,4 More important, those studies also concur with our recent investigations demonstrating the presence of an intact and sustained ET-1–induced venoconstriction mediated by ETA (but not ETB) receptors in DOCA-salt hypertension.19 The latter findings argue for a shift in the balance of ET-1–induced contractions that predominate in veins versus arteries in this model, which might contribute to a rise in blood pressure by increasing cardiac output and shifting blood to the arteries.5–7 Consistent with this notion, studies in both animal and human hypertension have demonstrated that a decrease in venous capacitance due to venoconstriction occurs preferentially in systemic veins and favors an increase in cardiac output and blood pressure.5,24,25 Accordingly, an increase in venoconstriction might significantly affect cardiac output and blood pressure. Therefore, increased O2− might contribute to the alteration in blood pressure by exaggerating the ET-1–induced venoconstriction in DOCA-salt hypertension.
There are 3 main enzymatic sources of O2− formation in the blood vessel wall, including NADPH oxidase, xanthine oxidase, and uncoupled NOS.26–28 In DOCA-salt hypertensive rats, aortic NADPH oxidase activity is significantly increased compared with that of normotensive controls.12,20 In the present study, we examined whether ET-1 stimulates venous O2− production through NADPH oxidase, xanthine oxidase, or NOS. An important finding of our study is the demonstration that NADPH oxidase activity was significantly higher in vena cavas of DOCA-salt rats compared with those of sham rats. Our results also showed that DPI (a flavoprotein inhibitor) and apocynin, but not l-NAME or allopurinol, inhibited the increased O2− levels in both ET-1–stimulated vena cavas of normal rats and vena cavas of DOCA-salt rats, suggesting that NADPH oxidase, but not xanthine oxidase or NOS, plays a major role in the ET-1–induced venous O2− production in DOCA-salt hypertensive rats. The selectivity of apocynin, a methoxy-substituted catechol, on NADPH oxidase has been well characterized, as it impedes the assembly of the p47phox and p67phox subunits within the membrane NADPH oxidase complex.29
In conclusion, the findings of the present study demonstrate, for the first time, that ET-1 elevates venous O2− levels via its ETA receptors and enhanced venous NADPH oxidase, resulting in augmented ET-1–induced venoconstriction in DOCA-salt hypertension. These findings might provide a novel mechanistic insight on O2−-induced venous dysfunction in hypertension.
This work was supported in part by American Heart Association grants 9806347X and 0130537Z, American Diabetes Association research award 7-01-RA-10, Juvenile Diabetes Research Foundation innovative grant 5-2001-311, and Michigan State University intramural research grants program grant No. 41140 to Dr A.F. Chen. Dr Lixin Li is an awardee of American Heart Association/Midwest Affiliate Physician-Scientist Postdoctoral Fellowship (0225408Z).
- Received October 16, 2002.
- Revision received November 4, 2002.
- Accepted June 24, 2003.
Schnackenberg CG, Welch WJ, Wilcox CS. Normalization of blood pressure and renal vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic: role of nitric oxide. Hypertension. 1998; 32: 59–64.
Nishiyama A, Fukui T, Fujisawa Y, Rahman M, Tian RX, Kimura S, Abe Y. Systemic and regional hemodynamic responses to tempol in angiotensin II–infused hypertensive rats. Hypertension. 2001; 37: 77–83.
Martin DS, Rodrigo MC, Appelt CW. Venous tone in the developmental stages of spontaneous hypertension. Hypertension. 1998; 31: 139–144.
Monos E, Berczi V, Nadasy G. Local control of veins: biomechanical, metabolic and humoral aspects. Physiol Rev. 1995; 75: 611–666.
Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II–induced but not catecholamine-induced hypertension. Circulation. 1997; 95: 588–593.
Somers MJ, Mavromatis K, Galis ZS, Harrison DG. Vascular superoxide production and vasomotor function in hypertension induced by deoxycorticosterone acetate-salt. Circulation. 2000; 101: 1722–1728.
Beswick RA, Dorrance AM, Leite R, Webb RC. NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. Hypertension. 2001; 38: 1107–1111.
Li LX, Crockett E, Wang DH, Galligan JJ, Fink GD, Chen AF. Gene transfer of endothelial NO synthase and manganese superoxide dismutase on arterial vascular cell adhesion molecule-1 expression and superoxide production in deoxycorticosterone acetate-salt hypertension. Arterioscler Thromb Vasc Biol. 2002; 22: 249–255.
Li L, Fink GD, Watts SW, Northcott CA, Galligan JJ, Pagano PJ, Chen AF. ET-1 increases vascular superoxide via ETA-NADPH oxidase pathway in low renin hypertension. Circulation. 2003; 107: 1053–1058.
Li LX, Galligan JJ, Fink GD, Chen AF. Vasopressin enhances vascular superoxide production by activating endothelin-1 in mineralocorticoid hypertension. Hypertension. 2003; 41 (pt 2): 663–668.
Gavras H, Brunner HR, Laragh JH, Vaughan ED Jr, Koss M, Cote LJ, Gavras I. Malignant hypertension resulting from deoxycorticosterone acetate and salt excess: role of renin and sodium in vascular changes. Circ Res. 1975; 36: 300–309.
Lariviere R, Thibault G, Schiffrin EL. Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension. 1993; 21: 294–300.
Matsumura Y, Hashimoto N, Taira S, Kuto T, Kitano R, Ohkita M, Opqenorth TJ, Takaoka M. Different contribution of endothelin-A and endothelin-B receptors in the pathogenesis of deoxycorticosterone acetate-salt-induced hypertension in rats. Hypertension. 1999; 33: 759–765.
Xu H, Fink GD, Galligan JJ. Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in DOCA-salt rats. Am J Physiol: Heart Circ Physiol. 2002; 283: H885–H892.
Cosentino F, Sill JC, Katusic ZS. Role of superoxide anions in the mediation of endothelium-dependent contractions. Hypertension. 1994; 23: 229–235.
Wedgwood S, McMullan M, Bekker JM, Fineman JR, Black SM. Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res. 2001; 89: 357–364.
Landmesser U, Harrison DG. Oxidant stress as a marker for cardiovascular events: Ox marks the spot. Circulation. 2001; 104: 2638–2640.