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. Author manuscript; available in PMC: 2015 Nov 24.
Published in final edited form as: Life Sci. 2013 Dec 29;118(2):226–231. doi: 10.1016/j.lfs.2013.12.021

Functional Heterogeneity of NAPDH Oxidase-Mediated Contractions to Endothelin with Vascular Aging

Matthias R Meyer a,*, Matthias Barton b, Eric R Prossnitz a
PMCID: PMC4074571  NIHMSID: NIHMS552840  PMID: 24382462

Abstract

Aims

Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1.

Main methods

Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1–100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp9Ids-tat (3 µmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 µmol/L).

Key findings

In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (p<0.001); inhibition of NADPH oxidase by gp9 Ids-tat reduced responses to ET-1 by 50% and 72% in the renal artery and aorta, respectively (p<0.05). Aging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, p<0.01 vs. young) and became insensitive to gp9Ids-tat.

Significance

The results suggest an age-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds.

Keywords: age, atherosclerosis, artery, endothelium, gp9Ids-tat, NADPH, Nox, kidney, physiology, oxidative stress, renal, superoxide, vasoconstriction

Introduction

Endothelin-1 (ET-1) is the predominant isoform of three distinct isopeptides constitutively secreted by endothelial and other vascular cells, and the most potent endogenous vasoconstrictor known [1, 2]. The renal artery is particularly sensitive to ET-1 [3–5], and an increase in renal artery tone may lead to reduced kidney perfusion and subsequent activation of the renin-angiotensin system, which contributes to ET-1-dependent regulation of basal vasomotor tone and blood pressure [2, 6, 7]. However, ET-1 also induces vascular oxidative stress, inflammation and remodeling [8, 9]. Indeed, ET-1 contributes to vascular stiffening and calcification with aging, which are all major independent cardiovascular risk factors and associated with cardiovascular complications such as myocardial infarction, stroke, and renal injury [10].

ET-1 activates two G protein-coupled receptors, ETA and ETB [2]. In the vascular wall, smooth muscle cells predominantly express ETA receptors to mediate vasoconstriction, although contractions in response to ETB receptor activation have also been reported for some vascular beds [2]. However, ETB receptors are predominantly found on endothelial cells, where their activation results in the release of the vasodilators nitric oxide (NO) and prostacyclin; moreover, ETB receptors are important for ET-1 clearance [2]. ET-1 acts in concert with other endothelium-derived contracting factors to balance the activity of endothelium-derived relaxing factors [11]. However, vascular aging impairs endothelial cell function favoring the production of contracting factors, including ET-1 [12, 13]. We have previously shown that aging increases circulating ET-1 levels, functional endothelin converting enzyme activity in the aorta, as well as ET-1 expression in conduit and renal arteries of otherwise healthy, normotensive animals [14, 15]. Accordingly, aging augments endothelial ET-1 expression [16] and ET-1-dependent vascular tone in human arteries [17–19]. These findings point towards an increase in ET-1 bioactivity with vascular aging, as also evidenced from the increased exocytotic ET-1 release in aged endothelial cells [20].

Many of the detrimental effects of vascular aging have been attributed to the increased generation of oxygen-derived free radicals, particularly superoxide [12, 13, 21, 22]. Although reactive oxygen species can stimulate ET-1 production, ET-1 on the other hand may also induce superoxide generation by activating NAPDH oxidase [23]. In young rats, ET-1 enhances NAPDH oxidase activity in carotid arteries [24], and induces contractions of renal arteries and aorta that are partly mediated by NADPH oxidase-derived superoxide [25, 26]. Moreover, activation of vascular NADPH oxidase is likely involved in impaired endothelium-dependent vasodilation and vascular remodeling due to ET-1 overproduction in transgenic mice [8]. Likewise, NADPH oxidase has been identified as an important source of ET-1 stimulated superoxide production in mammary arteries and saphenous veins of patients with coronary artery disease [27]. These findings suggest that generation of NAPDH oxidase-derived superoxide may contribute to ET-1-dependent regulation of vascular homeostasis in physiology and disease.

It is however not known whether vascular aging affects contractile responses to ET-1 mediated by NADPH oxidase. Given the physiological importance and high sensitivity of the renal vasculature to ET-1 [2–5], the present study was therefore designed to determine whether age affects ET-1-induced contractions, particularly through NADPH oxidase, in the renal artery. Parallel experiments were conducted in the aorta, which has previously been shown to be sensitive to ET-1-related vascular aging [14, 15].

Materials and Methods

Materials

ET-1 was from American Peptide (Sunnyvale, CA, USA), the NADPH oxidase-selective inhibitor gp9Ids-tat [28] from Anaspec (Fremont, CA, USA), and the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) from Cayman Chemical (Ann Arbor, MI, USA). All other drugs were from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were prepared according to the manufacturer’s instructions, and diluted in physiological saline solution (PSS, composition in mmol/L: 129.8 NaCl, 5.4 KC1, 0.83 MgSO4, 0.43 NaH2PO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose; pH 7.4) to the required concentrations before use.

Animals

Young and old male C57BL6 mice (4 and 24 months of age, mean body weight 29±1 g and 31±1 g, respectively, Harlan Laboratories, Indianapolis, IN) were bred and housed at the animal research facility of the University of New Mexico Health Sciences Center. Animals had free access to standard rodent chow and water, with a 12 hour light-dark cycle. All procedures were approved by the University of New Mexico Institutional Animal Care and Use Committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Isolated vessel preparation

After mice were euthanized by intraperitoneal injection of sodium pentobarbital (2.2 mg/g body weight), renal arteries and the abdominal aorta were immediately excised and transferred into cold (4 °C) PSS. Vessels were carefully dissected free from adherent connective tissue and fat, cut into rings 2 mm in length, and transferred to organ chambers of a Mulvany-Halpern myograph (620M Multi-Channel Myograph System, Danish Myo Technology, Aarhus, Denmark) containing PSS. Renal artery rings were mounted using two 25 µm tungsten wires threaded through the vessel lumen and secured to mounting jaws, whereas abdominal aorta rings were transferred onto 200 µm stainless steel pins. The jaws or pins were connected either to a micropositioner or to a force transducer for recording of isometric tension.

Vascular pharmacology studies

After equilibrating for 30 min in PSS (37°C, pH 7.4, bubbled with 21% O2, 5% CO2 and balanced N2), vascular rings were stretched stepwise until the optimal passive tension for generating force during isometric contraction was reached. Vessels were equilibrated for an additional period of 30 min (renal artery) or 45 min (abdominal aorta), and repeatedly exposed to K+ (PSS with equimolar substitution of 60 mmol/L potassium for sodium) to confirm vascular smooth muscle integrity and to determine maximal contractile responses. The role of NADPH oxidase was studied by randomly treating the left or right renal artery as well as one of two neighboring rings of the abdominal aorta with the Nox-selective inhibitor gp9Ids-tat (3 µmol/L for 30 min) [28–30]. Gp91ds-tat consists of a 9-amino acid peptide of the Noxl/Nox2 catalytic subunits of NAPDH oxidase (at the interface with p47phox, which is essential for activity) linked to the 11 -amino acid HIV-tat peptide, which facilitates cellular entry [28, 31]. After the incubation period, vessels were exposed to cumulative concentrations of ET-1 (0.1 – 100 nmol/L). All experiments were performed following inhibition of NO synthase by L-NAME (300 µmol/L for 30 min) to unmask contractions in the aorta [5], and to exclude ETB receptor-stimulated NO release [2] as well as potential differences in NO bioavailability between vascular beds and age groups [12, 13].

Data calculation and statistical analyses

Data are expressed as mean±SEM; n equals the number of animals used. Contractions to ET-1 are given relative to K+ (60 mmol/L)-induced responses. Fitting of dose-response curves to calculate area under the curve (AUC), EC50 values (as negative logarithm, pD2) and maximal responses was performed as described by deLean et al [32]. Data was analyzed using two-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test (Prism version 5.0 for Macintosh, GraphPad Software, San Diego, CA, USA). A p<0.05 value was considered significant.

Results

The renal artery is resistant to ET-1-related functional aging

To study the functional effects of aging on ET-1-dependent vascular tone, we first determined contractile responses in young and old mice (4 and 24 months of age). ET-1 induced potent contractions in the renal artery of young animals that were 6-fold stronger compared to the abdominal aorta (102±4% vs. 18±4%, n=4–8, p<0.001, Figure 1A). In the aorta, aging reduced contractions to ET-1 by 78% (from 18±4% to 4±1%, n=5–8, p<0.01, Figure 1A), whereas there was no change in the renal artery (102±4% vs. 92±8%, n=4–5, p=n.s. vs. young, Figure 1A). Consistent with these findings, the sensitivity to ET-1 was slightly but significantly greater in the renal artery compared to the abdominal aorta of young and old mice (n=4–8, p<0.05, Figure 1B). Age-dependent differential effects on responses to ET-1 were likely not due to altered contractile function of the smooth muscle, since the force response to K+ (60 mmol/L) in either vascular bed was unaffected by aging (Figure 1C). Taken together, these findings indicate that responses to ET-1 in the renal artery are highly potent and resistant to vascular aging.

Figure 1. Role of aging for contractions to endothelin-1 and K+ in the renal artery and abdominal aorta.

Figure 1

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Maximal effects (A) and the sensitivity (pD2 values, B) of endothelin-1-induced contractions in young (4 months) and old (24 months) mice were calculated by fitting of dose-response curves (0.1–100 nmol/L) [32]. K+ (60 mmol/L) was utilized to determine maximal smooth muscle contractile capacity for generating force (C). *p<0.01 vs. young animals; †p<0.05 vs. renal artery (n=4–2).

Local activity of NADPH oxidase regulates ET-1-induced contractions

We next studied whether contractions to ET-1 depend on functional NADPH oxidase with vascular aging, a condition characterized by increased oxidative stress [12, 13, 21, 22]. In renal arteries of both young and old animals, the NAPDH oxidase-selective inhibitor gp9Ids-tat [28] potently and equally reduced ET-1-induced contractions (50% reduction, n=4–6, p<0.001, Figure 2), consistent with the preserved response to ET-1 with aging in this vessel. The sensitivity to ET-1 (pD2 values) remained unaffected by gp9Ids-tat (not shown). Similarly, contractile responses to the predominantly α1-adrenergic agonist phenylephrine (1 µmol/L) did not differ between young and old animals (106±5% vs. 102±3%, n=5, p=n.s.) and were equally reduced by gp9Ids-tat, independent of age (25% and 22% reduction, n=5, p<0.01). These findings further corroborate the observation that NADPH oxidase-dependent and -independent responses in the renal artery are resistant to functional aging.

Figure 2. NAPDH oxidase-dependent contractions to endothelin-1 (ET-1) in the renal artery during vascular aging.

Figure 2

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Concentration-dependent responses to ET-1 were determined in young (4 months) and old (24 months) mice in the presence and absence of the NADPH oxidase-selective inhibitor gp9Ids-tat (3 µmol/L). Area under the curve (AUC) of endothelin-1-induced contractions is expressed as arbitrary units (AU). *p<0.001 vs. untreated (n=4–6).

In the abdominal aorta, however, inhibition of NADPH oxidase activity in young animals reduced responses to ET-1 to the level seen in old animals (4-fold, from 18±4% to 5±1%, n=4–8, p<0.05, Figure 3). In contrast, the blunted response to ET-1 in aged abdominal aortas was unaffected by inhibition of NADPH oxidase (4±1% vs. 4±2%, n=5, p=n.s., Figure 3), indicating that aging reduces contractions to ET-1 in the abdominal aorta by abolishing the contribution of NADPH oxidase.

Figure 3. Effect of aging on NADPH oxidase-dependent contractions to endothelin-1 (ET-1) in the abdominal aorta.

Figure 3

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Concentration-dependent responses to ET-1 were determined in young (4 months) and old (24 months) mice in the presence and absence of the NADPH oxidase-selective inhibitor gp9Ids-tat (3 µmol/L). Area under the curve (AUC) of endothelin-1-induced contractions is expressed as arbitrary units (AU). *p<0.05 vs. untreated; †p<0.05 vs. young animals (n=4–8).

Discussion

The present study investigated how NADPH oxidase and the physiological aging process affect ET-1-dependent contractions in the renal artery and abdominal aorta of healthy mice. We show that ET-1 induces highly potent NADPH oxidase-dependent and -independent responses in the renal artery that are resistant to vascular aging. In contrast, ET-1-induced contractions in the abdominal aorta are weak and further reduced by aging due to loss of NADPH oxidase activity. These findings are the first demonstration of an age-dependent, localized role of NADPH oxidase in specific vascular beds that determines ET-1-dependent arterial tone and suggest that the renal artery is resistant to NADPH oxidase-related functional aging.

An augmented release of ET-1 and other contracting factors by endothelial cells plays a significant role in the pathophysiology of vascular aging [12, 13]. In addition to enhanced vasomotor tone [17–19], age-dependent increases in the bioactivity of ET-1 have been implicated in vascular oxidative stress, inflammation and remodeling [8, 9], which in turn promote arterial stiffening and calcification [10]. Although ET-1-induced contractions display a marked heterogeneity between vascular beds and are generally less potent in mice compared to other species [5, 33], the renal vascular bed and particularly the main renal artery are highly sensitive to ET-1 as shown in the present and previous studies [2–5]. We now demonstrate that the potent responses to ET-1 are preserved in old mice, suggesting that the responsiveness to ET-1 in the renal vasculature, unlike the aorta, remains intact with aging. It is, however, important to note that acute, exogenous application of ET-1 might not necessarily reflect its chronic autocrine and paracrine actions within the vascular microenvironment [2]. In fact, aging has been found to increase endogenous vascular and renal ET-1 bioavailability [15, 16, 34], which in turn can down-regulate ETA receptor expression [34–36], potentially leading to reduced responsiveness to ET-1 as observed in other vascular beds [14, 37–39]. However, the preserved, potent contractions to ET-1 in the aged renal artery argue against such a compensatory change in vascular ETA receptor function. Despite the fact that the kidney already displays the greatest ET-1 concentration of all tissues under physiological conditions [40] that increases further with aging [34], other mechanisms known to potentiate ET-1-induced responses such as cross-talk with the renin-angiotensin or the adrenergic systems [2] might also contribute to the maintained high responsiveness to ET-1 in the aged renal artery.

The finding that ET-1-induced contractions remain unaffected with vascular aging in the renal artery but not in the abdominal aorta is strengthened by the fact that responses are independent of NO bioavailability, which may be affected by both aging and superoxide production [12, 13]. Thus, since all experiments were performed in the presence of the NO synthase inhibitor L-NAME, the observed age-dependent differences in ET-1-dependent contractility between vascular beds are unrelated to basal or endothelin ETB receptor-stimulated NO release as previously found in rat coronary arterioles [38]. The use of the NO synthase inhibitor also excludes potential confounding effects on vascular reactivity resulting from altered expression levels of endothelial and inducible NO synthase with aging [15]. Moreover, it is unlikely that the blunted response to ET-1 in the abdominal aorta of aged animals was due to a non-specific decline in smooth muscle contractile function, since the responsiveness to K+ did not change with age. Furthermore, K+-induced contractions in abdominal aortas and renal arteries varied by only 1.5-fold and thus cannot account for the observed marked differences in ET-1-dependent contractility between those vascular beds.

Increased production of oxygen-derived free radicals by NADPH oxidase, uncoupled endothelial NO synthase, and xanthine oxidase has been implicated in the physiology of vascular aging [12, 13, 21, 22]. Since vascular responses to ET-1 partly depend on its ability to stimulate superoxide production by NADPH oxidase [8, 23–26], we hypothesized that NADPH oxidase function might, at least in part, determine contractile responses to ET-1 with aging. In arteries of young mice, we found that the NADPH oxidase-selective inhibitor gp9Ids-tat [28] largely reduces contractions to ET-1. Gp9Ids-tat was originally designed to be a selective inhibitor for the Nox2 catalytic subunit [28], but likely also inhibits the assembly of Noxl due to its high sequence homology to the Nox2 isoform [31, 41]. Thus, the data from the present study suggest that ET-1-dependent contractions depend on the activity of the inducible, superoxide-generating Noxl or Nox2 isoform [31]. In line with our observations, a previous study in rats demonstrated that apocynin attenuates ET-1-dependent reductions in renal blood flow by 35% [26], although apocynin might not be considered a specific NADPH oxidase-specific inhibitor, since it may also exert potent antioxidant and other effects [31]. In the present study we now demonstrate that contractions to ET-1 are indeed NADPH oxidase-dependent, and that the NADPH-oxidase dependent contribution to the ET-1 response remains unaffected by aging in the renal artery. In contrast, NADPH oxidase-dependent contractions to ET-1 are abolished in the abdominal aorta of otherwise healthy aged animals, indicating an age-dependent, localized role of NADPH oxidase in the regulation of ET-1-dependent responses in the arterial vascular tree. Of note, these findings are not inconsistent with previous reports showing increased vascular ROS activity with aging [12, 13, 21, 22], since agonists other than ET-1 may differentially regulate functional NADPH oxidase activity. Furthermore, alternative vascular sources of ROS might become activated in the murine renal artery and abdominal aorta with aging [12, 13].

To the best of our knowledge, the present study is the first demonstration of a specific role of NADPH oxidase activity for the regulation of vasomotor tone in different vascular beds. However, the underlying mechanisms remain unclear. Different regulation of expression or activity of components of the NADPH oxidase multienzyme complex that are sensitive to vascular aging, such as Nox2 and p47phox [21, 22, 42], might contribute to the observed heterogeneous responsiveness to ET-1 in different vascular beds. Furthermore, activity of NADPH oxidase might be locally regulated by factors known to contribute to oxidative stress-driven vascular aging, such as the aging-associated genes klotho [43] and silent information regulator 1 (SIRT1) [44], or the transcription factor JunD [45].

Conclusions

The present study demonstrates that the vascular response to ET-1 in healthy young mice to a substantial degree depends on NADPH oxidase activity, one of the major vascular sources of reactive oxygen species. With aging, localized regulation of NADPH oxidase activity appears to determine the functional response to ET-1 in different vascular beds. Of note, the preserved, highly potent and partly NADPH oxidase-dependent reactivity to ET-1 in the aged renal artery might have clinical implications. Indeed, both oxidative stress and increased ET-1 bioactivity have been implicated in age-dependent impaired endothelial cell function [12, 13], which is associated with arterial stiffening and sclerosis [10], and consecutive renal injury [46]. Antagonizing ET-1-dependent effects have previously been found to improve endothelial function in individuals with early coronary artery disease [47], and even to reverse renal aging and glomerular vascular injury [48]. Moreover, treatment with an ETA receptor antagonist reduces arterial stiffness in patients with chronic kidney disease [49]. Thus, similar treatment strategies might be suitable to protect from age-induced changes in the renal vascular bed, such as impaired renal hemodynamics, renal arterial sclerosis and subsequent renal ischemia that are critically involved in progressive loss of kidney function with aging [46].

Acknowledgments

We thank Dr. Chelin Hu and Daniel F. Cimino for expert technical assistance. This study was supported by the National Institutes of Health (R01 CA127731 and CA163890 to ERP), Dedicated Health Research Funds from the University of New Mexico School of Medicine allocated to the Signature Program in Cardiovascular and Metabolic Diseases (to ERP), and the Swiss National Science Foundation (grants 135874 & 141501 to MRM and grants 108258 & 122504 to MB).

Footnotes

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Chemical compounds studied in this article:

Endothelin-1 (PubChem CID: 16212950); L-NG-nitroarginine methyl ester (PubChem CID: 39836)

Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

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