Abnormal nitric oxide production in aged rat mesenteric arteries is mediated by NAD(P)H oxidase-derived peroxide

Abstract

Previous work in our laboratory showed increased basal periarterial nitric oxide (NO) and H₂O₂ concentrations in the spontaneously hypertensive rat, characterized by oxidant stress, as well as impaired flow-mediated NO production that was corrected by a reduction of periarterial H₂O₂. Aging is also associated with an increase in vascular reactive oxygen species and results in abnormal vascular function. The current study was designed to assess the role of H₂O₂ in regulating NO production during vascular aging. In vivo, real-time NO and H₂O₂ concentrations were measured by microelectrodes in mesenteric arteries of retired breeder (aged; 8–12 mo) and young (2 to 3 mo) Wistar-Kyoto rats under conditions of altered flow. The results in aged rats revealed elevated basal NO (1,611 ± 286 vs. 793 ± 112 nM, P < 0.05) and H₂O₂ concentrations (16 ± 2 vs. 9 ± 1 μM, P < 0.05) and a flow-mediated increase in H₂O₂ but not NO production. Pretreatment of aged rats with the antioxidant apocynin lowered both basal H₂O₂ (8 ± 1 μM) and NO (760 ± 102 nM) to young levels and restored flow-mediated NO production. Similar results were obtained with the NAD(P)H oxidase inhibitor gp91ds-tat. In addition, acute incubation with topical polyethylene-glycolated catalase lowered the baseline NO concentration and restored flow-mediated NO production. Taken together, the data indicate that elevated baseline and suppressed flow-mediated NO production in aged Wistar-Kyoto rats are mediated by NAD(P)H oxidase-derived H₂O₂.

the balance between reactive oxygen species (ROS) and nitric oxide (NO) has been shown to be a key factor in age-related vascular dysfunction. Altered vascular tone (31, 43, 46, 52) and impaired compensatory remodeling (35, 49) during aging are thought to be due in part to increased superoxide generation and decreased NO bioavailability. Although many studies addressing mechanisms of NO regulation and endothelial dysfunction have focused on the role of superoxide in scavenging NO (21, 25, 52) and depleting tetrahydrobiopterin (1, 11, 23), the potential in vivo effects of hydrogen peroxide (H₂O₂) on NO production have largely been ignored.

Most in vitro studies of H₂O₂ effects on endothelial NO synthase (eNOS) activity have indicated stimulatory actions (9, 17, 38, 47, 48). However, a recent in vitro study (24) suggests peroxide increases eNOS activity in the short term but decreases its activity following chronic exposure. To our knowledge, no studies have addressed the impact of long-term peroxide elevation on NO signaling in resistance arteries in vivo. Recent results from our laboratory showed in young spontaneously hypertensive rats (SHR) that NAD(P)H oxidase-derived peroxide was responsible for elevated basal arterial NO concentration and impaired flow-mediated NO production (56). It is not known whether this effect of H₂O₂ on NO is unique to early stages of hypertension or is typical of pathological conditions characterized by chronically elevated oxidative stress. However, evidence from studies in humans with vascular disease shows that H₂O₂ is elevated in plasma (27) and in arterioles under conditions of elevated blood flow (33, 37). It is known that NO function, as assessed by bioassay methods, is suppressed in aging models where vascular ROS are chronically elevated (6, 46, 52), but the role of peroxide has not been investigated.

The current study was undertaken to address the hypothesis that an increased production of H₂O₂ occurs with aging and mediates abnormal NO production in resistance vessels under in vivo conditions of resting and elevated flow. We used the Wistar-Kyoto (WKY) retired breeder rat as a model of vascular aging. Our group has found this rat strain at 8–12 mo of age to have impaired mesenteric collateral artery growth (32), similar to that previously demonstrated in the aged Wistar rat (49) and analogous to older humans (35) and other models of elevated oxidative stress and endothelial dysfunction (3, 22, 50). The results from in vivo, real-time measurements of NO and H₂O₂ in mesenteric arteries of retired breeder rats indicated that, similar to previous results in the SHR, early senescence resulted in an NAD(P)H oxidase-mediated increase in peroxide causing abnormal regulation of both basal and flow-mediated NO production. The data suggest that peroxide may be an important regulator of NO-related functions in the vasculature characterized by chronic oxidative stress.

METHODS

Animals.

Male young WKY (∼200 g, 2 to 3 mo old) and retired breeder (aged) WKY (∼400 g, 8–12 mo old) rats were obtained from Harlan (Indianapolis, IN). All procedures were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. To test for the effects of antioxidant treatment on periarterial NO and H₂O₂ concentrations, aged rats were randomly designated for apocynin treatment or control groups. Apocynin (acetovanillone; Fisher Scientific, Hampton, NH) was given in drinking water (3 mM) for 1 wk before NO/H₂O₂ measurements. Subsequent experiments to more clearly delineate the mechanisms utilized the specific NAD(P)H oxidase inhibitor gp91ds-tat (39) [1.0 μM, 40 min topical incubation (36); synthesized by EZBiolab, Westfield, IN] or polyethylene-glycolated (PEG) catalase (250 U/ml, 20 min topical incubation, Sigma, St. Louis, MO). We previously demonstrated (56) that apocynin and gp91ds-tat had no effect on flow, NO, or H₂O₂ production in young WKY rats; thus these control experiments were not repeated in the current study.

Rats were anesthetized with subcutaneously administered thiopental sodium (250 mg·ml⁻¹·kg⁻¹). The trachea was cannulated and animals were mechanically ventilated at 70 breaths/min and at a sufficient tidal volume to generate oxygen saturation of hemoglobin in the 92–95% range. The right femoral artery was cannulated, and lactated Ringer solution was infused (∼0.5 ml·h⁻¹·100 g⁻¹) to maintain a stable arterial blood pressure. Animals were maintained at body temperature by placing them on a heating manifold (35–37°C).

Acute arterial occlusion model creation and flow and diameter measurements.

The mesenteric model used in the current study has been previously described in detail (56) and is identical, except that a low-flow condition was not generated. Briefly, the small intestine and mesentery were prepared for in vivo observation by using a standardized technique that maintained innervation and all arterial and venous connections (4). The antimesenteric edges of the bowel wall were secured with small sutures to stabilize the bowel and to allow the mesentery to be gently spread out in the bath solution. The mesenteric artery bath solution consisted of bicarbonate-buffered (pH 7.35–7.45) physiological saline equilibrated with 5% O₂-5% CO₂-90% N₂ (5). Isoproterenol and norepinephrine (<10⁻⁷ M) were added to the media to suppress visceral smooth muscle spontaneous contractions and to minimize bowel motility (55, 56).

A section (3 mm) of mesenteric artery ∼2 cm from the bowel was isolated from its companion vein for placement of a precalibrated 0.5-mm-diameter flow probe (0.5 PSB, Transonic Systems, Ithaca, NY) as previously described (56). This location was chosen to ensure a distance of ≥10 mm from the site of NO and H₂O₂ measurements to prevent electrical interference. The zero flow value was set with the flow probe suspended in the bath above the artery. To assess the modulation of NO and H₂O₂ by increased flow, two microvascular clamps were placed sequentially on adjacent mesenteric arteries to increase flow ∼50% and ∼100%, respectively, over basal flow as previously described and illustrated (56). Videotape recordings of mesenteric arteries were acquired with a dissecting microscope and camera, and luminal diameters were estimated by measuring arterial red cell columns with image analysis software (Image-1/AT).

NO measurement.

Periarterial NO concentrations were measured by NO-selective microelectrodes based on established techniques (5). Microelectrodes were calibrated against NO gas concentrations (Matheson, Joliet, IL) up to 1,200 nM. A Keithley model 6517A electrometer (Cleveland, OH) was used to polarize NO microelectrodes relative to a silver-silver chloride electrode to the voltage (0.7 or 0.9 V) most sensitive to NO concentrations. Electronic drift was compensated mathematically using a virtual baseline adjusted for time and the linear rate of drift (2). Zero NO concentration was obtained by elevating the electrode tip ∼200 μm above the artery in the tissue bath. To obtain periarterial NO measurements, the microelectrode tip penetrated the mesentery beside the mesenteric artery and the tip was advanced into the outer connective tissue over the artery wall. During the placement or removal of the microvascular clamps on or from adjacent arteries, the microelectrode was briefly removed (1 to 2 min) from the perivascular region to prevent tip breakage. However, the previous penetration track and vessel/tissue landmarks were used to ensure the return of the microelectrode to the exact same vascular site. Within the 1- to 2-min interval of clamp placement and microelectrode repositioning, new steady-state levels of NO and H₂O₂ were established so that the data traces reflected only the difference in concentrations between basal and elevated flow states. To verify NO electrode specificity, the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (1 mM) was added to the bath solution at the end of some experiments, and as previously shown for young WKY rats (56), periarterial NO concentrations were significantly reduced. A similar reduction in NO concentration was also found for aged WKY rats (data not shown). NO electrode current was not influenced by in vivo H₂O₂ because the electrodes were unresponsive to topical H₂O₂ (≤100 μM), added to the bath as previously described (56).

H₂O₂ measurement.

Perivascular H₂O₂ concentrations were measured with a World Precision Instruments (Sarasota, Florida) Apollo 4000 System using the ISO-HPO-100 Sensor (100 μm diameter, and 2 mm length). This proprietary system has been previously used for both in vitro and in vivo studies (10, 40, 56). The electrode was calibrated against freshly diluted H₂O₂ in Ringer solution in the range of 0–100 μM at 37°C and was insensitive to NO gas (1,200 nM). For measurements of perivascular H₂O₂, the exposed carbon fiber sensor was placed on the vessel wall at a shallow angle to ensure that the entire sensor tip length of 2 mm touched the outside wall of the artery. The procedure for H₂O₂ electrode manipulation during clamping was the same as for the NO measurements. H₂O₂ electrode specificity was verified by the addition of PEG catalase (250 U/ml) to the bath solution at the end of the experiments, as previously shown (56).

Statistical analyses.

Data analyses were performed by one- or two-way ANOVA (SigmaStat 3.0; Systat Software, San Jose, CA) with or without repeated measures, as indicated in the figure legends. When the ANOVA indicated statistical differences between groups, multiple pairwise comparisons were performed with the Student-Newman-Keuls test. Data are presented as group averages with means ± SE, and the criterion for significance was P < 0.05.

RESULTS

Basal NO and H₂O₂ concentration measurements.

The initial experiments revealed that by in vivo direct measurement, the baseline periarterial NO concentration was significantly greater (∼200%, P = 0.01, Fig. 1A) in aged than in young rats (1,611 ± 286 vs. 793 ± 112 nM, respectively). Pretreatment of aged WKY rats with apocynin (3 mM for 7 days) reduced the baseline perivascular NO concentration to a concentration range similar to that of young WKY rats (760 ± 102 nM) (Fig. 1A).

Fig. 1.

Periarterial nitric oxide (NO) and H₂O₂ measurements in young and aged Wistar-Kyoto (WKY) rats at rest (basal blood flow). A: basal periarterial NO measurements. Aged WKY rats had a significantly higher mesenteric artery basal NO concentration compared with young rats. Apocynin (Apo) pretreatment (3 mM × 7 days) in aged rats restored the NO concentration to the range of young WKY rats (n = 6 to 7/group). Statistical analyses were performed with one-way ANOVA. B: basal periarterial H₂O₂ measurements. Aged rats had significantly higher basal H₂O₂ concentrations compared with young rats. Apo pretreatment significantly decreased the H₂O₂ concentration in aged rats to a level similar to that of young rats. One-way ANOVA was used for statistical analyses; n = 4 in each group.

Measurements of in vivo periarterial H₂O₂ concentrations obtained with a H₂O₂-sensitive electrode under basal conditions are reported in Fig. 1B. Basal peroxide in aged rats (16 ± 2 μM) was increased 78% compared with that in young WKY (9 ± 1 μM). Pretreatment with apocynin decreased the H₂O₂ concentration in aged rats to a range similar to young rats (8 ± 1 μM).

Modulation of periarterial NO concentration by flow.

The baseline arterial blood flow and arterial diameters were similar between young, aged, and apocynin-pretreated aged rats (0.27 ± 0.06, 0.31 ± 0.03, and 0.29 ± 0.05 ml/min and 240 ± 15, 224 ± 9 and 234 ± 18 μm for young, aged, and apocynin-pretreated aged rats, respectively). Changes in blood flow due to the sequential application of two vascular clamps are illustrated in Fig. 2. Two-way repeated-measures ANOVA indicated significant (P < 0.001) differences in flow for each clamping status but no differences between groups and no interaction between group and clamping status. The changes in flow were similar between all groups and experimental conditions.

Fig. 2.

Relationship between blood flow and clamp condition. Significant changes occurred in flow that correlated with sequential arterial clamping (P < 0.001) in young, aged, and aged + Apo rats, but there was no statistical significance (P = 0.12) in flow changes between rat groups and clamp status (two-way repeated-measures ANOVA).

The relationship between the percent change in NO concentration and blood flow compared with the basal level is shown in Fig. 3A. In young rats, the periarterial NO concentration significantly increased with changes in flow. However, in aged rats, there was no increase in NO concentration at either level of increased blood flow, demonstrating a profound impairment in flow-modulated NO production. Pretreatment of aged rats with the antioxidant apocynin completely restored the correlation between elevated flow and NO concentration. To determine whether the redox regulation of flow-mediated NO production was related to NAD(P)H oxidase activity, an acute incubation of aged arteries with gp91ds-tat was done in an additional set of rats. As seen in Fig. 3B, acute gp91ds-tat both lowered basal NO and restored flow-mediated NO production.

Fig. 3.

Flow-mediated NO production in young and aged WKY rat mesenteric arteries. A: relationship between percent change in NO concentration and blood flow in young, aged, and Apo-treated aged WKY rats. Periarterial NO concentrations significantly increased with elevated flow (induced by sequential clamping) in young and aged + Apo groups (P < 0.05) but not in the aged group. The increase in NO was more significant in the aged + Apo than the young group (P < 0.05 in both clamp conditions). Two-way repeated-measures ANOVA was used for statistical analyses; n = 6 to 7/group. B: effect of NAD(P)H oxidase inhibition on periarterial NO production. Acute topical application of gp91ds-tat (ds-tat; 1 μM) significantly lowered the basal NO concentration (P < 0.05, paired t-test; n = 4/group) and restored flow-mediated NO production in the aged rats (two-way repeated-measures ANOVA; n = 6 to 7/group).

Flow-modulated H₂O₂ concentration and impact on NO production.

As shown in Fig. 4A, compared with basal flow, the increased flow in the aged rat arteries resulted in a significantly increased periarterial H₂O₂ concentration, whereas no increase was detected in young arteries. In fact, the tendency in young arteries was for H₂O₂ to decrease with elevated flow. This positive relationship between flow and H₂O₂ concentration in the aged rats was abolished by the pretreatment with apocynin. The results for acute incubation with gp91ds-tat in aged arteries (Fig. 4B) showed that both the basal and flow-mediated increases in H₂O₂ concentration were significantly decreased, suggesting that the majority of peroxide was derived from NAD(P)H oxidase.

Fig. 4.

Flow-mediated H₂O₂ production in young and aged WKY rat mesenteric arteries. A: relationship between percent change in H₂O₂ concentration and blood flow in young, aged, and Apo-treated aged WKY rats. There was a significant increase of H₂O₂ with increased flow (induced by sequential clamping) in aged rats compared with young for both clamp conditions, and Apo pretreatment prevented the increase in H₂O₂ production. Two-way ANOVA was used for statistical analyses; n = 5 to 6/group. B: effect of NAD(P)H oxidase inhibition on periarterial H₂O₂ production. Topical application of gp91ds-tat (1 μM) significantly lowered the basal H₂O₂ concentration and prevented flow-mediated peroxide production in the aged rats. Two-way repeated-measures ANOVA was used for statistical analyses; n = 4/group.

Experiments were also performed with an acute incubation of PEG catalase to determine whether abnormal NO production in aged rats was a result of the elevated H₂O₂ concentration. The results illustrated in Fig. 5 show that topical PEG catalase not only decreased the basal periarterial NO concentration but also restored the flow-mediated NO production in aged rats.

Fig. 5.

Role of H₂O₂ in NO production. Acute incubation with PEG catalase (PEG-Cat; 250 U/ml topically) resulted in a significant decrease of the basal NO concentration and restored flow-mediated NO production in aged rat mesenteric arteries. Two-way repeated-measures ANOVA was used for statistical analyses; n = 4/group.

DISCUSSION

This study tested the hypothesis that mesenteric arteries of aged WKY rats are characterized by elevated concentrations of H₂O₂ that mediate abnormal NO production. Similar to the results obtained previously with the SHRs (56), direct in vivo measurements of perivascular NO and H₂O₂ in aged rats indicated an environment of excessive ROS, associated with an elevated basal NO concentration, but suppressed endothelial NO production in response to increased blood flow. Treatment with apocynin, gp91ds-tat, or PEG catalase completely restored normal NO production. These novel observations indicate a significant role for NAD(P)H oxidase-derived peroxide in NO dysfunction during the aging process.

Basal elevation of NO and H₂O₂.

In our study the aged WKY rat was found to have chronically increased basal H₂O₂ and NO concentrations compared with the young WKY rat (Fig. 1). The elevated periarterial H₂O₂ is consistent with the common view of increased ROS during aging (6, 31, 52). Our observation of elevated NO with aging is consistent with other studies reporting increased eNOS expression and/or activity in aged mesentery artery from Sprague-Dawley rats (6) or renal and femoral arteries of aged Fischer 344 rats (51). We also found that apocynin pretreatment decreased the basal H₂O₂ and NO concentrations in aged rat arteries to levels similar to those in young rats (Fig. 1). This result implied that NO increased as a result of stimulation by ROS. The ability of PEG catalase to lower the basal NO concentration in aged arteries (Fig. 5) indicated that H₂O₂ was stimulating NO production. Consistent with this observation, Harrison's group has demonstrated that H₂O₂ increases eNOS expression levels chronically both in vitro (17) and in vivo (29). A 7-day treatment with ebselen, a mimic of glutathione peroxidase, significantly decreased the eNOS expression levels in C57BL/6J mice and in transgenic mice overexpressing p22^phox (29). One study (24) suggests an inhibitory effect on eNOS when H₂O₂ is used in extended cell culture incubations (8 h), but this result was obtained using a supraphysiological (500 μM) concentration of peroxide. In support of the differential concentration effects of peroxide, results with cultured endothelial cells (53) demonstrate that chronic incubation with a relatively low concentration of peroxide (12 μM), similar to that measured in the aged WKY rat, stimulates eNOS protein expression but that higher concentrations (50–100 μM) significantly decrease the expression.

Flow modulation of H₂O₂ and NO production.

The results of in vivo, real-time measurements indicated that acutely increased arterial blood flow elevated the NO concentration in young, but not aged, rats (Figs. 3 and 5). This finding is consistent with the results from Sun et al. (46), who observed decreased shear stress-induced dilation and NO production (based on nitrite generation) in isolated mesenteric arteries of 24-mo-old Fischer 344 rats, which are characterized by vascular oxidative stress. However, contrary to our results in retired breeder rats, Sun et al. (46) show that basal NO decreases with age and that both suppressed basal and shear-mediated NO production are not restored to young levels by antioxidant treatment. Their data demonstrate that NO scavenging is the primary mechanism responsible for impaired dilation in aged Fischer 344 rats. One factor that may account for the different mechanism of NO regulation we observed is that retired breeder WKY rats are at a relatively early stage of aging compared with the more advanced age of animals used by Sun et al. (46) and in other studies (6, 13). This age difference may account for our observation of an elevated basal concentration of NO, rather than a depressed level as would be expected if extensive scavenging was occurring. The age-related differential expression of eNOS and/or NO production, if true, would suggest that redox regulation of NO function may be dependent on different mechanisms during the development of endothelial dysfunction. Other differences potentially impacting NO regulation are the type of animal model and/or vessel studied. We have previously shown that flow-mediated collateral growth is dependent on the rat genetic background (41), and different results for changes in eNOS expression/activity with age have been reported in various rat strains and vessel types (6, 13, 14, 42, 46, 51, 52).

We also observed that elevated blood flow further increased periarterial H₂O₂ over basal levels in the aged rats (Fig. 1). Apocynin pretreatment for 1 wk suppressed basal and flow-mediated H₂O₂ production and corrected the high-resting NO concentration in aged animals to that of young rats (Fig. 1) while simultaneously restoring blood flow-mediated NO production (Fig. 3). This result suggests that excessive ROS were responsible for the abnormal flow regulation of NO. An acute incubation of PEG catalase normalized not only basal but also flow-mediated NO production in aged WKY rat artery (Fig. 5), indicating that H₂O₂ itself was regulating the production of NO. While destruction by oxygen radicals is a widely accepted mechanism for decreasing the bioavailability of NO (21, 44), there is also clear evidence that H₂O₂ acutely stimulates NO production. Previous studies showed that H₂O₂ increased the phosphorylated eNOS-to-eNOS ratio within 30 min through the ERK1/2 (8) or PK3-Akt pathway in endothelial cells in vitro (24, 47, 48) and therefore increased eNOS activity. In a recent study (56), we found that acutely administered exogenous H₂O₂ (20–100 μM) increased NO production in young WKY rat arteries by two- to threefold. Another study (17) demonstrated a near twofold acute increase of the phosphorylated eNOS-to-eNOS ratio in vitro by H₂O₂. Coincidently, our results showed that acute PEG catalase treatment decreased periarterial NO levels by about twofold in the aged WKY rats (Fig. 5). Collectively, these results, along with the current data showing effects of acute gp91ds-tat and PEG catalase exposure on flow-mediated NO (Figs. 3 and 5), illustrate that the peroxide-mediated regulation of abnormal NO production, at least under increased flow conditions, is due to increased eNOS activity, likely via phosphorylation.

The flow-mediated elevation of H₂O₂ in aged, but not young, WKY rats is consistent with observations by Gutterman and colleagues (33, 37) from studies using human resistance vessels. They found that the normal function of NO in flow-mediated dilation was replaced by H₂O₂ in both coronary and adipose arterioles from patients with coronary artery disease. Thus humans and rats with compromised vascular regulation and age as a risk factor may have a specific type of ROS dysregulation in common. PEG catalase used acutely in the current study had essentially the same effect as gp91ds-tat, with both agents restoring abnormal basal and flow-mediated NO production in aged arteries to young levels (Figs. 3 and 5). These results, along with the ability of gp91ds-tat to inhibit H₂O₂ production, suggest that NAD(P)H oxidase is the enzymatic source of elevated H₂O₂ in the aged WKY rat. At least one study with human coronary artery has shown that flow-induced dilation is mediated by peroxide derived from mitochondrial respiration (30). However, NAD(P)H oxidase has been suggested to be the source of ROS in other vessel types, including both human internal mammary arteries and saphenous veins (20). Very recent work (28) demonstrates that NAD(P)H oxidase can in fact be a significant source of peroxide in human coronary arterioles under certain conditions. NAD(P)H oxidase is considered by many to be the primary source of pathologically elevated vascular ROS (6, 7, 14, 21, 34), and there is evidence that mitochondrial sources of ROS may be trigged by increased NADPH oxidase (16). This possibility is consistent with the results from our current study showing an age-related increase in NAD(P)H oxidase-derived peroxide. Our previous study in the SHR (56) also provides support for NAD(P)H oxidase-derived peroxide in the context of vascular dysfunction, because PEG catalase and gp91ds-tat decreased periarterial H₂O₂ equivalently. Although the elevated H₂O₂ we detected may result from the dismutation of superoxide, recent evidence (15) demonstrates that peroxide may also be produced directly by Nox4 under certain conditions. The parallel results for SHR (56) and now aged rats linked through oxidant stress provide compelling evidence that some resistance vessels operate at elevated, not reduced, NO concentration and that the impaired ability to appropriately regulate NO production results from excessive H₂O₂ stimulation.

Many in vitro studies assessing NO bioavailability have based conclusions on the results of experiments measuring arterial relaxation/dilation in response to NO-generating agonists but have not considered the hemodynamic factors regulating actual in vivo NO levels or the possibility that vascular sensitivity to NO might be reduced. The relationship between NO production and flow velocity/shear rate is important to consider, because 60–80% of the resting arterial NO concentration is due to flow-mediated mechanisms (5). Results from Stasch et al. (45) indicate that a type of oxidant stress-related endothelial dysfunction exists in humans and rodents which is characterized by receptor level NO resistance due to a nonfunctional, NO-insensitive soluble guanylate cyclase (sGC) variant. Elevated ROS and NO can inactivate and downregulate sGC (18, 19, 45, 54), and the downregulation of sGC expression has been shown to occur in the aorta of aged WKY (26) and Fischer 344 (12) rats. An early stage of senescence/aging may thus be associated with limited NO scavenging that generates sufficient ROS to initiate endothelial dysfunction characterized by elevated NO and sGC insensitivity to NO, which then progresses to a later stage where extensive scavenging and the resultant decreased NO bioavailability is the dominant mechanism.

Our current findings are significant in that they suggest that during aging and under in vivo conditions of hemodynamic and humoral stimuli, eNOS is activated by H₂O₂ to the extent that a further flow-mediated production of NO cannot occur. Although many studies to date indicate superoxide scavenging of NO and the resultant decrease in NO bioavailability as an important mechanism regulating endothelial dysfunction, there may be other mechanisms that significantly impact the disposition of NO during different stages of progressive vascular aging. Future studies are warranted to investigate these possibilities and to elucidate the signaling mechanisms involved in the occurrence of age-related vascular dysfunction due to NO dysregulation.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-42898 (to J. L. Unthank) and HL-20605 (to H. G. Bohlen) and American Heart Association Midwest Affiliate Grant-In-Aid 0855658G (to S. J. Miller).

DISCLOSURES

No conflicts of interest are declared by the author(s).