Amelioration of salt-induced vascular dysfunction in mesenteric arteries of Dahl salt-sensitive rats by missense mutation of extracellular superoxide dismutase

Abstract

Superoxide dismutase (SOD) enzymes, including extracellular SOD (ecSOD), are important for scavenging superoxide radicals (O₂^·−) in the vasculature. This study investigated vascular control in rats [SS-Sod^3m1Mcwi (ecSOD^E124D)] with a missense mutation that alters a single amino acid (E124D) of ecSOD that produces a malfunctioning protein in the salt-sensitive (Dahl SS) genetic background. We hypothesized that this mutation would exacerbate endothelial dysfunction due to elevated vascular O₂^·− levels in SS, even under normal salt (NS; 0.4% NaCl) conditions. Aortas of ecSOD^E124D rats fed standard rodent chow showed enhanced sensitivity to phenylephrine and reduced relaxation to acetylcholine (ACh) vs. SS rats. Endothelium-dependent dilation to ACh was unaffected by the mutation in small mesenteric arteries of ecSOD^E124D rats fed NS diet, and mesenteric arteries of ecSOD^E124D rats were protected from endothelial dysfunction during short-term (3–5 days) high-salt (HS; 4% NaCl) diet. ACh-induced dilation of mesenteric arteries of ecSOD^E124D rats and SS rats fed NS diet was inhibited by N^G-nitro-l-arginine methyl ester and/or by H₂O₂ scavenging with polyethylene glycol-catalase at higher concentrations of ACh. Total SOD activity was significantly higher in ecSOD^E124D rats vs. SS controls fed HS diet, most likely reflecting a compensatory response to loss of a functional ecSOD isoform. These findings indicate that, contrary to its effect in the aorta, this missense mutation of ecSOD in the SS rat genome has no negative effect on vascular function in small resistance arteries, but instead protects against salt-induced endothelial dysfunction, most likely via compensatory mechanisms involving an increase in total SOD activity.

oxidative stress, with one of its most common forms being elevated levels of superoxide anion (O₂^·−), plays a central role in vascular dysfunction in numerous diseases, including atherosclerosis, ischemia-reperfusion injury, diabetes, hyperlipidemia, and hypertension (3, 15, 16, 34). As a result, strategies to reduce the effects of superoxide have gained increasing attention for the treatment of cardiovascular disease.

The deactivation of O₂^·− occurs in two main steps. In the first, O₂^·− is converted to O₂ and H₂O₂, a reactive species that not only has considerable oxidant activity, but can also be converted to highly toxic radicals, such as hydroxyl radical and HOCl under certain conditions. Deactivation of O₂^·− is catalyzed by a group of crucially important antioxidant enzymes, the superoxide dismutases (SODs). H₂O₂ is then further processed to water and oxygen by catalase. To date, three different isoforms of SOD have been described: Cu/Zn SOD (SOD1), MnSOD (SOD2), and extracellular SOD (ecSOD or SOD3). SOD1 and SOD3 are copper- and zinc-containing proteins. These SOD isoforms participate in the steady-state regulation of O₂^·− in the cytosol and nucleus, and in the extracellular space, respectively. MnSOD is a mitochondrial enzyme that disposes of O₂^·− generated by respiratory chain activity (1, 29, 35).

SODs, in conjunction with H₂O₂ scavengers, play an important role in the regulation of oxidant stress by scavenging superoxide and H₂O₂ in the vasculature (24, 29, 36). In many species, including mouse and humans, ecSOD, a primary antioxidant enzyme secreted to the extracellular space, is highly expressed in a number of organs, including blood vessels, heart, lungs, kidney, placenta, and extracellular fluids (11, 12, 21, 40). As such, this enzyme is an important component in regulating blood pressure and vascular tone because of its ability to modulate endothelial function by controlling the levels of extracellular O₂^·− and nitric oxide (NO) availability in the vasculature (14, 17). However, the importance of ecSOD in the rat vasculature is controversial, because ecSOD in rats has a very low affinity for binding to heparan sulfate proteoglycans on cell surfaces, including the endothelial cell membrane (2). The ratio of ecSOD in the interstitium vs. the extracellular fluids and plasma is much lower in rats than in other mammalian species, and, as a result, ecSOD content is very low in rat tissues (2, 20, 21). Because of these alterations in the binding affinity of ecSOD, rats exhibit a very low level of vascular ecSOD compared with other species (2, 20). As a result, the rat interstitial space is poorly protected against oxidant stress, and enzymatic protection of the endothelium is potentially compromised (2).

Increased levels of superoxide (and other reactive oxygen species) are present during insult with high-salt (HS) diet, high-fat diet, and other external stressors. In Dahl salt-sensitive (SS) rats, endothelial function is severely impaired because of elevated vascular O₂^·− levels, even under normal salt (NS; 0.4% NaCl) baseline conditions (5, 9). The present study investigated vascular control in SS-Sod^3m1Mcwi (hereafter called ecSOD^E124D) rats with a missense mutation that alters a single amino acid (E124D) of ecSOD in the SS genetic background. The gene with a point mutation is located in the second exon of the gene and is computationally predicted to produce a malfunctioning protein by changing the structure of the active binding site.

Despite the current uncertainty regarding the importance of ecSOD in contributing to vascular oxidant defenses in the rat, Xu et al. (39) reported that monocrotaline-induced pulmonary hypertension, pulmonary vascular remodeling, right ventricular hypertrophy, and right ventricular fibrosis are exacerbated in ecSOD^E124D rats vs. wild-type controls. Those authors also verified that the enzymatic activity of EC SOD was significantly reduced (by ∼60%) in lung tissue of ecSOD^E124D rats compared with SS controls (39). In light of those findings, we hypothesized that the missense mutation in the ecSOD gene in the SS genetic background would exacerbate vascular dysfunction in systemic arteries ecSOD^E124D rats, making them more susceptible to increased oxidative stress and prone to vascular dysfunction induced by HS diet and other external stressors.

To test our hypothesis, we examined responses to vasodilator stimuli in the aorta and in small mesenteric arteries from ecSOD^E124D rats fed NS diet or challenged with HS (4% NaCl) diet for 3–5 days. As expected, we found that vascular reactivity in the aorta was altered in a manner consistent with enhanced oxidant stress and reduced NO availability on the ecSOD^E124D rats. However, contrary to our original hypothesis, the missense mutation in ecSOD led to a compensatory increase in total SOD activity and improved endothelial function in mesenteric resistance arteries of ecSOD^E124D rats fed HS diet.

MATERIALS AND METHODS

Generation of SS-SOD3^m1Mcwi (ecSOD^E124D) strain.

The ecSOD^E124D mutant strain (SS-SOD3^m1Mcwi) and the Dahl SS control strain (SS/JrHsdMcwi) utilized in the present study came from colonies at maintained the Medical College of Wisconsin (MCW) and are the same strains utilized by another group in an earlier study (39). The two strains are described in the PhysGen strains section of the PhysGen Program for Genomic Applications (PGA) website at the MCW (http://pga.mcw.edu).

In brief, SS/JrHsdMcwi (Dahl SS) male founders were injected with N-ethyl-N-nitrosourea and bred to females. The pups were genetically screened using the TILLING assay (an enzyme-based heteroduplex cleavage assay) and confirmed by nucleotide sequencing to identify and characterize target genes possessing N-ethyl-N-nitrosourea-induced mutations. The ecSOD^E124D was identified to harbor a G→T transversion in codon 124, resulting in a glutamic acid (GAG) to aspartic acid (GAT) amino acid encoding change. SIFT (http://sift.jcvi.org) predicted that this amino acid change would affect protein function. The founder animal was backcrossed to the parental strain before intercrossing, and the rats were maintained as a homozygous colony, similar to the study of Xu et al. (39). All animals used in this study were generation N2F9-F15.

Mean arterial pressures in the PGA phenotyping protocols (standard rodent chow) were not significantly different [123 ± 4.2 mmHg for SS rats (n = 32) and 116 ± 4.9 mmHg for the ecSOD^E124D rats (n = 10)]. Anesthetized blood pressures, which run higher with the anesthetic regimen used in these experiments, were not significantly different in SS and ecSOD^E124D rats fed NS (139 ± 8 mmHg, n = 6 in SS; 132 ± 5 mmHg, n = 16 in ecSOD^E124D) or short-term HS (144 ± 5 mmHg, n = 8 in SS; 136 ± 7 mmHg, n = 10 in ecSOD^E124D) diet.

Experimental animal groups.

SS or ecSOD^E124D rats were maintained on a NS (0.4% NaCl, Dyets, Bethlehem, PA) or switched to a HS (4.0% NaCl, Dyets, Bethlehem, PA) diet 3–5 days before the isolated vessel experiment to suppress plasma ANG II levels. All rats were housed with free access to food and water in an animal care facility at the MCW that is approved by the American Association for Accreditation of Laboratory Animal Care. All protocols were approved by the MCW Institutional Animal Care and Use Committee.

Evaluation of vascular reactivity in aortas and small mesenteric arteries.

To determine whether other vascular beds are affected in a similar fashion by the ecSOD^E124D mutation as the pulmonary circulation (39), we also evaluated vascular function in isolated small arteries from the intestinal mesenteric circulation and compared those results with the findings of the vascular phenotyping protocol for aortic rings posted on the PhysGen web site (http//pga.mcw.edu) for the Program for Genomic Applications at the MCW.

For studies of the mesenteric arteries, rats were anesthetized with an intramuscular injection containing ketamine (75.0 mg/kg), acepromazine (2.2 mg/kg), and xylazine (10 mg/kg) or with pentobarbital sodium (60 mg/kg ip). Small arteries (∼300-μm internal diameter, 350-μm external diameter) supplying the small intestine were isolated from the clear mesentery, transferred to a heated (37°C) chamber, and cannulated with tapered glass micropipettes in either a heated (37°C) tissue culture myograph system (Danish Myo-Technology, Aarhus, Denmark), or in an isolated vessel chamber similar to those employed in previous studies by our laboratory (27, 28, 33). The vessel was stretched to approximate its in situ length and continuously perfused and superfused with physiological salt solution having the following ionic composition (in mM): NaCl (119.0), KCl (4.7), CaCl₂ (1.6), NaH₂PO₄ (1.18), MgSO₄ (1.17), NaHCO₃ (24.0), d-glucose (5.5), and ethylenediaminetetraacetic acid (EDTA) (0.03). Intraluminal pressure was set at 75 mmHg, and the artery was allowed to equilibrate for 30 min with continuous superfusion and perfusion of the lumen with physiological salt solution equilibrated with a 21% O₂, 5% CO₂, 74% N₂ gas mixture. Vessel diameters were measured by video microscopy. Responses to vasodilator stimuli (see below) were determined in arteries precontracted with 4 μM norepinephrine (NE), and expressed as %increase from control (NE-precontracted) diameter or absolute change (Δ μm) from the NE-precontracted diameter.

Responses of the mesenteric arteries to the endothelium-dependent vasodilator agonist acetylcholine (ACh) and the NO donor diethylenetriamine (DETA)-NONOate (1 mM) in mesenteric arteries were evaluated in SS and ecSOD^E124D rats fed NS and HS diet. We also evaluated the responses of the arteries of the ecSOD^E124D rats to ACh in the presence of N^G-nitro-l-arginine methyl ester (l-NAME) (100 μM), indomethacin (1 μM), polyethylene glycol (PEG) catalase (200 U/ml) alone or in combination to assess the relative contributions of NO, cyclooxygenase metabolites, and H₂O₂ to ACh-induced dilation in resistance arteries of the mutant rats, where endothelium-dependent dilation was preserved in the presence of elevated dietary salt intake. In a final series of studies, we evaluated the effects of l-NAME, PEG-catalase, and PEG-catalase + l-NAME on arterial responses to ACh in SS rats fed NS diet, where ACh-induced dilation was still present in our initial experiments.

Western blots.

The expression of ecSOD, Cu/Zn SOD, and endothelial NO synthase (eNOS) proteins and phosphorylated eNOS (p-eNOS) (Ser1177) was evaluated by Western blotting. Briefly, samples containing 2.5 μg of protein were separated by electrophoresis using 4–20% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated at 4°C overnight with primary antibodies in 2% nonfat dry milk in Tris-buffered saline-Tween (TBST) buffer: 1) ecSOD (Santa Cruz) at 1:3,000; 2) Cu/Zn SOD (Enzo Life Sciences) at 1:6,000; 3) eNOS (BD Biosciences) at 1:3,000; 4) p-eNOS (BD Biosciences) at 1:1,000; and 5) β-actin (Sigma) at 1:35,000. Secondary horseradish peroxidase-conjugated antibodies in 2% nonfat dry milk in TBST buffer were applied at room temperature for 2 h. These included goat anti-rabbit (Sigma) at 1:6,000 for Cu/Zn SOD; goat anti-mouse (Sigma) at 1:35,000 for β-actin; and goat anti-mouse (Sigma) at 1:2,000 for ecSOD, Cu/Zn SOD, eNOS, and p-eNOS. Bands were visualized using enhanced chemiluminescence SuperSignal West Pico (Thermo Scientific), quantitated utilizing densitometry software (Un-Scan It-Orem UT), and normalized to either β-actin (eNOS, Cu/Zn SOD, ecSOD, MnSOD) or total eNOS (p-eNOS).

Measurement of SOD activity.

Total SOD activity was measured in homogenates of mesenteric arteries using the Cayman Chemical SOD-KIT (Cayman Chemical, Ann Arbor, MI). Samples were prepared and handled according to the manufacturer's protocol, with the modification of the lysis buffer containing 0.25 M sucrose, 0.1 M EDTA, 0.01 M KH₂PO₄, and 0.01 M K₂HPO₄.

Statistical methods.

Data are presented as means ± SE. For all concentration-response curves, differences between groups at each concentration were determined using a two-way, repeated-measures ANOVA. A post hoc Tukey test was used for comparison of more than two means following ANOVA. A probability value of P < 0.05 was considered to be statistically significant.

RESULTS

Aortic vascular reactivity.

Table 1 summarizes vascular phenotypes obtained in isolated aortic rings of SS rats vs. ecSOD^E124D mutants fed NS diet as part of the PhysGen Program for Genomic Applications at the MCW (http://pga.mcw.edu), and Fig. 1 compares concentration-response curves to phenylephrine and ACh in aortas of ecSOD^E124D rats and SS parental rats. In those experiments, aortas of the ecSOD^E124D rats were significantly more sensitive to phenylephrine and showed a reduced relaxation in response to ACh compared with SS rats. These findings are consistent with the predicted effects of loss of SOD activity, increased oxidant stress, and lower levels of NO to mediate ACh-induced dilation and modulate phenylephrine-induced contractions.

Table 1.

Aortic ring phenotyping studies from PhysGen Program for Genomic Applications website

	SS Rats	ecSOD Mutants
Body weight, g	299 ± 5.3 (11)	313 ± 7.3 (10)
MAP, mmHg	123 ± 4.2 (32)	116 ± 4.9 (10)
Maximum force, g/g wt	1.40 ± 0.91 (10)	1.17 ± 0.68 (9)
Fast slope, g/min	1.58 ± 0.29 (6)	1.42 ± 0.28 (8)
Slow slope, g/min	0.1 ± 0.03 (5)	0.07 ± 0.024 (9)
PE, −log EC50	7.697 ± 0.07 (11)	8.111 ± 0.07* (9)
ACh, −log EC50	6.95 ± 0.14 (10)	6.66 ± 0.57 (9)
Maximum relaxation to ACh, %	71.6 ± 6.0 (9)	54 ± 2.03* (9)

Values are means ± SE; nos. in parentheses, no. of animals. SS, salt sensitive; ecSOD, extracellular superoxide dismutase; MAP, mean arterial pressure; PE, phenylephrine; ACh, acetylcholine. Maximum force is maximum force developed by aorta in response to 80 mM K⁺ + 10⁻⁵ M PE. Fast slope is fast slope of PE-induced contraction. Slow slope is slow slope of PE-induced contraction.

^*P < 0.05 vs. SS.

Fig. 1.

Response of aortic rings from normal salt (NS)-fed extracellular superoxide dismutase (ecSOD) E124D mutant rats and salt-sensitive (SS) rats to phenylephrine (A) and acetylcholine (ACh; B). Values are summarized as means ± SE; n ≥ 8. *P < 0.05 SS vs. ecSOD^E124D.

Response of mesenteric arteries to ACh and DETA-NONOate in SS and ecSOD^E124D rats.

In contrast to the reduced response to ACh in the aorta, the magnitude of the ACh-induced dilation was not significantly different in small mesenteric arteries of SS and ecSOD^E124D rats fed NS diet (Fig. 2A). Contrary to our initial hypothesis, the ecSOD^E124D mutation had an unexpected protective effect to preserve vascular relaxation to ACh in mesenteric arteries of ecSOD^E124D rats fed HS diet compared with those from SS rats, where endothelium-dependent dilation to ACh was significantly impaired with HS diet (Fig. 2C). Vasodilator responses to the NO donor DETA-NONOate were significantly larger in mesenteric arteries of ecSOD^E124D rats fed HS diet vs. SS controls, but not in animals fed NS diet (Fig. 2, B and D).

Fig. 2.

Effect of ecSOD^E124D mutation on response of mesenteric arteries to the endothelium-dependent dilator ACh (A and C) and the nitric oxide donor diethylenetriamine (DETA)-NONOate (1 mM; B and D) in rats fed NS (A and B) or high-salt (HS; C and D) diet. Values are summarized as means ± SE; n ≥ 8. #P < 0.05 vs. SS.

In separate experiments, we evaluated potential mediators of ACh-induced dilation in mesenteric resistance arteries of ecSOD^E124D rats. Similar to the initial experiments [and contrary to studies in Sprague-Dawley rats (27, 33), mice (25), hamsters (26), and humans (32)], endothelium-dependent dilation to ACh was unaffected by HS diet in arteries of the ecSOD^E124D mutant rats (Fig. 3A). In ecSOD^E124D mutant rats fed either NS or HS diet, inhibition of NOS with l-NAME alone caused a significant reduction in the response of the arteries to 10⁻⁷ M ACh, but had no effect on dilation in response to higher concentrations of ACh (Fig. 3A). In SS rats fed NS diet, where vasodilator responses to ACh were still present, NOS inhibition with l-NAME eliminated dilation of the arteries in response to both 10⁻⁷ M and 10⁻⁶ M ACh (Fig. 3B). Inhibition of cyclooxygenase with indomethacin had no significant effect on the responses of arteries of ecSOD^E124D rats to ACh, either alone or in the presence of l-NAME (not shown). Addition of PEG-catalase to the tissue bath to scavenge H₂O₂ caused a partial inhibition of endothelium-dependent dilation to 10⁻⁷ M ACh in ecSOD^E124D rats fed either NS or HS diet and SS rats fed NS diet, with no effect on dilation of the artery in response to higher doses of ACh (Fig. 3, C–E). Combined addition of PEG-catalase and l-NAME to the tissue bath eliminated vascular relaxation in response to 10⁻⁷ M ACh in both groups of ecSOD^E124D rats and to 10⁻⁶ M ACh in HS-fed ecSOD^E124D rats only (Fig. 3, C and D). Similar to the effect of l-NAME alone (Fig. 3B), addition of l-NAME to PEG-catalase-treated arteries of SS rats fed NS diet eliminated vasodilator responses to 10⁻⁷ M ACh and 10⁻⁶ M ACh, but not 10⁻⁵ M ACh (Fig. 3E).

Fig. 3.

Endothelium-dependent dilation to ACh in small mesenteric arteries of ecSOD^E124D rats fed NS or HS diet ± N^G-nitro-l-arginine methyl ester (l-NAME; 100 μM; A); SS rats fed NS diet ± l-NAME (100 μM) (*P < 0.05 vs. NS control; B); ecSOD^E124D rats fed NS diet ± polyethylene glycol (PEG)-catalase (200 U/ml) alone or PEG catalase (200 U/ml) + l-NAME (100 μM) (*P < 0.05 vs. NS control; ‡P < 0.05 vs. NS+PEG-catalase; C); ecSOD^E124D rats fed HS diet ± PEG-catalase (200 U/ml) alone or PEG catalase (200 U/ml) + l-NAME (100 μM) (*P < 0.05 vs. HS control; ‡P < 0.05 vs. HS+PEG-catalase; D); and SS rats fed NS diet ± PEG-catalase (200 U/ml) alone or PEG catalase (200 U/ml) + l-NAME (100 μM) (*P < 0.05 vs. NS control; ‡P < 0.05 vs. NS+PEG-catalase; E). Values are mean change ± SE in diameter (μm) from norepinephrine preconstricted diameter.

Expression of Cu/Zn SOD, MnSOD, and immunoreactive ecSOD protein.

The expression of Cu/Zn SOD (Fig. 4A), MnSOD (Fig. 4B), and immunoreactive ecSOD protein (Fig. 4C) were not significantly different in mesenteric arteries of ecSOD^E124D rats fed NS diet or HS diet, compared with corresponding groups of SS rats.

Fig. 4.

Expression of Cu/Zn SOD (A), MnSOD (B), and immunoreactive ecSOD protein (C) in mesenteric arteries of SS and ecSOD^E124D rats fed NS or HS diet. Values are normalized to β-actin and expressed as means ± SE for n = 6 (Western blots).

Total SOD activity in mesenteric arteries.

Total SOD activity in mesenteric arteries of ecSOD^E124D mutant rats fed NS and HS diet are summarized in Fig. 5. SOD activity in ecSOD^E124D rats fed NS diet (16.6 ± 3.5 U/μg protein, n = 15) tended to be higher than that in SS rats fed NS diet (7.2 ± 0.87 U/μg protein, n = 9). Consistent with the restored dilation to ACh in arteries of ecSOD^E124D rats fed HS diet, increased dietary salt intake caused an approximately fivefold increase in SOD activity in mesenteric arteries of HS-fed ecSOD^E124D rats. In direct contrast to the response in vessels from the ecSOD^E124D rats, switching SS rats to HS diet caused a significant reduction in SOD activity (Fig. 5).

Fig. 5.

Total SOD activity in homogenates of mesenteric arteries from SS and ecSOD^E124D rats fed NS or HS diet. Values are means ± SE for N ≥ 5–15 per group. *P < 0.05 vs. NS diet in the same strain. #P < 0.05 vs. SS, same diet.

eNOS expression and phosphorylation.

Total eNOS expression was similar in mesenteric arteries of ecSOD^E124D rats compared with those of SS rats fed NS or HS diet (Fig. 6A). Phosphorylation of eNOS at S1177 was not significantly different in ecSOD^E124D rats and SS rats fed either diet (Fig. 6B).

Fig. 6.

Western blots comparing expression of endothelial nitric oxide synthase (eNOS) (%β-actin; A) and phosphorylation of eNOS (p-eNOS) (S1177) (%total eNOS; B) in mesenteric arteries of SS and ecSOD^E124D rats fed NS or HS diet. Values are means ± SE for n = 6.

DISCUSSION

SODs are extremely important in free radical management in the vasculature and in other tissues. Decreased levels of SODs are usually associated with increased superoxide levels and vascular dysfunction. The present study evaluated vascular reactivity to vasodilator stimuli in mesenteric resistance arteries of a strain of rats carrying a missense mutation for ecSOD^E124D in the Dahl SS genetic background.

In a recent study, Xu et al. (39) reported that monocrotaline-induced pulmonary hypertension, pulmonary vascular remodeling, right ventricular hypertrophy, and right ventricular fibrosis are exacerbated in ecSOD^E124D rats vs. wild-type controls. Those authors also verified that the enzymatic activity of ecSOD was dramatically reduced in lung homogenates of ecSOD^E124D rats carrying a whole body mutation of the ecSOD gene compared with wild-type SS controls. Because this functional mutation in the ecSOD protein is not tissue specific, the study of Xu et al. (39) and the results of the present study indicate that the functional mutation in the ecSOD protein has effects on the vasculature that are manifest, despite the low affinity of ecSOD for the endothelial cell glycocalyx and low levels of ecSOD in rat blood vessels and interstitium (2).

In our experiments, the changes in aortic vascular reactivity that occurred in the ecSOD^E124D rats, namely increased sensitivity to phenylephrine and reduced relaxation to ACh, are consistent with the predicted deleterious effects of the mutation to increase vascular oxidant stress. However, contrary to our original hypothesis, endothelium-dependent vascular relaxation in response to ACh was unaffected by the ecSOD^E124D mutation in mesenteric arteries of animals fed NS diet. Strikingly, and in contrast to multiple studies in rats (27, 33), mice (25), hamsters (26), and humans (32), switching the animals to a HS diet actually unmasked a protective effect of the mutation on endothelium-dependent vasodilation. In the latter case, HS diet caused abrogated endothelium-dependent vasodilation to ACh in the SS parental strain, but not in the ecSOD^E124D mutant rats.

One important question that arises from our observations is the identity of the mediator of ACh-induced dilation in mesenteric arteries of ecSOD^E124D mutant rats, where ACh-induced dilation is preserved in arteries of animals fed HS diet. While endothelium-dependent dilation to ACh is NO dependent in the aorta and in many other vessel types, there is increasing evidence that other compounds, including H₂O₂, acting to hyperpolarize the vascular smooth muscle cells, may serve as mediators of endothelium-dependent relaxation in response to ACh and other endothelium-dependent vasodilator stimuli in animals and humans. For example, a substantial component of endothelium-dependent dilation to ACh in mouse mesenteric arteries and to bradykinin in human mesenteric arteries (and porcine coronary microvessels) can be blocked by scavenging H₂O₂ with catalase (30).

Based on our experiments with l-NAME and catalase, and consistent with existing reports in the literature, (23, 30), it appears that NO, H₂O₂, and another as yet unidentified vasodilator compound contribute to the relaxation of mesenteric arteries in response to different concentrations of ACh. In these experiments, NO synthase alone appeared to play a greater role in mediating ACh-induced dilation of arteries of SS rats fed NS diet than those of mutant rats fed either diet, as l-NAME blocked dilation to both 10⁻⁷ M ACh and 10⁻⁶ M ACh in SS rats, but only to 10⁻⁷ M ACh in ecSOD^E124D rats fed either diet. Consistent with a strong evidence implicating it as an endothelium-dependent vasodilator (30), H₂O₂ appears to contribute to the relaxation of mesenteric arteries to 10⁻⁷ M ACh in all three groups. In the presence of PEG-catalase, the effect of NOS inhibition with l-NAME was similar to the effect of l-NAME alone in SS rats and in ecSOD^E124D rats fed NS diet. However, NO appears to play a more important role in mediating ACh-induced dilation in the presence of PEG-catalase in ecSOD^E124D rats fed HS diet, as l-NAME eliminated vasodilator responses to both 10⁻⁶ M and 10⁻⁷ M ACh in that group. The latter report is consistent with reports that the inhibitory effect of l-NAME on carbachol-induced relaxation of small mesenteric arteries of Wistar rats is greatly enhanced in the presence of catalase (37). However, dilation of mesenteric arteries to 10⁻⁵ M ACh was unaffected by l-NAME, PEG-catalase, or PEG-catalase + l-NAME in any of the groups, suggesting the presence of an additional mediator of ACh-induced vasodilation, most likely an endothelium-derived hyperpolarizing factor in both the SS rats and the ecSOD^E124D mutant rats.

In our estimation, the most intriguing finding of the present study is that exposure to HS diet increases the contribution of NO to ACh-induced dilation in small mesenteric arteries of ecSOD^E124D rats, which were protected from HS-induced endothelial dysfunction (Fig. 2). The latter observation is consistent with the approximately fivefold compensatory increase in total SOD activity that we observed in this ecSOD^E124D rats fed HS diet (Fig. 5).

The dramatic increase in total SOD activity that we observed in ecSOD^E124D rats fed HS diet supports the hypothesis that this mutation leads to compensatory responses to protect these vessels against the salt-induced vascular dysfunction and oxidant stress that occurs in mesenteric arteries of Sprague-Dawley rats (27, 41), and multiple other vascular beds of animals (25, 26, 33) and humans (32). This compensatory response to upregulate SOD activity would, in turn, result in increased NO availability and would, therefore, have a protective effect on microvascular function that is normally compromised in SS rats (4–10, 28). It is also possible that upregulation of antioxidant defenses contributes to the greater NO sensitivity that we observed in mesenteric arteries of the HS-fed ecSOD^E124D rats (Fig. 2), in light of reports that increased levels of reactive oxygen species can lower guanylyl cyclase expression and activity (13, 18, 19) and that the expression and activity of soluble guanylyl cyclase is reduced in salt-fed spontaneously hypertensive rats as a function of dietary salt intake and not elevated blood pressure. By contrast, and consistent with their impaired response to ACh, SOD activity was significantly lower in mesenteric arteries of SS rats fed HS diet compared with values measured in arteries of SS rats fed NS diet (Fig. 5).

We believe that the most likely mechanism for the dramatic increase in total SOD activity observed in the present study is a compensatory response to reduced ecSOD activity, as the ecSOD protein with the E124D mutation is predicted to generate a malfunction in the enzyme, as suggested by the studies of Xu et al. (39), who reported that eSOD activity was reduced by ∼60% in the ecSOD^E124D mutant rats. One limitation of that study was that the specificity and efficiency of the immunoprecipitation of ecSOD in samples from whole lung homogenates was not clear. In this regard, it would be important to evaluate ecSOD activity specifically in arteries of the ecSOD^E124D mutant rats, utilizing a highly specific and targeted assay, namely ConA sepharose, to isolate ecSOD from the samples taken from the vasculature (22, 31). Nonetheless, consistent with the results of our study, and supporting the hypothesis that compensatory upregulation of other antioxidant defense mechanisms plays an important role in preventing salt-induced vascular dysfunction in the ecSOD^E124D mutant rats, Gongora et al. (14) reported that Cu/Zn SOD activity is upregulated and vascular function is preserved in the aorta during ANG II infusion in mice with a genetic deletion of ecSOD (14, 17). One important finding in that study was that the increase in Cu/Zn SOD activity in response to ANG II infusion in the ecSOD knockout mice occurred without a change in Cu/Zn SOD protein expression, apparently due to an upregulation of the copper chaperone protein for Cu/Zn SOD and/or restoration of copper levels in the enzyme, which are depleted under conditions of the elevated oxidant stress (14) similar to those that exist in mesenteric arteries during HS diet (41).

With this experimental model, we have shown that creating a missense mutation in ecSOD leads to a compensatory upregulation of other mechanisms to preserve endothelium-dependent vasodilation in mesenteric resistance arteries, eventually resulting in a protective effect on vascular relaxation and endothelial function in these vessels. This paradoxical improvement of endothelium-dependent vascular relaxation in mesenteric resistance arteries of ecSOD^E124D mutant rats contrasts with the more predictable effects of the mutation on vascular reactivity in the aorta (Fig. 1 and Table 1) and the pulmonary vasculature of these animals (39), which exhibit alterations that are consistent with the predicted deleterious effects of the ecSOD^E124D mutation on endothelium-dependent vascular relaxation and NO availability.

The current findings employing the ecSOD^E124D strain with a mutation leading to a dysfunctional ecSOD raise some intriguing questions regarding the role of various SOD isoforms in contributing to antioxidant defense mechanisms in the vasculature. In an editorial commentary, Wolin (38) noted that the localized activities of different SOD forms in the vessel wall may be a key factor that influences vascular function during normal conditions and in disease processes. He postulated that the intracellular SOD isoform, Cu/Zn SOD, which regulates superoxide levels in the cytosolic region, may be the primary regulator of superoxide levels to preserve NO regulation in the vessel wall under normal conditions, while ecSOD may play a more important role in preserving NO under pathophysiological conditions where oxidases are activated and superoxide escapes the intracellular environment (38).

In their review article, Shimokawa and Morikawa (30) noted that Cu/Zn SOD activity accounts for 50–80% of all SOD activity in the vascular wall and concluded that, while ecSOD dismutates extracellular superoxide to protect the diffusion of NO, endothelial Cu/Zn SOD activity plays an important role in generating H₂O₂ from the endothelial cell membrane, to act as an endothelium-derived hyperpolarizing factor. Consistent with our hypothesis that compensatory upregulation of the activity of other antioxidant enzymes plays an important role in preventing salt-induced vascular dysfunction in the mutant rats, Gongora and coworkers (14) showed that Cu/Zn SOD activity was significantly increased in the aorta of ecSOD knockout mice, even though expression of Cu/Zn SOD protein was similar in the two strains.

In ecSOD^−/− null mice, Gongora et al. (14) reported that ANG II infusion had different effects on Cu/Zn SOD expression in the aorta (upregulated) vs. mesenteric resistance arteries (unaffected), and concluded that there are basic differences in the regulation of Cu/Zn SOD function in conduit arteries and resistance arteries of the ecSOD^−/− mice. Our findings indicate that this initial generalization is not universal to conduit vs. resistance arteries, but that there may be differences in the compensatory upregulation of Cu/Zn SOD among specific vascular beds and under different levels of oxidant stress. In this regard, an increased understanding of the mechanisms regulating the expression and activity of SOD isoforms and other antioxidant enzymes in resistance arteries and the interaction of these mechanisms under different physiological and pathophysiological conditions could improve our understanding of the environmental, genetic, and physiological factors that affect oxidant stress in pathophysiological conditions, such as stroke, hypertension, and atherosclerosis, and lead to better strategies for the treatment or modification of these factors.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-65289 (J. H. Lombard), HL-72920 (J. H. Lombard), HL-92026 (J. H. Lombard), P30–101353 (A. M. Geurts), and U01-HL-66579 (H. J. Jacob).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.M.B., A.M.G., H.J.J., M.R.D., and J.H.L. conception and design of research; A.M.B., G.R., B.D.W., K.F., M.S.R., A.M.G., and M.R.D. performed experiments; A.M.B., G.R., B.D.W., K.F., M.S.R., M.R.D., and J.H.L. analyzed data; A.M.B., G.R., B.D.W., A.M.G., H.J.J., M.R.D., and J.H.L. interpreted results of experiments; A.M.B., G.R., B.D.W., K.F., and J.H.L. prepared figures; A.M.B. and J.H.L. drafted manuscript; A.M.B., G.R., B.D.W., A.M.G., H.J.J., and J.H.L. edited and revised manuscript; A.M.B., G.R., B.D.W., A.M.G., H.J.J., M.R.D., and J.H.L. approved final version of manuscript.