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Am J Physiol Heart Circ Physiol 287: H1141-H1148, 2004; doi:10.1152/ajpheart.01215.2003
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Vasomotor responses in MnSOD-deficient mice

Jon J. Andresen,1 Frank M. Faraci,1,2 and Donald D. Heistad1,2,3

Departments of 1Internal Medicine and 2Pharmacology, University of Iowa, Roy J. and Lucille A. Carver College of Medicine; and 3Veterans Administration Medical Center, Iowa City, Iowa 52242

Submitted 22 December 2003 ; accepted in final form 2 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MnSOD is the only mammalian isoform of SOD that is necessary for life. MnSOD–/– mice die soon after birth, and MnSOD+/– mice are more susceptible to oxidative stress than wild-type (WT) mice. In this study, we examined vasomotor function responses in aortas of MnSOD+/– mice under normal conditions and during oxidative stress. Under normal conditions, contractions to serotonin (5-HT) and prostaglandin F2{alpha} (PGF2{alpha}), relaxation to ACh, and superoxide levels were similar in aortas of WT and MnSOD+/– mice. The mitochondrial inhibitor antimycin A reduced contraction to PGF2{alpha} and impaired relaxation to ACh to a similar extent in aortas of WT and MnSOD+/– mice. The Cu/ZnSOD and extracellular SOD inhibitor diethyldithiocarbamate (DDC) paradoxically enhanced contraction to 5-HT and superoxide more in aortas of WT mice than in MnSOD+/– mice. DDC impaired relaxation to ACh and reduced total SOD activity similarly in aortas of both genotypes. Tiron, a scavenger of superoxide, normalized contraction to 5-HT, relaxation to ACh, and superoxide levels in DDC-treated aortas of WT and MnSOD+/– mice. Hypoxia, which reportedly increases superoxide, reduced contractions to 5-HT and PGF2{alpha} similarly in aortas of WT and MnSOD+/– mice. The vasomotor response to acute hypoxia was similar in both genotypes. In summary, under normal conditions and during acute oxidative stress, vasomotor function is similar in WT and MnSOD+/– mice. We speculate that decreased mitochondrial superoxide production may preserve nitric oxide bioavailability during oxidative stress.

superoxide dismutase 2; oxidative stress; mitochondria; hypoxia

HOMOZYGOUS MICE that are deficient for MnSOD die within days of birth (29), whereas mice deficient for Cu/ZnSOD (44) or extracellular SOD (ECSOD) (4) appear to be normal. Thus, of the three mammalian SOD isoforms, only the mitochondrial isoform, MnSOD, is essential for life. Mice heterozygous for MnSOD (MnSOD+/–) appear to be normal, but mitochondria from these mice are dysfunctional and exhibit diminished enzymatic activity that is indicative of oxidative stress (25, 29, 58, 64). In addition, MnSOD+/– mice are more susceptible than wild-type (WT) mice to oxidative stress, including cerebral (24, 36) and myocardial (2) ischemia-reperfusion and mitochondrial inhibition by neurotoxins (1).

Under normal conditions, mitochondria are the major cellular source of reactive oxygen species (ROS) (5), and inhibition of mitochondrial respiration increases mitochondrial ROS production. Antimycin A, an inhibitor of ubiquinol-cytochrome c reductase (complex III), increases mitochondrial superoxide levels (43). In blood vessels, antimycin A reduces contraction (50, 63) and impairs endothelium-dependent relaxation by reducing nitric oxide (NO·) bioavailability (12). We tested the hypothesis that oxidative stress from antimycin A may increase superoxide and impair endothelium-dependent relaxation to a greater extent in MnSOD+/– mice than in WT mice.

Diethyldithiocarbamate (DDC) is a copper chelating agent that inhibits Cu/ZnSOD and ECSOD by chelating the active site copper from each enzyme (15, 34). In vessels, DDC increases superoxide and impairs endothelium-dependent relaxation (10,30,31,35,38,40). ROS scavengers reduce superoxide levels and restore endothelium-dependent relaxation in vessels treated with DDC (9, 38, 40, 61). We tested the hypothesis that, during inhibition of Cu/ZnSOD and ECSOD, DDC may impair endothelial function and increase superoxide to a greater extent in MnSOD+/– mice than in WT mice.

Hypoxia relaxes systemic arteries in vivo and in vitro (16, 60). Hypoxia increases mitochondrial ROS production, especially from complex III (62) [and possibly complex II (39)] of the electron transport chain. Reoxygenation after hypoxia also increases ROS production from mitochondria (27, 28). We tested the hypothesis that oxidative stress generated by hypoxia may impair vasomotor function to a greater extent in MnSOD+/– mice than in WT mice.

Thus the purpose of this study was to examine vasomotor function and superoxide levels in aortas of WT and MnSOD+/– mice under normal conditions and during acute oxidative stress. Under control conditions, we anticipated that vasomotor function may be normal in MnSOD+/– mice. Generating oxidative stress with antimycin A, DDC, and hypoxia were expected to impair vasomotor function to a greater extent in MnSOD+/– mice than in WT mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Breeding pairs of MnSOD knockout mice (CD-1 SOD2 tmlCje) were kindly provided by Dr. Pak Chan of Stanford University. Male and female mice were studied at 8–16 wk of age. Mice were killed by an intraperitoneal injection of pentobarbital sodium (150 mg/kg). Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of the University of Iowa.

Genotype was determined by PCR analysis of DNA extracted from tail snips. Three primers designed by Tsan et al. (56) were used for PCR: one common primer (5'-CGA GGG GCA TCT AGT GGA GAA GT-3'), one mutant primer (5'-TTT GTC CTA CGC ATC GGT AAT GAA-3'), and one WT primer (5'-AGG GCT CAG GTT TGT CCA GAA AAT-3'). After a hot start at 95°C for 4 min, PCR was performed under the following conditions: 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. Bands for WT and mutant MnSOD genes ran at ~500 and ~350 bp, respectively.

Vascular function. Thoracic aortas were carefully removed and placed in cold, oxygenated Krebs solution [containing (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 12 glucose]. Aortas were then gently cleaned of excess fat and connective tissue, with care taken not to remove the adventitia.

Aortic segments 3–4 mm in length were suspended on wire hooks in individual water-jacketed organ baths containing Krebs solution with 95% O2-5% N2 and kept at 37°C. Resting tension was increased from 0 to 0.5 g over 1 h, and, after this equilibration period, the vessels were twice contracted with 80 mM KCl for 5 min. Vessels were precontracted with prostaglandin F2{alpha} (PGF2{alpha}) (Pharmacia & Upjohn; Kalamazoo, MI) to ~1.0 g (~60–70% of the maximum response to PGF2{alpha}), and concentration-response curves to ACh (10–9–3 x 10–6 M, Sigma; St. Louis, MO), sodium nitroprusside (SNP; 10–9–10–4 M, Sigma), and papaverine (10–7–3 x 10–5 M, Sigma) were carried out as indicated. Contractions to serotonin (5-HT; 10–8–3 x 10–6 M, Sigma) and PGF2{alpha} (10–6–10–4 M) were also examined. Vessels were rinsed before and after each concentration-response curve and every 30 min between curves.

Vessels were treated with 0.02 µg/ml antimycin A (Sigma) for 15 min before responses were examined to ACh and SNP. Separate vessels were incubated with 1 mM DDC for 1 h before the concentration-response curves were performed. Mild hypoxia was generated by bubbling the organ bath with 9% O2-86% N2-5% CO2, and severe hypoxia was generated with 95% N2-5% CO2. In some studies, vessels were pretreated with 1 mM 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron, Sigma) for 10 min before other interventions. In some of the studies of severe hypoxia, vessels were incubated with 1 µM glybenclamide (Sigma) for 10 min or 100 µM NG-nitro-L-arginine (L-NNA, Sigma) for 30 min before determination of vasomotor responses.

Superoxide levels. Lucigenin-enhanced chemiluminescence was used to detect superoxide as previously described (33, 37). Aortas were prepared as above, and the rings were kept in ice-cold Krebs solution until use. Bis-N-methylacridinium nitrate (lucigenin, 5 µM, Sigma) was prepared fresh daily in PBS at least 30 min before use and kept in the dark at room temperature.

Vessel rings were studied under control conditions and after treatment with antimycin A (0.02 µg/ml) for 15 min. In other experiments, DDC (1 mM) or an equivalent volume of PBS was applied, and the rings were incubated for 1 h at 37°C before measurements of superoxide were made. In some experiments, vascular rings were incubated with 100 µM N{omega}-nitro-L-arginine methyl ester (L-NAME, Sigma) for 30 min, with or without DDC, before superoxide determination. After superoxide measurement, vessels were cut open longitudinally and dried flat on plastic plates. The surface area of the vessels was measured using digital photos of the vessels and the supplied tools in Scion Image (NIH, v4.0.2). Data are expressed as relative light units per second per millimeter squared.

Immunoblotting. Aortas were removed and cleaned as described above. Aortas were frozen in liquid N2 and pulverized with a pestle, followed by homogenization in 100 µl of room temperature homogenization buffer (1% SDS, 10 mM EDTA, and protease inhibitor cocktail, Roche; Basel, Switzerland). Samples were then boiled for 15 min and centrifuged at 15,000 g for 15 min. The protein concentration of the supernatant was determined using the Lowry method (Bio-Rad; Hercules, CA).

Samples were diluted 1:1 with 2x Laemmli buffer and boiled for 15 min before being loaded (5–10 µg) into wells of 4–20% SDS gels (Ready Gel, Bio-Rad). After electrophoresis at a constant 150 V for 1.5 h, proteins were transferred to nitrocellulose membranes using the semidry cell transfer method (Bio-Rad) at a constant 40 mA/gel for 45 min. After transfer, equal loading was assured by staining the nitrocellulose blots for 5 min using 0.1% Ponceau S (Sigma). The blots were then blocked in cold blocking solution (5% nonfat milk and 1% BSA) for 1 h. Blots were then incubated overnight at 4°C with the MnSOD primary rabbit antibody, which has activity toward both human and mouse MnSOD (65) (1:1,000, a gift from Dr. Larry Oberley, University of Iowa, and now available from Upstate Biotechnology; Lake Placid, NY).

Blots were then rinsed in PBS, blocked for 1 h at room temperature, and then exposed to the horseradish peroxidase-conjugated secondary antibody (1:10,000 anti-rabbit) for 1 h at room temperature. After blots were rinsed in PBS, bands were detected by chemiluminescence (Supersignal West Femto Maximum Sensitivity, Pierce; Rockford, IL). Densiometric analysis was performed using a Bio-Rad Flour-S MultiImager and the supplied software. Densiometric data are expressed as optical density units per millimeter squared.

Immunostaining. Aortas were removed and cleaned as described above. Vessels were then frozen and cut in a cryostat at 8 µm. After overnight storage at –20°C, the sections were fixed with 2% paraformaldehyde for 15 min. The sections were then washed for 15 min with PBS and air dried for 45 min at room temperature. Sections were then blocked with 8% BSA and 2% normal goat serum for 1 h before the primary antibody (anti-MnSOD made in rabbit, 1:1,000) was applied and incubated overnight at 4°C.

The sections were then rinsed with PBS for 15 min, incubated in 0.3% hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase activity, and washed in distilled H2O for 5 min. Sections were then rinsed for another 15 min in PBS and blocked in 8% BSA for 1 h. MnSOD was then visualized using a Vectastain Elite ABC kit (PK-6101, Vector Labs; Burlingame, CA) following the manufacturer's directions. Sections were then counterstained with hematoxylin and eosin, dehydrated, cleared, and coverslipped. Specificity of the antibodies was demonstrated by a lack of staining in conditions where no primary antibody was applied (data not shown).

SOD activity. Total SOD activity in aortic homogenates was measured using the nitroblue tetrazolium (NBT; Sigma) assay (47). Two aortas were pooled for each measurement. Aortic homogenates (0.5 µg/µl final concentration) were added to either standard assay buffer (1 mM diethylenetriamine pentaacetic acid, 0.13 mg/ml BSA, 0.1 mM xanthine, 0.05 mM bathocuproine disulfonic acid, 0.056 mM NBT, and 1 U/ml catalase, all from Sigma) or buffer containing 1.0 mM DDC as well. After incubation at room temperature for 1 h, xanthine oxidase was added to the samples and the rate of absorbance change at 560 nm was followed for 5 min. SOD activity was calculated as described by Spitz et al. (47).

Statistical analysis. Data are expressed as mean ± SE. Differences were analyzed with two-factor ANOVA followed by Bonferroni's multiple-comparisons posttest or two-tailed paired t-tests where appropriate. Statistical analyses were computed using InStat (version 3.05), Prism 4 (version 4.00), and Statmate (version 1.01i), all from GraphPad Software (San Diego, CA). Significance was set at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Control conditions. MnSOD is present throughout the vascular wall, and immunoreactivity was particularly intense in the endothelium of aortas from both WT (not shown) and MnSOD+/– mice (Fig. 1A). Consistent with the genotype, MnSOD protein content was reduced by 45% in aortas of MnSOD+/– mice (Fig. 1B). In the aorta, total SOD activity was 248 ± 12 U/mg in WT mice and 331 ± 41 U/mg in MnSOD+/– mice (P > 0.05, n = 7 each).



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  		Fig. 1. MnSOD in the aorta. A: aorta from a MnSOD-deficient (MnSOD+/–) mouse. MnSOD immunoreactivity is present throughout the vascular wall and is especially dense in endothelium (arrow). Magnification, x100. B: Western blot (top) for MnSOD in aortas of wild-type (WT) and MnSOD+/– mice. Bottom, densiometric analysis of lanes loaded with 5 µg protein for both WT and MnSOD+/– mice (*P < 0.05, n = 6 each). OD, optical density. Data are expressed as means ± SE.

 
Under control conditions, contractions to PGF2{alpha} (Fig. 2) and 5-HT (Fig. 3) were comparable in aortas of WT and MnSOD+/– mice. Under control conditions, responses to ACh also were similar in aortas of WT and MnSOD+/– mice (Figs. 4 and 5). Endothelium-independent relaxation to SNP and papaverine was also similar in aortas of both genotypes (not shown). Under control conditions, superoxide levels were similar in aortas of WT and MnSOD+/– mice (Fig. 6).



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  		Fig. 2. Contraction to PGF2{alpha} in aortas of WT and MnSOD+/– mice. A: responses in WT mice in the presence of vehicle (control, n = 8) or 1 mM diethyldithiocarbamate (DDC; n = 8). B: responses in MnSOD+/– mice in the presence of vehicle (control, n = 7) or 1 mM DDC (n = 8). Data are expressed as means ± SE.

 


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  		Fig. 3. Contraction to serotonin (5-HT) in aortas of WT and MnSOD+/– mice. A: responses in WT mice in the presence of vehicle (control, n = 6), 1 mM DDC (n = 6), or DDC and 1 mM tiron (n = 6). B: responses in MnSOD+/– mice in the presence of vehicle (control, n = 7), 1 mM DDC (n = 7), or DDC and 1 mM tiron (n = 6). *P < 0.05 vs. control. Data are expressed as means ± SE.

 


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  		Fig. 4. Relaxation to ACh after treatment with antimycin A. A: responses in WT mice (n = 3) in the presence of vehicle (control) and after 0.02 µg/ml antimycin A treatment. B: responses in MnSOD+/– mice (n = 5) in the presence of vehicle (control) and after 0.02 µg/ml antimycin A treatment. *P < 0.05 vs. control. Data are expressed as means ± SE.

 


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  		Fig. 5. Relaxation to ACh after treatment with DDC. A: responses in WT mice in the presence of vehicle (control, n = 11), 1 mM DDC (n = 11), or DDC and 1 mM tiron (n = 5). B: responses in MnSOD+/– mice in the presence of vehicle (control, n = 10), 1 mM DDC (n = 10), or DDC and 1 mM tiron (n = 5). *P < 0.05 vs. control. Data are expressed as means ± SE.

 


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  		Fig. 6. Aortic superoxide levels. Superoxide levels in aortas of WT (n = 11) and MnSOD+/– (n = 13) mice are shown. DDC (1 mM) increased superoxide levels (*P < 0.05 each vs. control), but the increase was greater in WT mice than in MnSOD+/– mice ({dagger}P < 0.05). Tiron (1 mM) reduced superoxide levels in DDC-treated aortas of both genotypes to control levels (P < 0.05 each vs. DDC treatment). RLU, relative light units. Data are expressed as means ± SE.

 
Effect of antimycin A. In control vessels, precontraction was 1.1 ± 0.1 g in WT mice and 1.0 ± 0.1 g in MnSOD+/– mice. Antimycin A reduced contraction to PGF2{alpha} by ~50% in both genotypes (P < 0.05 each vs. control). Antimycin A impaired endothelium-dependent relaxation to ACh similarly in WT and MnSOD+/– mice (P < 0.05 each vs. control; Fig. 4). Relaxation to SNP was not impaired by antimycin A in either genotype (not shown). Antimycin A did not detectably increase aortic superoxide levels in WT or MnSOD+/– mice (not shown).

Effect of DDC. Contraction to 5-HT (Fig. 3) but not PGF2{alpha} (Fig. 2) was enhanced by DDC in aortas of WT mice (P < 0.05 vs. control). Interestingly, in DDC-treated aortas, the maximum response to 5-HT tended to be greater in WT (43%, P > 0.05) mice than in MnSOD+/– mice. Enhanced contraction to 5-HT in DDC-treated vessels was prevented by tiron pretreatment (Fig. 3). Tiron alone did not alter contraction in control vessels (not shown).

DDC impaired maximum relaxation to ACh by ~20% in aortas of both WT and MnSOD+/– mice (P < 0.05 each vs. control; Fig. 5). Tiron pretreatment prevented DDC from impairing relaxation to ACh in aortas of both genotypes (Fig. 5). Relaxation to papaverine was not affected by DDC, tiron, or a combination of DDC and tiron in aortas of either WT or MnSOD+/– mice (not shown).

DDC increased aortic superoxide levels 2.5-fold in WT mice and 1.9-fold in MnSOD+/– mice (P < 0.05 each vs. control; Fig. 6). Surprisingly, the increase in superoxide after DDC treatment was greater in aortas of WT mice than in MnSOD+/– mice (P < 0.05). Tiron restored superoxide to control levels in DDC-treated aortas of both genotypes (Fig. 6). Treatment with L-NAME did not alter superoxide levels in control aortas or in those treated with DDC in either WT or MnSOD+/– mice (not shown).

Consistent with the increase in superoxide levels and previous studies (35, 61), DDC reduced total SOD activity from 248 ± 12 to 103 ± 20 U/mg in WT aortas and from 331 ± 41 to 128 ± 21 U/mg in MnSOD+/– aortas (P < 0.05 each vs. control). Total SOD activity in DDC-treated aortas was similar in aortas of WT and MnSOD+/– mice.

Effect of hypoxia. Severe (PO2 = 30 ± 0.4 mmHg) but not mild hypoxia (PO2 = 83 ± 2 mmHg) reduced contraction to 5-HT by a maximum of ~35% in aortas of WT and MnSOD+/– mice (P < 0.05 each vs. control, not shown). Severe hypoxia also markedly attenuated contraction to PGF2{alpha} to a similar extent in WT and MnSOD+/– mice (not shown). Maximum contraction to PGF2{alpha} under control conditions (PO2 = 596 ± 15 mmHg) was ~1.4 g in aortas of WT and MnSOD+/– mice, and severe hypoxia reduced maximum contraction by ~80% in both genotypes (P < 0.05 each vs. control, n = 4 each).

In response to mild hypoxia, precontracted aortas of both WT and MnSOD+/– mice relaxed by ~35% and regained tension after restoration of control conditions (not shown). In precontracted aortas, severe hypoxia elicited a triphasic response characterized by a transient relaxation and contraction, followed by a large, sustained relaxation (Fig. 7A). Severe hypoxia induced a transient relaxation of ~25%, followed by a transient contraction of 25 ± 4% above the level of precontraction, followed by sustained relaxation, all of which were similar in aortas of WT and MnSOD+/– mice (Fig. 7B). There was also no difference between WT and MnSOD+/– mice in the recovery of tension after reoxygenation (Fig. 7B).



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  		Fig. 7. Responses to severe hypoxia. A: response to severe hypoxia (PO2 = 30 ± 0.4 mmHg) in aortic rings precontracted with PGF2{alpha}. There are three phases to the response: transient relaxation (TR), transient contraction (TC), and maximum relaxation (MR). Recovery (Rec) of tension occurs after reoxygenation. B: summary of responses to severe hypoxia and reoxygenation in naïve (i.e., first response to hypoxia) WT ( n = 12) and MnSOD+/– (n = 15) mice. There is no significant difference in any of the responses between WT and MnSOD+/– mice. Data are expressed as means ± SE.

 
A second exposure to hypoxia did not alter vasomotor responses to mild or severe hypoxia in precontracted aortas of either WT or MnSOD+/– mice (not shown). L-NNA eliminated the transient responses to severe hypoxia in aortas of both genotypes. Neither L-NNA nor the ATP-sensitive K+ (KATP) channel inhibitor glybenclamide altered maximal relaxation to severe hypoxia in either WT or MnSOD+/– mice (not shown). In precontracted aortas of both genotypes, tiron pretreatment did not change the vasomotor response to severe hypoxia in naïve vessels or in those previously exposed to severe hypoxia (not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to examine vascular function in MnSOD-deficient mice. The major new findings of the study are as follows: 1) under normal conditions, vasomotor function and superoxide levels are similar in aortas of WT and MnSOD+/– mice; 2) the mitochondrial inhibitor antimycin A impairs endothelium-dependent relaxation to ACh to a similar degree in aortas of WT and MnSOD+/– mice without detectably increasing superoxide levels; 3) inhibition of Cu/ZnSOD and ECSOD with DDC enhances contraction to 5-HT in aortas of WT but not MnSOD+/– mice, whereas relaxation to ACh was similarly impaired by DDC in aortas of both genotypes; 4) after DDC treatment, aortic superoxide levels were greater in WT mice than in MnSOD+/– mice; and 5) contraction to 5-HT and PGF2{alpha} was impaired during hypoxia similarly in both genotypes, and vasomotor responses to hypoxia were similar in aortas of WT and MnSOD+/– mice. Overall, these finding suggest that aortas of MnSOD+/– mice are not more susceptible to endothelial dysfunction caused by acute oxidative stress.

MnSOD location and content. In aortas of WT and MnSOD+/– mice, MnSOD immunostaining was intense in the endothelium and was diffuse in the smooth muscle and adventitia. Consistent with a previous study (13), aortic MnSOD protein expression was reduced by 45% in MnSOD+/– mice. Total SOD activity was similar in aortas of the two genotypes. Because MnSOD comprises 10% (or less) of total SOD activity in the mouse aorta (49), measurement of total SOD activity may not be sufficiently sensitive to detect a reduction in SOD activity in MnSOD+/– mice. Likewise, it is difficult to determine whether the other isoforms of SOD have compensated for the reduction of MnSOD. When considered with previous studies (13, 21, 29), however, it is probable that Cu/ZnSOD and ECSOD are not upregulated in aortas of MnSOD+/– mice. We cannot rule out the possibility that changes in other proteins may influence vascular function and/or superoxide levels in MnSOD-deficient mice.

Vasomotor function and superoxide levels under normal conditions. As we hypothesized, contraction, endothelium-dependent relaxation, and superoxide levels were similar in aortas of WT and MnSOD+/– mice under normal conditions. Previous studies suggest that oxidative stress is confined to mitochondria of MnSOD+/– mice and that subjecting MnSOD+/– mice to oxidative stress is required to observe a more severe phenotype in MnSOD+/– mice (2, 25, 29, 58, 64).

Effects of antimycin A. In isolated mitochondria, antimycin A stimulates superoxide production (43, 48), but, as with other electron transport chain inhibitors, this may not occur in intact tissues (32, 39). In the present study, antimycin A impaired contraction to PGF2{alpha}, which is concordant with effects in other models (50, 63). Antimycin A alters Ca2+ handling (50) and decreases ATP levels (3, 8, 68), each of which could explain the reduction of contraction to PGF2{alpha} observed in the present study.

Antimycin A impairs relaxation to ACh in rabbit aortic strips (12), and there is an association between endothelial dysfunction and ATP depletion (51, 52). It is conceivable that ATP depletion and not superoxide production per se is the primary mechanism for endothelial dysfunction and reduced contraction produced by antimycin A seen in this study.

Effects of DDC. Contrary to our hypothesis, there was no difference in the response to ACh in aortas of WT and MnSOD+/– mice treated with DDC. Previous studies have also shown that DDC impairs endothelium-dependent relaxation in the aorta (10, 14, 30, 31, 35, 40). DDC did, however, significantly enhance contraction to 5-HT in aortas of WT mice but not MnSOD+/– mice. To our knowledge, the present data are the first to demonstrate that DDC enhances contraction to 5-HT. Interestingly, Cu/ZnSOD-deficient mice also display endothelial dysfunction typified by impaired relaxation to ACh and enhanced contraction to 5-HT (11).

DDC may act as a NO· spin trap (59), but our data are consistent with the concept that DDC produces endothelial dysfunction by increasing superoxide. The superoxide scavenger tiron restored relaxation to ACh and contraction to 5-HT to control levels in aortas of both WT and MnSOD+/– mice treated with DDC. Others have also found that scavengers of superoxide protect vessels from DDC-induced endothelial dysfunction (9, 38, 40, 41, 61). Because reducing superoxide with tiron restores endothelial function in the presence of DDC, the effects of DDC appear to be mediated primarily by elevated superoxide levels and not direct interactions between NO· and DDC.

Similar to previous studies, we found that DDC reduced total aortic SOD activity and increased superoxide (19, 35, 38, 40, 61). Surprisingly, superoxide levels in DDC-treated aortas were greater in WT mice. This unexpected result may provide an explanation for the finding that contraction to 5-HT was greater in DDC-treated aortas of WT mice, because contraction to 5-HT is modulated by NO· levels (26) and is increased by oxidative stress (17, 18, 55). Nevertheless, it is surprising that WT mice have higher levels of superoxide than MnSOD+/– mice. Two explanations were considered: increased production of NO· or reduced production of superoxide under basal conditions in MnSOD+/– mice.

Consistent with the findings that relaxation to ACh and contraction to 5-HT are similar in control aortas of WT and MnSOD+/– mice, inhibition of NO· synthase with L-NAME did not increase superoxide in vehicle- or DDC-treated aortas of either WT or MnSOD+/– mice. Thus there is evidence showing that release of NO· is not greater in the aortas of MnSOD+/– mice than in WT mice. The possibility that basal superoxide production is reduced in MnSOD+/– mice is considered below.

Effect of hypoxia. Similar to previous studies in other models (23, 46), we found that severe hypoxia attenuated contractile responses in aortas of both WT and MnSOD+/– mice. Because impairment of contraction was not greater in MnSOD+/– mice than in WT mice, the primary mechanism for impairment may not be directly related to superoxide. It is possible that other events during hypoxia, such as increased K+ conductance (53, 54) or altered responses to Ca2+ in smooth muscle (46), underlie the reduced contraction during hypoxia observed in this study.

Hypoxia produces relaxation in systemic arteries, although specific mechanism(s) are not completely defined. Similar to previous studies (45, 53, 54), we demonstrated that responses of precontracted vessels differ depending on the severity of the hypoxia. Mild hypoxia simply relaxed aortic rings, whereas the response to severe hypoxia was triphasic.

As in previous studies (7, 67), we found that the first two phases of the response to severe hypoxia were dependent on NO· synthesis, but maximum relaxation was NO· independent. Consistent with some (23, 46) but not all studies (53, 54), we did not find that glybenclamide-sensitive KATP channels are involved in the sustained relaxation to hypoxia. Mechanisms that mediate the large, sustained relaxation, while almost certainly not NO· or KATP channel dependent, are not clear.

In some (6, 32) but not other (7, 42, 46, 66) models, generation of ROS from mitochondria contributes to the vascular response to hypoxia. Although levels of MnSOD are reduced, the aortas of WT and MnSOD+/– mice respond similarly to hypoxia, and the ROS scavenger tiron did not alter vasomotor responses to hypoxia in either genotype. These findings suggest that increased ROS levels may not be important for vasomotor response to hypoxia in the mouse aorta.

In summary, levels of MnSOD are reduced in aortas of MnSOD+/– mice, and, under normal conditions, vasomotor function and superoxide levels are similar in aortas of WT and MnSOD+/– mice. Interestingly, after DDC treatment, contraction to 5-HT and superoxide levels were greater in aortas of WT mice than in MnSOD+/– mice, suggesting that superoxide is reduced in aortas of MnSOD+/– mice under normal conditions.

Previous studies have shown that mitochondrial oxygen consumption is reduced in MnSOD+/– mice (20, 29). Mitochondrial oxygen consumption is directly related to superoxide production (22, 57). We speculate that decreased oxygen consumption by mitochondria could contribute to the finding that the aortas of MnSOD+/– mice have lower superoxide levels than those of WT mice. In support of this hypothesis, impairment of vasomotor function was not greater in aortas of MnSOD+/– mice than in WT mice, and less superoxide was observed in aortas of MnSOD+/– mice after DDC treatment than in WT mice. Therefore, it seems likely that under normal conditions, aortic mitochondria produce less superoxide in MnSOD+/– mice than in WT mice.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants HL-16066, NS-24621, HL-62984, and HL-38901, the Department of Veterans Affairs Office of Research and Development, and funds from the Carver Trust for Medical Research. J. J. Andresen was also supported by fellowships AG-00214 and NS-42502.


   ACKNOWLEDGMENTS
 
We thank Dr. Yi Chu for assistance with genotyping and immunoblotting, Dr. Douglas Spitz for assistance with the SOD activity assay, Dr. William Sivitz for support and advice regarding mitochondrial function, and Pamela Tompkins for assistance with immunostaining.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. D. Heistad, Dept. of Internal Medicine, Univ. of Iowa, Roy J. and Lucille A. Carver College of Medicine, 200 Hawkins Dr., Iowa City, IA 52242 (E-mail: donald-heistad{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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