Published online before print April 25, 2002, doi: 10.1161/01.RES.0000019757.57344.D5
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© 2002 American Heart Association, Inc.
Integrative Physiology |
Antioxidant Improves Smooth Muscle Sarco/Endoplasmic Reticulum Ca2+-ATPase Function and Lowers Tyrosine Nitration in Hypercholesterolemia and Improves Nitric Oxide–Induced Relaxation
From the Vascular Biology Unit, Whitaker Cardiovascular Institute, Department of Medicine and Cardiothoracic Surgery, Boston University Medical Center (T.A., R.M., S.X., M.K., H.L.L., R.A.C.), Boston, Mass, and the Department of Pharmaceutical Chemistry, University of Kansas School of Pharmacy (V.S.S., C.S.), Lawrence, Kan.
Correspondence to Richard A. Cohen, MD, Director, Vascular Biology Unit, Boston University Medical Center X708, 650 Albany St, Boston, MA 02118-2393. E-mail racohen{at}medicine.bu.edu
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Abstract |
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Antioxidants improve endothelial function in hypercholesterolemia (HC); however, whether this includes improvement of the vascular smooth muscle response to NO is unknown. NO relaxes arteries, in part, by stimulating Ca2+ uptake via sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in aortic smooth muscle, and HC impairs SERCA function and the response to NO. HC induces oxidative stress, which could impair SERCA function. To study the effect of antioxidants, which are known to improve endothelium-dependent relaxation in HC, smooth muscle SERCA activity and NO-induced relaxation were studied in rabbits fed normal chow or a 0.5% cholesterol diet for 13 weeks. The antioxidant t-butylhydroxytoluene (BHT, 1%) was mixed with the HC diet in the last 3 weeks. HC impaired acetylcholine- and NO-induced relaxation, and these were restored by BHT. After inhibiting SERCA with thapsigargin, no difference existed in NO-induced relaxation among the three groups. Reduced aortic SERCA activity in HC was restored by BHT without changing SERCA protein expression. 3-Nitrotyrosine was notably increased in the media of the HC aorta, where it colocalized with SERCA. Tyrosine-nitrated SERCA protein was immunoprecipitated in the aortas of HC rabbits, where it was decreased by BHT, and it was also detected in the aortas of atherosclerotic humans. Thus, the antioxidant reverses impaired smooth muscle SERCA function in HC, and this is correlated with the improved relaxation to NO. These beneficial effects may depend on reducing the direct effects on SERCA of reactive oxygen species that are augmented in HC.
Key Words: t-butylhydroxytoluene • nitric oxide • sarco/endoplasmic reticulum Ca2+-ATPase • atherosclerosis • nitrotyrosine
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Introduction |
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Hypercholesterolemia (HC) and other vascular diseases, including hypertension,1–3 diabetes,4,5 and hyperhomocysteinemia,6,7 may increase oxidative stress and decrease NO bioactivity, which may be associated with promoting atherosclerosis.8 Antioxidants, including ß-carotine,9 vitamin E,9,10 vitamin C,11 and probucol,12,13 improve endothelium-dependent relaxation in HC. The effects of these antioxidants do not depend on lowering plasma cholesterol levels,12,13 suggesting that oxidative stress in HC is related to vascular dysfunction.14 However, the protective mechanism of antioxidants on NO bioactivity has not yet been elucidated.
Oxidative stress may impair NO release15 or endothelial NO synthase function,16 although increased NO release in HC has also been reported.17 Increased scavenging of NO by superoxide may decrease NO bioactivity; however, a few hours of incubation with antioxidants18,19 or adenovirus-mediated gene transfer of superoxide dismutase20 fails to normalize endothelium-dependent relaxation, suggesting that a decrease in superoxide generation is not enough to immediately restore NO bioactivity in HC.
Another possible factor is that oxidative stress impairs the function of target proteins of NO in aortic smooth muscle. NO activates guanylyl cyclase and relaxes arteries, and its activity is preserved at least during the early stages of HC.19,21,22 Thus, sodium nitroprusside–induced relaxations that depend on the activation of guanylyl cyclase and those caused by 8-bromo-cGMP are preserved in HC.19 K+ channels are directly activated by NO23; however, K+ channel–mediated relaxation by NO is preserved in HC carotid arteries.22 We have provided evidence that NO induces relaxation, in part, by stimulating the refilling of intracellular Ca2+ stores via smooth muscle sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which thereby decreases intracellular Ca2+ levels and inhibits store-dependent Ca2+ influx via plasma membrane ion channels.24,25 This process is, at least partially, cGMP independent.19,25 Moreover, we have shown that HC impairs SERCA function and decreases the cGMP-independent SERCA-dependent smooth muscle response to NO.19
The reduced SERCA activity in HC is not due to decreased SERCA protein levels.21 Rather, SERCA2, which is the primary isoform in vascular smooth muscle, is sensitive to oxidative stress.26 For example, hydroxyl radical,27 peroxide,28 hypochlorous acid,29 and peroxynitrite30 all decrease SERCA activity. Therefore, SERCA activity and NO-induced relaxation might be impaired by oxidative stress in HC.
We hypothesized that chronic increases in oxidative stress can impair SERCA function and decrease the smooth muscle response to NO in HC. To test this hypothesis, the antioxidant t-butylhydroxytoluene (BHT, 1%) was mixed with a cholesterol-enriched diet,31,32 and its effects on relaxation to NO, SERCA activity, and oxidative modifications in SERCA were tested.
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Materials and Methods |
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Reagents
NO was obtained from Matheson Gas. A saturated NO solution (
3 mmol/L) was prepared at 4°C and depleted of anionic oxidation products of NO with an anion exchange resin, as described previously.18,19,22,33,34 Monoclonal SERCA2 antibody was from Affinity Bioreagents (clone IID8). Polyclonal antibody for 3-nitrotyrosine was from Upstate Biochemicals. Anti-mouse IgG and anti-rabbit IgG antibody were from Pierce. Rhodamine red and fluorescein-conjugated secondary antibody were purchased from Jackson Immuno-Research Laboratories. 45CaCl2 was obtained from New England Nuclear. All other reagents were from Sigma Chemical Co.
Animals
Male New Zealand White rabbits were assigned to control and cholesterol-fed groups randomly, and they received a diet (Agway Prolab, Agway) or chow supplemented with 0.5% cholesterol (wt/wt) and 4% peanut oil (wt/wt) for 13 weeks, as previously reported.18,19,22,34 In some rabbits, BHT was mixed with the cholesterol-enriched diet during the last 3 weeks.
Human Aortic Biopsies
Biopsies of atherosclerotic human aortas were obtained at the time of coronary artery bypass surgery from the sites of insertion of vein grafts. Atherosclerosis was diagnosed by coronary artery angiogram in each of the 3 male and 2 female patient samples analyzed in the present study. The average age of the patients was 64±3 years. On curettage from the aorta, the biopsies were placed in saline and transported immediately to the laboratory, where they were frozen for later homogenization and analysis as for the rabbit aortic samples. The protocol was approved by the Institutional Review Board for Human Research.
Plasma and Aortic Cholesterol Levels
Plasma cholesterol levels were measured with a chemiluminescent assay kit (Sigma). Rabbit aortic cholesterol levels were measured as described.35
Isometric Tension Measurement
The detailed methods have previously been reported. The portion of the descending thoracic aorta 3 to 5 cm from top of the arch was studied. In some rings, the endothelium was removed mechanically. Rings were mounted at optimal resting tension (6 g) at which contractions to phenylephrine or elevated K+ were found to be not significantly different in normal and HC rabbits.12 Some rings without endothelium were incubated with a specific SERCA inhibitor, thapsigargin (10 µmol/L),36 for 1 hour. Thereafter, rings were contracted by adding phenylephrine to cause a 40% to 50% maximal contraction. Rings without endothelium were relaxed by logarithmic increments of NO concentration, and rings with endothelium were relaxed by half-logarithmic increments of acetylcholine concentration.18,19,22
SERCA Activity by 45Ca2+ Uptake
SERCA activity was measured by 45Ca2+ uptake in a postnuclear supernatant fraction, as previously reported.19,26 The midportion of the descending aorta (5 to 7 cm from the top of the arch) was homogenized in sucrose buffer (100:1 [wt/vol]; 0.34 mol/L sucrose, 1 mmol/L phenylmethylsulfonyl fluoride, and 2 mmol/L dithiothreitol) with a sintered glass-on-glass homogenizer on ice. The homogenate was centrifuged at 4000 rpm for 2 minutes at 4°C, and the supernatant was assayed. To determine SERCA-dependent uptake, paired samples were treated with thapsigargin (10 µmol/L, 15 minutes) at 37°C. 45Ca2+ uptake buffer (30 mmol/L Tris-HCl, pH 7.0, 100 mmol/L KCl, 5 mmol/L NaN3, 6 mmol/L MgCl2, 0.15 mmol/L EGTA, 0.12 mmol/L CaCl2, and 10 mmol/L oxalate) was mixed with 1 µCi 45Ca2+ and 2 mmol/L ATP at 37°C. The reaction was started by adding 100 µL protein (250 to 400 µg, total volume 500 µL). Aliquots of this mixture (100 µL) were filtered through Whatman GF/C glass filters at 10, 30, and 60 minutes. The filters were rinsed 2 times with 2.5 mL wash buffer (30 mmol/L imidazole, 250 mmol/L sucrose, and 0.5 mmol/L EGTA). 45Ca2+ uptake was calculated by counting the radioactivity collected by the filters and standardized by protein concentration determined by the Bradford method.
Western Blot Analysis
The remaining rabbit descending aorta was homogenized in Tris-sucrose buffer with 1% Triton X-100 and protease inhibitors. After centrifugation at 14 000 rpm for 15 minutes at 4°C, the supernatant was mixed with Laemmli buffer and applied to 7.5% SDS-PAGE gels (10 µg protein per lane) for separation. Proteins were transferred to nitrocellulose paper, and the membrane was incubated with anti-SERCA2 (clone IID8 [1:2500, overnight], Affinity Bioreagents) or 
-actin (1:5000, 2 hours, Sigma) antibody. The secondary anti-mouse IgG (1:50 000, Pierce) was incubated, and bands were detected by enhanced chemiluminescence.19
Immunoprecipitation With Anti-Nitrotyrosine or Anti-SERCA Antibody
Approximately 1 mg protein of aortic extract was diluted in 500 µL lysis buffer. After a preclearing with protein A or G agarose (30 µL, Santa Cruz), the supernatant was mixed with 10 µL polyclonal anti-nitrotyrosine or SERCA2 antibody and incubated overnight at 4°C. Prewashed protein A or G agarose (50 µL) was then added to the samples, and after 1-hour further incubation, the immunocomplex was resuspended in Laemmli sample buffer (50 µL) containing 5% mercaptoethanol and loaded onto SDS-PAGE gels.37
Immunohistochemistry for 3-Nitrotyrosine
Some rings from thoracic aortas were placed in 4% formalin overnight and embedded in paraffin. After removal of paraffin and rehydration, slides were treated with 10 mmol/L citric acid (pH 6) and were microwave-heated (2 minutes, 3 times at 700 W) to recover antigenicity. Nonspecific binding was blocked with 10% normal goat serum in PBS (pH 7.4) for 30 minutes before incubation with polyclonal anti-nitrotyrosine antibody (1 µg/mL, Upstate Biotechnology) in PBS with 1% BSA overnight at 4°C. Tissue sections were then incubated for 30 minutes at room temperature with a biotinylated anti-rabbit IgG (1:800) secondary antibody with use of the Vectastain ABC kit (Vector). Vector red alkaline phosphate substrate was used to visualize positive immunoreactivity for 3-nitrotyrosine. Preincubation of the anti–3-nitrotyrosine antibody with free 3-nitrotyrosine (10 mmol/L) nearly eliminated staining in HC, indicating that the staining is specific (data not shown).3,38 Three observers who were blinded to the sample identity performed semiquantitative analysis of tissue immunoreactivity for 3-nitrotyrosine in the smooth muscle layer by using an arbitrary grading system from 0 to 3 to estimate the degree of positive medial staining.3,38,39
Sections of rabbit aorta labeled with fluorescent antibodies were imaged by using a 2-photon scanning confocal microscope designed and constructed in collaboration with the laboratory of Dr Peter So (Massachusetts Institute of Technology, Cambridge, Mass). The light source was a diode laser–pumped titanium sapphire laser (Coherent). The excitation pulses were centered in wavelength at 800 nm, and the average power was reduced to 100 mW by using polarizing optics. Fluorescence emission wavelengths were separated by a 565-nm dichroic mirror, and the emission filters were centered at 605 nm with a 55-nm bandwidth and at 535 nm with a 30-nm bandwidth for the long- and short-emitted wavelengths, respectively (Chroma Technology). Images were 512x512 pixels and were analyzed by using NIH Image J and Adobe Photoshop.
3-Nitrotyrosine Chemical Analysis
Amino acid analysis was performed as described40 on SERCA immunoprecipitated from rabbit and human aortic samples, run on SDS-PAGE gels, transferred, and then eluted from polyvinylidine difluoride (PVDF) membranes. The phenylthiocarbamate-derivatized amino acids were separated on a 250x4.6-mm Waters Pherisorb S5 ODS2 C18 column by using the following gradient (for mobile phase A, 10 mmol/L potassium phosphate, pH 6.5; for mobile phase B, 30% A/70% acetonitrile [vol/vol]): equilibration of the column with 100% A; linear gradients increasing B from 0% to 11% between 0 and 15 minutes, increasing B to 14% between 15 to 17 minutes, increasing B to 36% between 17 and 39 minutes, and increasing B to 100% between 39 to 42 minutes, followed by isocratic elution with 100% B between 42 and 48 minutes. Results are expressed as moles of 3-nitrotyrosine per mole of 110-kDa protein, which was shown by mass spectrometry to contain SERCA in rabbit samples.
Statistical Analysis
Relaxations to NO were analyzed by the maximum response after adding each concentration and expressed as a percentage of the contraction to phenylephrine. Data are expressed as mean±SEM. Statistical evaluation among three groups was performed by using a repeated-measures ANOVA (SAS, Inc). The bands on Western blots were quantified by densitometry (Molecular Analyzer). Differences between two groups at individual concentrations of NO and band densities were analyzed with the Student t test. A value of P<0.05 was considered to be statistically significant.
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The HC diet increased plasma cholesterol levels, and BHT did not significantly affect them (Figure 1A). HC also increased aortic cholesterol levels, and BHT partially decreased them, although the levels remained significantly 12-fold higher than normal (Figure 1B).
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P<0.05 vs HC.HC significantly decreased acetylcholine- and NO-induced relaxation, both of which were restored by BHT treatment (Figures 2A and 2B). Thapsigargin significantly inhibited NO-induced relaxation in the normal chow group (Figure 2C; at 1 µmol/L NO, 59±4% for control versus 31±5% for thapsigargin); however, thapsigargin did not significantly inhibit NO-induced responses in the HC group (28±6% for control versus 24±5% for thapsigargin). Thapsigargin inhibited NO-induced relaxation in aortas of BHT-treated rabbits similar to that in normal rabbit aortas (59±3% for control versus 24±2% for thapsigargin). After treatment with thapsigargin, the residual relaxation to NO was not significantly different in the three groups (Figure 2C), suggesting that HC selectively inhibits SERCA-dependent relaxation to NO and that BHT restores it.
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Total 45Ca2+ uptake in aortic homogenates was markedly decreased in HC and recovered with BHT treatment (Figure 3A). Thapsigargin-insensitive 45Ca2+ uptake was not different among the three groups (Figure 3A). SERCA activity, defined as thapsigargin-sensitive 45Ca2+ uptake, was decreased to 30% of control in HC and recovered to 70% of control with BHT treatment (Figure 3B).
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To test whether the improvement of SERCA function caused by BHT was due to increased SERCA2 protein expression, Western blotting for SERCA2 and -actin was performed in HC and BHT-treated groups. Densitometry showed that the expression of SERCA2 did not change significantly with BHT (18±4 U [HC, n=4] versus 17±2 U [BHT, n=4], P=0.81). The ratio of SERCA2 to -actin expression by Western blot also did not significantly change with BHT (Figure 4B; 0.6±0.1 [HC] versus 0.6±0.1 [BHT], P=0.74).
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The similar expression of SERCA2 protein suggested that posttranslational modification of SERCA could explain its impaired activity in HC and restoration by BHT. SERCA2 activity is known to be inhibited by peroxynitrite30,40,41 or hypochlorous acid,29 both of which are generated in atherosclerotic human and rabbit arteries, where they could possibly generate 3-nitrotyrosine protein moieties.38,39,42 Therefore, we performed immunohistochemistry for 3-nitrotyrosine in the aortas of rabbits from the three groups. Compared with normal arteries (Figure 5A), 3-nitrotyrosine staining increased in HC not only in the endothelium, plaque, and adventitia but also, notably, in medial smooth muscle (Figure 5B). In the BHT-treated group, staining for 3-nitrotyrosine was nearly eliminated in medial smooth muscle, although less intense staining remained in the plaque, endothelium, and adventitia (Figure 5C). Semiquantitative analysis of the 3-nitrotyrosine staining in the smooth muscle region showed a visibly significant increase in 3-nitrotyrosine staining score in HC that was significantly decreased by BHT treatment (0.3±0.1 units for normal versus 2±0.5 for HC and 0.9±0.3 for HC+BHT).
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To determine whether 3-nitrotyrosine is colocalized with SERCA in HC rabbit aorta, double staining with anti-SERCA2 (Figure 6A, rhodamine red) and nitrotyrosine (Figure 6B, fluorescein) antibodies was performed and examined by 2-photon confocal microscopy. Staining for 3-nitrotyrosine and SERCA clearly colocalized in the smooth muscle. Control studies indicated that secondary antibodies alone resulted in minimal staining and that negligible crossover occurred between the two fluorescence indicators.
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To further establish whether increased 3-nitrotyrosine was formed on SERCA protein in HC, immunoprecipitates of aortic proteins obtained with polyclonal anti-nitrotyrosine antibody were separated by SDS-PAGE and stained with anti-SERCA antibody. Despite the similar expression of SERCA2 protein among the groups, HC markedly increased SERCA in the 3-nitrotyrosine immunoprecipitates, and BHT decreased it (Figure 7A). If we eliminated the anti-nitrotyrosine antibody or aortic proteins during the immunoprecipitation, the immunostaining of SERCA was absent. We also confirmed that the immunoprecipitate was not stained with the secondary antibody used to detect SERCA (data not shown). Densitometry showed significant increases in SERCA protein present in the 3-nitrotyrosine immunoprecipitate obtained from HC rabbit aortic samples, and the amount was decreased by BHT (Figure 7B).
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Because 3-nitrotyrosine was noted previously by immunohistochemistry in human atherosclerotic lesions, 3-nitrotyrosine in SERCA derived from atherosclerotic human aortic biopsies was assessed. Immunoprecipitated SERCA2 was stained positively with the polyclonal anti-nitrotyrosine antibody, and the staining was prevented with preabsorption of the antibody with 10 mmol/L 3-nitrotyrosine (Figure 7C).
To confirm the increase in 3-nitrotyrosine in SERCA2, after the protein was immunoprecipitated from HC rabbit and human aortic homogenates, the 110-kDa protein band was eluted from the PVDF membranes, and amino acid analysis was performed by protein hydrolysis and high-performance liquid chromatography (HPLC).14 In 4 normal rabbit aortic samples, there was 0.08±0.06 mol 3-nitrotyrosine/mol protein. In contrast, the 3-nitrotyrosine content in aortas from 4 HC rabbits was increased 20-fold to 1.6±0.7 mol/mol protein (P<0.01, Figure 7D). SERCA that was immunoprecipitated from human aortas also contained 3-nitrotyrosine (n=5, 1.6±0.1 mol/mol protein). These measurements verify that immunoprecipitates containing SERCA obtained from rabbit and human aortas contain authentic 3-nitrotyrosine.
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Discussion |
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A number of studies indicate that antioxidants can improve endothelium-dependent relaxation in HC.9–13 HC not only decreases endothelium-dependent relaxation but also decreases the smooth muscle response to NO that is inherently important in mediating endothelium-dependent relaxation to agents such as acetylcholine. The antioxidant BHT dramatically improved the smooth muscle response to NO, and this was mirrored in improved endothelium-dependent relaxation to acetylcholine, suggesting that the beneficial effects of antioxidant may depend not only on endothelial cell function but also on improvement in the smooth muscle response to NO. Acceleration of sarcoplasmic reticulum (SR) Ca2+ uptake via SERCA is the principle mechanism by which NO decreases intracellular Ca2+ levels contributing to smooth muscle relaxation.27 In other studies,19 we have shown that HC impairs the relaxation to NO in a SERCA-dependent fashion. That is, after the inhibition of SERCA with either thapsigargin (the present study) or cyclopiazonic acid (CPA),19 there was no difference in relaxation between normal and HC rabbit aortas. In contrast, relaxations to sodium nitroprusside were found to depend to a negligible extent on SERCA function and were preserved in HC.19 These findings suggest that HC selectively impairs relaxation to authentic NO via impairment of SR Ca2+ uptake and that BHT reverses this. Moreover, BHT improved the impaired thapsigargin-sensitive SR Ca2+ uptake in HC rabbit aortas. There was no change in SERCA protein expression, suggesting that SERCA activity is reduced in HC by oxidant mechanisms and that the effect of oxidants on SERCA can account not only for the reduced relaxation caused by NO but also for the improvement caused by antioxidants such as BHT. The evidence that Ca2+ uptake via SERCA is required for a normal response to NO is admittedly indirect, on the basis of the use of thapsigargin and CPA19 and kinetic arguments.25 However, the impairment of SERCA activity in HC and the fact that either thapsigargin or CPA masks the impairment of NO-induced relaxation in HC also represent evidence that SERCA is involved in the response.
HC increases superoxide generation not only in the endothelium and adventitia but also in medial smooth muscle.20 An increase in superoxide anion generation in smooth muscle has also been reported in streptozocin-induced diabetes.5,43 NO has a long enough half-life to diffuse to all layers of the blood vessel wall, unless it reacts with superoxide and forms peroxynitrite. Detection of 3-nitrotyrosine in both rabbit and human arteries has been presented as evidence of peroxynitrite formation. 3-Nitrotyrosine can also be generated from hypochlorous acid and nitrite by myeloperoxidase.42 An increase in these reactive species in HC smooth muscle is suggested in the present study by a marked increase in 3-nitrotyrosine immunostaining, which was dramatically decreased with BHT. SERCA represents at least one of the 3-nitrotyrosine–modified proteins as shown by immunochemical colocalization as well as immunoprecipitation and HPLC techniques. We also detected 3-nitrotyrosine on SERCA derived from human atherosclerotic aortas by immunoblotting and HPLC. The fact that BHT reduced 3-nitrotyrosine associated with SERCA suggests that BHT treatment reduces or prevents the actions of the reactive nitrogen species responsible.
SERCA2, the major smooth muscle isoform, is an oxidant-sensitive protein, and numerous oxidants have been reported to impair its activity, including hydroxyl radical,27 peroxide,28 hypochlorous acid,29 and peroxynitrite.30 In the present study, SR Ca2+ uptake activity was measured under strong reducing conditions provided by dithiothreitol. Therefore, any oxidative changes in the protein responsible for the reduced activity were not easily reversible. Peroxynitrite was shown to inhibit SERCA activity in skeletal muscle, and the reduced function was also not recovered with dithiothreitol.30 We have also found that treatment of aortic homogenates with peroxynitrite or hypochlorous acid inhibits SERCA activity (data not shown); thus, it is possible that these oxidants are involved in vivo. In addition, thiol oxidation inhibits the activity of SERCA,30,41 and peroxynitrite can oxidize Met, Lys, Phe, Thr, Ser, and Leu on SERCA protein30 as well. Thus, although it is not clear whether tyrosine nitration is directly responsible for the impairment of SERCA activity, 3-nitrotyrosine does serve as a molecular marker of exposure of proteins to oxidants in vivo. Furthermore, the association of 3-nitrotyrosine with SERCA, its decrease in function in HC, and its restoration during treatment with an antioxidant strongly suggest that oxidants are responsible for the alteration in protein function.
BHT has been reported to decrease lipid peroxidation and to improve atherosclerosis in hypercholesterolemic animals.31 In the present study, treatment during the final 3 weeks of administration of a high cholesterol diet did not change plasma cholesterol levels, although it did modestly decrease aortic cholesterol levels. This indicates that accumulation of cholesterol, a marker of atherogenesis, was significantly decreased by this relatively brief treatment. Therefore, we cannot completely exclude the possibility that the improvement of SERCA function may partially depend on the amount of cholesterol or atherosclerosis. However, the cholesterol content remained 12-fold higher than normal in the BHT-treated group, whereas acetylcholine- and NO-induced relaxations were completely restored. Previously, Simon et al12 and Keaney et al13 showed that short-term treatment with probucol, structurally a dimer of BHT, improved endothelium- dependent relaxation in rabbits, which was unexplained by a decrease in aortic cholesterol. Probucol was also shown to decrease aortic superoxide levels.13 Thus, reduced levels of superoxide anion might explain not only the restoration of function observed in the present study but also the reduced presence of 3-nitrotyrosine on SERCA in the aortic media. The oxidative modifications demonstrated in the present study allow us to speculate that oxidant mechanisms acting directly on SERCA can account for the reduction in vascular function associated with HC and its restoration by BHT.
Improvement of endothelium-dependent relaxation precedes the regression of atherosclerosis caused by cholesterol lowering44 or antioxidant treatment, suggesting that improvement of NO bioactivity can limit the progression of atherosclerosis.8 The present study suggests that a major effect of antioxidants, ie, improvement of endothelium-dependent relaxation, is mediated by improvement in smooth muscle responsiveness to NO. This improvement in smooth muscle function was associated with reduced oxidative modification of SERCA protein and improved thapsigargin-sensitive Ca2+ uptake. This suggests that preservation of SERCA function may be regarded as a new target for the treatment of impaired vascular function, which precedes atherosclerosis caused by HC.
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Acknowledgments |
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This study was supported by NIH grants HL-31607, HL-55993, HL-55620, and P01 AG-12993 and grants from the Juvenile Diabetes Foundation and the American Heart Association (AHA). Dr Adachi was supported by an AHA Postdoctoral Fellowship from the New England Affiliate.
Received August 3, 2001; revision received April 15, 2002; accepted April 16, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. J Clin Invest. 1996; 97: 1916–1923.[Medline] [Order article via Infotrieve]
2.
Wang HD, Hope S, Du Y, Quinn MT, Cayatte AJ, Pagano PJ, Cohen RA. Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II–induced hypertension. Hypertension. 1999; 33: 1225–1232.
3.
Wang HD, Xu S, Johns DG, Du Y, Quinn MT, Cayatte AJ, Cohen RA. Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice. Circ Res. 2001; 88: 947–953.
4. Cohen RA. Dysfunction of vascular endothelium in diabetes mellitus. Circulation. 1993; 87: V67–V76.
5.
Lund DD, Faraci FM, Miller FJJr, Heistad DD. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation. 2000; 101: 1027–1033.
6. Loscalzo J. The oxidative stress of hyperhomocyst(e)inemia. J Clin Invest. 1996; 98: 5–7.[Medline] [Order article via Infotrieve]
7. Eberhardt RT, Forgione MA, Cap A, Leopold JA, Rudd MA, Trolliet M, Heydrick S, Stark R, Klings ES, Moldovan NI, Yaghoubi M, Goldschmidt-Clermont PJ, Farber HW, Cohen RA, Loscalzo J. Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia. J Clin Invest. 2000; 106: 483–491.[Medline] [Order article via Infotrieve]
8. Cohen RA. The role of nitric oxide and other endothelium-derived vasoactive substances in vascular disease. Prog Cardiovasc Dis. 1995; 38: 105–128.[CrossRef][Medline] [Order article via Infotrieve]
9.
Keaney JFJr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Dietary antioxidants preserve endothelium-dependent vessel relaxation in cholesterol-fed rabbits. Proc Natl Acad Sci U S A. 1993; 90: 11880–11884.
10.
Keaney JFJr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Low-dose -tocopherol improves and high-dose -tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest. 1994; 93: 844–851.[Medline]
[Order article via Infotrieve]
11.
Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JFJr, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996; 93: 1107–1113.
12. Simon BC, Haudenschild CC, Cohen RA. Preservation of endothelium-dependent relaxation in atherosclerotic rabbit aorta by probucol. J Cardiovasc Pharmacol. 1993; 21: 893–901.[Medline] [Order article via Infotrieve]
13. Keaney JFJr, Xu A, Cunningham D, Jackson T, Frei B, Vita JA. Dietary probucol preserves endothelial function in cholesterol-fed rabbits by limiting vascular oxidative stress and superoxide generation. J Clin Invest. 1995; 95: 2520–2529.[Medline] [Order article via Infotrieve]
14. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial SO production. J Clin Invest. 1993; 91: 2546–2551.[Medline] [Order article via Infotrieve]
15. Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest. 1999; 103: 897–905.[Medline] [Order article via Infotrieve]
16.
Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001; 103: 1282–1288.
17. Minor RLJr, Myers PR, Guerra RJr, Bates JN, Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest. 1990; 86: 2109–2116.[Medline] [Order article via Infotrieve]
18. Adachi T, Cohen RA. Decreased aortic glutathione levels may contribute to impaired nitric oxide-induced relaxation in hypercholesterolaemia. Br J Pharmacol. 2000; 129: 1014–1020.[CrossRef][Medline] [Order article via Infotrieve]
19.
Adachi T, Matsui R, Najibi S, Cohen RA. Reduced sarco/endoplasmic reticulum Ca2+ uptake activity can account for the reduced response to NO, but not sodium nitroprusside, in hypercholesterolemic rabbit aorta. Circulation. 2001; 104: 1040–1045.
20.
Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.
21. Schmidt K, Klatt P, Mayer B. Hypercholesterolemia is associated with a reduced response of smooth muscle guanylyl cyclase to nitrovasodilators. Arterioscler Thromb Vasc Biol. 1993; 12: 1159–1163.
22. Najibi S, Cohen RA. Enhanced role of potassium channels in relaxation of hypercholesterolemic rabbit carotid artery to nitric oxide and sodium nitroprusside. Am J Physiol. 1995; 269: H805–H811.[Medline] [Order article via Infotrieve]
23. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle cells. Nature. 1994; 368: 850–853.[CrossRef][Medline] [Order article via Infotrieve]
24.
Trepakova ES, Cohen RA, Bolotina VM. Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase–dependent refilling of Ca2+ stores. Circ Res. 1999; 84: 201–209.
25.
Cohen RA, Weisbrod RM, Gericke M, Yaghoubi M, Bierl C, Bolotina VM. Mechanism of nitric oxide–induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx. Circ Res. 1999; 84: 210–219.
26. Grover AK, Samson SE. Effect of superoxide radical on Ca2+ pumps of coronary artery. Am J Physiol. 1988; 255: C297–C303.[Medline] [Order article via Infotrieve]
27.
Xu KY, Zweier JL, Becker LC. Hydroxyl radical inhibits sarcoplasmic reticulum Ca2+-ATPase function by direct attack on the ATP binding site. Circ Res. 1997; 80: 76–81.
28. Grover AK, Samson SE, Fomin VP. Peroxide inactivates calcium pumps in pig coronary artery. Am J Physiol. 1992; 263: H537–H543.[Medline] [Order article via Infotrieve]
29.
Favero TG, Colter D, Hooper PF, Abramson JJ. Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. J Appl Physiol. 1998; 84: 425–430.
30. Viner RI, Hühmer AFR, Bigelow DJ, Schöneich C. The oxidative inactivation of sarcoplasmic reticulum Ca2+-ATPase by peroxynitrite. Free Radic Res. 1996; 24: 243–259.[Medline] [Order article via Infotrieve]
31.
Bjorkhem I, Henriksson-Freyschuss A, Breuer O, Diczfalusy U, Berglund L, Henriksson P. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arterioscler Thromb Vasc Biol. 1991; 11: 15–22.
32. Freyschuss A, Stiko-Rahm A, Swendenborg J, Henriksson P, Björkhem I, Berglund L, Nilsson J. Antioxidant treatment inhibits the development of intimal thickening after balloon injury of the aorta in hypercholesterolemic rabbits. J Clin Invest. 1993; 91: 1282–1288.[Medline] [Order article via Infotrieve]
33.
Weisbrod RM, Griswold MC, Du Y, Bolotina VM, Cohen RA. Reduced responsiveness of hypercholesterolemic rabbit aortic smooth muscle cells to nitric oxide. Arterioscler Thromb Vasc Biol. 1997; 17: 394–402.
34. Weisbrod RM, Griswold MC, Yaghoubi M, Komalavilas P, Lincoln TM, Cohen RA. Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide. Br J Pharmacol. 1998; 125: 1695–1707.[CrossRef][Medline] [Order article via Infotrieve]
35. Morin RJ. Rapid enzymatic determination of free and esterified cholesterol of serum and tissue. Clin Chim Acta. 1976; 71: 75–80.[CrossRef][Medline] [Order article via Infotrieve]
36. Inesi G, Sagara Y. Specific inhibitors of intracellular Ca2+ transport ATPases. J Membr Biol. 1994; 141: 1–6.[Medline] [Order article via Infotrieve]
37. Grover AK, Xu A, Samson SE, Narayanan N. Sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle is regulated by a novel pathway. Am J Physiol. 1996; 271: C181–C187.[Medline] [Order article via Infotrieve]
38.
Luoma JS, Stralin P, Marklund SL, Hiltunen TP, Sarkioja T, Yla-Herttuala S. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1998; 18: 157–167.
39. Beckman JS, Ye YZ, Anderson PG, Chen J, Accavitti MA, Tarpey MM, White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem. 1994; 375: 81–88.
40. Viner RI, Ferrington DA, Williams TD, Bigelow DJ, Schöneich C. Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle. Biochem J. 1999; 340: 657–669.[CrossRef][Medline] [Order article via Infotrieve]
41. Viner RI, Williams TD, Schöneich C. Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and s-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase. Biochemistry. 1999; 38: 12408–12415.[CrossRef][Medline] [Order article via Infotrieve]
42. Eiserich JP, Hristova M, Cross CS, Jones AD, Freeman BA, Halliwell B, van der Vliet A. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature. 1998; 391: 393–397.[CrossRef][Medline] [Order article via Infotrieve]
43. Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RAK, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T, Warnholtz A. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88: e14–e22.[Medline] [Order article via Infotrieve]
44. Harrison DG, Armstrong ML, Freiman PC, Heistad DD. Restoration of endothelium-dependent relaxation by dietary treatment of atherosclerosis. J Clin Invest. 1987; 80: 1808–1811.[Medline] [Order article via Infotrieve]
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