Vascular Function in the Vitamin E–Deprived Rat
An Interaction Between Nitric Oxide and Superoxide Anions
Abstract—We tested the hypothesis that oxidative stress, mediated by dietary vitamin E deprivation, would alter vascular function through the interaction of oxygen-derived free radicals and nitric oxide (NO). This interaction may play an important role in the vascular pathophysiology of many diseases associated with oxidative stress. Mesenteric arteries from control (n=12) and vitamin E–deprived (n=12) Sprague-Dawley rats were studied with a myograph. Superoxide dismutase, which scavenges superoxide anions, produced a significantly greater relaxation in the arteries from the vitamin E–deprived rats compared with the controls (P<.05). Superoxide dismutase and catalase produced results similar to superoxide dismutase alone. Pretreatment with an NO synthase inhibitor eliminated the superoxide dismutase–induced relaxation in arteries from both control and vitamin E–deprived rats. l-Arginine induced a greater relaxation in arteries of the vitamin E–deprived group (P<.05). Agonist-induced relaxation with methacholine was not altered by superoxide dismutase for either group of animals, indicating that stimulated release of NO was not influenced by superoxide anions. With the use of Western immunoblot analysis, nitrotyrosine residues were shown to be present in arteries from both the vitamin E–deprived and control rats, but the amount of nitrotyrosine observed was not different between the two groups. In summary, our data indicate that there is a greater inhibition of NO caused by superoxide anions in the vitamin E–deprived group. We speculate that in conditions of oxidative stress (reduced vitamin E levels), altered vascular function may be due to increased destruction of NO by oxygen-derived free radicals.
Oxidative stress, defined as an imbalance between prooxidant and antioxidant forces, has been proposed to be a pathophysiological feature of many vascular diseases, such as hypertension,1 2 atherosclerosis,3 and the pregnancy disorder preeclampsia.4 Furthermore, several epidemiological investigations have provided correlative evidence for lower levels of the antioxidant vitamin E and risk for cardiovascular disease.5 6 Although lowered antioxidant protection by vitamin E may have a role in vascular disease, little is known regarding the consequence of vitamin E deprivation on vascular function.
The pathophysiology of many vascular diseases include endothelial cell dysfunction.7 8 The endothelium, which lines the lumen of all blood vessels, has many functions including the maintenance of vascular integrity, regulation of intravascular coagulation, and modification of contractile responses of the underlying smooth muscle. The modulation of vascular smooth muscle tone is regulated, in part, through release of endothelium-derived vasoactive agents such as NO.
Endothelial cells are constantly being subjected to oxidative stress and are therefore a primary target of oxygen-derived free radical–mediated mechanisms. Alterations in endogenous free radical production under conditions of oxidative stress may play an important role in modulating spontaneous and agonist-stimulated NO production and thus alter vascular function. Superoxide anion is an oxygen-derived free radical that is known to interact with NO and reduce its ability to act as a vasodilator.9 10 Furthermore, the interaction of superoxide anions with NO results in the production of the cell damaging prooxidant peroxynitrite.10
We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deficiency would alter vascular function through the interaction of superoxide anions and NO.
General Animal Model
Ten-week-old female Sprague-Dawley rats were obtained from Harlan (Harlan, Ind) and divided into two groups. One group (n=9) received a diet deficient in vitamin E; the other (n=9) received an equivalent diet supplemented with 50 IU dl-α-tocopherol acetate (US Biochemical Corp)/kg diet. The animals were maintained on these diets for 10 weeks to adequately deplete plasma and tissue stores of α-tocopherol in the rats receiving the diet deficient in vitamin E.11 The rats were housed in the facilities at Magee-Womens Research Institute, which is accredited by the American Association for the Accreditation of Laboratory Animal Care.
Assessment of Oxidative State
Rats were killed while under light anesthesia with methohexital sodium (50 mg/kg body wt), and blood was collected by heart puncture. Samples were kept at 4°C until centrifugation, and sera were then stored frozen at −70°C until assayed. Serum vitamin E levels were analyzed by high-pressure liquid chromatography according to Bieri et al12 as modified by Chow and Omaye.13 The assay modification used the presence of the antioxidant butylated hydroxytoluene (0.024%) during the extraction with heptane. All samples were analyzed in one run of the assay to avoid interassay variation.
Lipid peroxidation is an important manifestation of oxidative stress that occurs when oxygen free radicals interact with polyunsaturated fatty acids in membranes or lipoproteins. We assayed for evidence of lipid peroxidation to determine if our model of vitamin E deprivation resulted in oxidative stress. Malondialdehyde, a product of lipid peroxides detectable in serum, was used as an indicator of lipid peroxidation. Malondialdehyde concentrations were determined by high-pressure liquid chromatography by use of the technique of Wong et al.14 The detection limit of this assay is 0.15 μmol of malondialdehyde per liter of serum. All samples were analyzed in one run of the assay to avoid interassay variation.
A section of the mesentery 5 to 10 cm distal to the pylorus was rapidly removed and placed in ice-cold HEPES-buffered PSS (HEPES-PSS). One mesenteric artery averaging 250 μm in diameter was dissected free from surrounding adipose tissue, cut into two 1.8-mm lengths, and threaded onto 20-μm wires. These wires were fastened to two stainless steel blocks that were mounted in an isometric myograph system. One block was attached to a Kulite strain gauge force transducer (Kulite Semiconductor Products), the other connected to a displacement device. The blocks rested in 5-mL glass-jacketed organ baths with HEPES-PSS solution kept at 37°C. Each experiment had two baths running simultaneously.
Resting Length-Tension Curve
After the arteries were mounted, they were stretched to ≈0.2 mN/mm vessel length (1 mN=102 mg) and allowed to equilibrate for 1 hour in HEPES-PSS buffer. The arteries were then given a conditioning stretch of ≈0.6 mN. A resting length-tension curve was generated for each vessel. The arterial circumference that was used to perform the dose-response curves was obtained by use of the Law of LaPlace. With this equation, L100 is calculated from the exponential curve fit of tension versus circumference. L100 is defined as the circumference the vessel would have at a transmural pressure of 100 mm Hg. We have found from our previous studies that dose-response curves obtained at .8 L100 is a point that provides maximum active force generation with minimum passive tension.
Solutions and Drugs
The HEPES-PSS solution used in these experiments contained (in mmol/L): sodium chloride 142; potassium chloride 4.7; magnesium sulfate 1.17; calcium chloride 1.56; potassium phosphate 1.18; HEPES 10; and glucose 5.5. The HEPES-PSS solution was maintained at a pH of 7.4. Stock solutions of phenylephrine (l-phenylephrine hydrochloride), methacholine (acetyl-β-methylcholine chloride) (all from Sigma), and meclofenamate (Warner Lambert) were prepared in HEPES-PSS at a concentration of 10 mmol/L for each experiment. A stock solution of 10 mmol/L of L-NMMA (Cayman Chemical Co) was prepared in water. Stock solutions of SOD (2000 U/mL) and catalase (50 000 U/mL) were prepared in HEPES-PSS. Appropriate dilutions of all stocks were obtained with the use of HEPES-PSS.
Two separate baths were used to study arterial segments simultaneously. Cumulative doses of phenylephrine (0.3 to 10 μmol/L) were administered to determine the dose that would give a 50% contraction for each individual artery. The EC50 of phenylephrine was used to constrict arteries to achieve a baseline from which subsequent relaxation responses were measured. After completion of each dose-response curve, a 30-minute recovery period was allowed, during which the baths were changed every 10 minutes with fresh HEPES-PSS. To determine whether superoxide anions modulated vascular function, SOD was used to reduce superoxide anions to hydrogen peroxide. In the presence of catalase, hydrogen peroxide is further reduced to water and molecular oxygen. Therefore, cumulative doses of SOD (1 to 10 U/mL) in the absence or presence of catalase (500 U/mL) were conducted. The curves were generated in the absence or presence of the NO synthase inhibitor L-NMMA (100 μmol/L) to determine a role for NO in the responses. Cumulative doses of the muscarinic agonist methacholine (0.01 to 1 μmol/L) in the absence or presence of SOD (50 U/mL) were administered to assess the potential role of superoxide anions on endothelial-dependent (NO-dependent) relaxation responses. The response of mesenteric arteries to an exogenous source of l-arginine or d-arginine (0.01 to 100 μmol/L) was also determined.
The studies performed involving the absence and presence of inhibitors were conducted sequentially on the same artery. The reproducibility of repeating curves for these experiments was determined in a preliminary set of experiments designed to test for tachyphylaxis.
Evidence of peroxynitrite formation in mesenteric arteries from control and vitamin E–deficient animals was evaluated indirectly by the presence of nitrotyrosine residues. Western immunoblot for nitrotyrosine residues was conducted with a monoclonal antinitrotyrosine antibody (Upstate Biotechnology Inc). A standard of nitrosylated proteins was prepared, using a heart perfused with 200 μmol/L of peroxynitrite. Mesenteric arteries were homogenized in Tris-Cl buffer containing protease inhibitors. For gel electrophoresis, samples were diluted by addition of an equal volume of 2× gel sample buffer (40 mmol/L Tris/Cl, pH 6.8, 2% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.02% bromphenol blue). Samples were boiled for 5 minutes and centrifuged at 10 000g for 2 minutes. Aliquots of 10 μL were loaded into individual wells formed within the stacking gel (5% acrylamide in stacking gel buffer, 25 mmol/L Tris-HCl, pH 6.8) overlaid on 12% acrylamide gels in Tris-HCl, pH 8.8, and separated by electrophoresis at 100 to 300 V for ≈1 hour in a mini–gel apparatus according to the method of Laemmli.15
After separation, samples were transferred to a membrane (Nylon NT, Magnagraph, Inc). Prestained standards (high and low molecular weight, BioRad) were included in separate lanes in each gel for identification of the approximate molecular weight of unknowns. Primary antibody (nitrotyrosine) was incubated for 2 hours; secondary antibody (horse radish peroxidase conjugated) was incubated for 1 hour, and ECL detection (Amersham LIFE Science) was used. Autoradiography was done with the use of scientific imaging film (X-OMAT AR, Kodak).
The Student’s two-tailed t test was used to determine the statistical difference of the parameters between the control and vitamin E–deprived rats shown in the Table⇓. Maximum response to superoxide dismutase and l-arginine was also evaluated with the Student’s two-tailed t test. The data from the methacholine dose-response curve was fitted to the Hill equation,16 from which a straight line was generated by linear least-squares regression analysis. The mean effective concentration that produced a 50% response (EC50) was determined from this line and expressed as the geometric mean±SEM. A two-way ANOVA with repeated measures was used to compare the response to a drug before and after SOD. Pairwise comparison was then conducted using the Student-Newman-Keuls test. Differences among means were considered significant at a value of P<.05.
The Table⇑ depicts the results for the assessment of the peroxidative state in the rats. There was no difference in body weights between the vitamin E–deprived and control rats, indicating that growth was not impaired by vitamin E deprivation. As expected, vitamin E levels were significantly less in the vitamin E–deprived group than in the controls. Serum malondialdehyde concentrations were elevated in the vitamin E–deprived rats.
Mesenteric arteries preconstricted with their EC50 of phenylephrine demonstrated a concentration-dependent relaxation response to SOD that was greater in the arteries from the vitamin E–deprived rats than in arteries of the control rats (Fig 1⇓). To determine whether hydrogen peroxide produced in the presence of SOD was responsible for the enhanced relaxation, the SOD dose-response curve was repeated in the presence of catalase. Catalase did not significantly affect the relaxation to SOD for either groups of animals (Fig 2⇓).
To determine whether SOD relaxation was dependent on NO, SOD was repeated in the presence of the NO synthase inhibitor L-NMMA. L-NMMA effectively blocked relaxation of SOD for both the arteries from control and vitamin E–deprived rats (Fig 3⇓).
Because the SOD response appeared to be NO dependent, we next tested whether increasing substrate availability for NO would result in a differential response in arteries of control and vitamin E–deprived rats (Fig 4⇓). l-Arginine relaxed arteries from the vitamin E–deprived rats to a greater extent than the arteries of the control animals. d-Arginine had no effect on vascular reactivity for either group of animals (data not shown).
There was a 100% relaxation response to methacholine in arteries from both the control and the vitamin E–deprived rats. Fig 5⇓ depicts the EC50 values for this relaxation response to methacholine alone and methacholine in the presence of SOD for arteries from both groups of rats. Methacholine produced a similar relaxation response in arteries from both the control and vitamin E–deprived rats (EC50=0.075±0.009 versus 0.077±0.006 μmol/L). Pretreatment with SOD did not alter the relaxation for arteries in either the control or the vitamin E–deprived groups. The majority of the relaxation response to methacholine was NO dependent. The NO synthase inhibitor L-NMMA effectively inhibited the relaxation response to methacholine, with the exception of the highest dose of methacholine (1 μmol/L).
A representative immunoblot for nitrotyrosine residues in mesenteric arteries from control and vitamin E–deprived rats is shown in Fig 6⇓. Nitrotyrosine residues were present in arteries from both the control and the vitamin E–deficient animals. There was no difference in the intensity of immunostaining between the two groups (in arbitrary units; control=89.7±5.8, vitamin E–deprived=67.4±14.6).
The purpose of this study was to investigate whether oxidative stress, mediated by a dietary vitamin E deficiency, would alter vascular function through an interaction of oxygen-derived free radicals and NO. We proposed that the reaction of superoxide anion with NO would reduce the availability of NO as a vasorelaxant and also result in the production of peroxynitrite, a potential cytotoxic oxidant. Our study demonstrated that arteries from the vitamin E–deprived rats relaxed to a greater extent to SOD compared with the control rats. The presence of catalase did not alter the relaxation response to SOD, indicating that the response was not due to hydrogen peroxide. Pretreatment of arteries with an NO synthase inhibitor eliminated the SOD-induced relaxation response in arteries from both the control and vitamin E–deprived rats. These data indicate that there is a greater inhibition of NO because of superoxide anions in the vitamin E–deprived group.
We observed that vitamin E deprivation resulted in greater basal NO production that was not available as a vasorelaxant because of superoxide anions. In contrast, however, relaxation responses to agonist-induced release of NO was not altered by SOD for either group of animals. It is evident that the balance between NO and superoxide anions is critical. From our study, an increased NO release overcame the inactivation by superoxide anions. The reactions and reaction rates of NO are complex and are not completely understood. One aspect of NO is that the half-life is not a constant value and is inversely proportional to the concentration of NO, so that the half-life becomes shorter as NO becomes more concentrated.10 Perhaps under the conditions of our experiments, the rate of superoxide production was too slow to consume the amount of NO produced under agonist-stimulated release and thus did not affect the ability of NO to produce a vasorelaxation response.
In our study, we used an animal model of vitamin E deprivation to determine the effect on vascular function, with specific interest in the interaction of superoxide anion and NO. Vitamin E is a mixture of compounds called tocopherols, the most potent of which is α-tocopherol.17 Low levels of vitamin E are related to a higher occurrence of cardiovascular disease, and intake of vitamin E lowers the risk of coronary heart disease.5 6 Vitamin E is well known as a lipid-soluble antioxidant that scavenges lipid peroxyl radicals within cellular membranes. Because of a hydroxyl group that is exposed to the aqueous phase, vitamin E has the capacity to scavenge oxygen radicals attacking from outside the membrane.17
Superoxide anion is an oxygen-derived free radical formed by univalent reduction of molecular oxygen. By dismutation with SOD, superoxide anions are reduced to hydrogen peroxide. Hydrogen peroxide may be further reduced to molecular oxygen and water by catalase. Superoxide anions may participate in the regulation of vascular tone in normal physiological conditions as well as vascular pathologies. In an animal model of hypertension, intravenous injection of SOD reduced blood pressure in spontaneously hypertensive rats but not in Wistar-Kyoto normotensive rats.18 Similar to our study, in the diabetic rat aorta, SOD produced a greater relaxation in diabetic aorta compared with control. The authors suggested that a greater release of spontaneous NO is masked by inactivation of NO by superoxide.19 Similar to the diabetic aorta, the addition of catalase did not alter vascular reactivity for mesenteric arteries from vitamin E–deprived rats, indicating that hydrogen peroxide was not contributing to the vascular response under these conditions. In contrast, Rubanyi and Vanhoutte20 demonstrated that hydrogen peroxide was vasoactive by causing a relaxation response in canine coronary arteries.
In our study, it appears that superoxide anions were responsible for inactivation of NO. This interaction of superoxide anions with NO could lead to an increase in peroxynitrite formation.10 Peroxynitrite is a stable and powerful prooxidant that is cytotoxic through direct oxidative mechanisms.10 In addition, peroxynitrite decomposition yields the hydroxyl radical, which is also a powerful oxidant that has important pathological actions on the vasculature.21
Another aspect of peroxynitrite is that it may interfere with pathways important to vascular function, such as modulating prostaglandin production. Landino et al22 demonstrated that peroxynitrite can activate prostaglandin endoperoxide synthase (PGHS), leading to increased eicosanoid synthesis. In a previous study, we observed an increased NO response along with an increased PGHS-dependent vasoconstrictor in arteries from vitamin E–deprived rats.23 More recently, we and others have demonstrated that NO can modulate prostaglandin synthesis in cells and tissue.24 25 26 27 28 Tsai et al29 reported that NO is not capable of stimulating isolated PGHS, which suggests that an intermediate pathway for NO may exist. Indeed, the study of Landino et al22 establishes that superoxide anion could serve as a link between NO and prostaglandin synthesis. In the vitamin E–deprived rat model, we have demonstrated an increase NO production that is inactivated by superoxide anions; perhaps the resulting product, peroxynitrite, is responsible for the previously observed increase in PGHS-dependent vasoconstriction.
Although peroxynitrite may have an active role in the vascular response in the vitamin E–deficient rat model, we were not able to demonstrate through immunoblotting evidence for increased peroxynitrite formation. Nitrotyrosine was used as a marker for peroxynitrite oxidation of proteins.30 Tyrosine nitration is not necessarily due to the formation of peroxynitrite, although peroxynitrite is thought to be the principle source.10 This method and antibody have been used to demonstrate that extensive nitration takes place around foamy macrophages in human atherosclerotic lesions.30 In our study, nitrotyrosine residues were present in arteries from both the vitamin E–deprived rats and control rats, but the amount of nitrotyrosine observed was not different between the two groups. In the vitamin E–deprived rat model, the role of peroxynitrite in vascular pathophysiology may be more subtle than that observed with atherosclerotic lesions and therefore may not be detected through immunostaining.
Alternatively, although the interaction of NO and superoxide anions is well documented, there is a possibility that superoxide anions inhibited the release and/or formation of NO. The lack of nitrotyrosine formation in the arteries of the vitamin E–deficient animals may support this mechanism.
In summary, our data indicate that arteries from the vitamin E–deprived rats, compared with control rats, release more spontaneous NO that is inhibited by superoxide anions. We speculate that in conditions of oxidative stress (lower vitamin E levels), altered vascular function may be due to increased destruction of NO by oxygen-derived free radicals.
Selected Abbreviations and Acronyms
|PSS||=||physiological saline solution|
Dr S.T. Davidge is supported by the Medical Research Council of Canada, the Heart and Stroke Foundation of Canada, and the Alberta Heritage Foundation for Medical Research. Dr M.K. McLaughlin is supported by National Institutes of Health grant HD30367. We thank Karen Schuler and Wahida Yasmin for their excellent technical assistance.
- Received August 1, 1997.
- Revision received September 11, 1997.
- Accepted November 5, 1997.
Beckman JS, Koppenol WH. Nitric oxide, superoxide and peroxynitrite: the good, the bad, and the ugly. Am J Physiol. 1996;271(5 Pt I): C1424–C1437.
Bieri JG, Tolliver TJ, Catignani GL. Simultaneous determination of alpha-tocopherol and retinal in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr. 1972;32:2143–2151.
Wong SHY, Knight JA, Hopfer SM, Zaharia O, Leach CN, Sunderman FW. Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin Chem. 1987;33:214–220.
Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;277:680–685.
Hill A. The combination of hemoglobin with oxygen and carbon monoxide. Biochem J. 1913;7:471–480.
Nakazono K, Watanabe N, Matsuno K, Sasaki J, Sato T, Inoue M. Does superoxide underlie the pathogenesis of hypertension? Proc Natl Acad Sci U S A.. 1991;88:10045–10048.
Langenstroer P, Pieper GM. Regulation of spontaneous EDRF release in diabetic rat aorta by oxygen free radicals. Am J Physiol. 1992;263:H257–H265.
Rubanyi GM, Vanhoutte PM. Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle. Am J Physiol. 1986;250:H815–H821.
Pfeiffer S, Gorren ACF, Schmidt K, Werner ER, Hansert B, Bohle DS, Mayer B. Metabolic fate of peroxynitrite in aqueous solution. J Biol Chem. 1997;272:3465–3470.
Landino LM, Crews BC, Timmons MD, Morrow JD, Marnett LJ. Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc Natl Acad Sci U S A.. 1996;93:15069–15074.
Davidge ST, Hubel CA, McLaughlin MK. Cyclooxygenase-dependent vasoconstrictor alters vascular function in the vitamin E-deprived rat. Circ Res. 1993;73:70–88.
Davidge ST, Baker PN, McLaughlin MK, Roberts JM. Nitric oxide produced by endothelial cells increases production of eicosanoids through activation of prostaglandin H synthase. Circ Res. 1995;77:274–283.
Franchi AM, Chaud M, Rettori V, Suburo A, McCann SM, Gimeno M. Role of nitric oxide in eicosanoid synthesis and uterine motility in estrogen-treated rat uteri. Proc Natl Acad Sci U S A. 1994;91:539–543.
Rettori V, Gimeno M, Lyson K, McCann S. M. Nitric oxide mediates norepinephrine-induced prostaglandin E2 release from the hypothalamus. Proc Natl Acad Sci U S A. 1992;89:11543–11546.
Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci U S A. 1993;90:7240–7244.