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Am J Physiol Heart Circ Physiol 286: H575-H583, 2004. First published October 2, 2003; doi:10.1152/ajpheart.00331.2003
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Effect of high-salt diet on NO release and superoxide production in rat aorta

Jiaxuan Zhu, Takefumi Mori, Tianjian Huang, and Julian H. Lombard

Department of Physiology, Medical College of Wisconsin. Milwaukee, Wisconsin 53226

Submitted 11 April 2003 ; accepted in final form 25 September 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sprague-Dawley rats were fed either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 3 days. Nitric oxide (NO) and superoxide production were assessed in the thoracic aorta by evaluating the fluorescence signal intensity from 4,5-diaminofluorescein (DAF-2DA) and dihydroethidine, respectively. Methacholine caused increased NO release in the aortas from rats on a LS but not HS diet. The SOD mimetic tempol restored methacholine-induced NO release in aortas from rats on a HS diet. Methacholine also caused superoxide production in the aortas of rats on a HS diet but not in the aortas of rats on a LS diet. Tempol and NG-monomethyl-L-arginine eliminated methacholine-induced superoxide production in the aortas of rats on a HS diet. Aortic rings from rats on the HS diet showed impaired methacholine-induced relaxation, which was improved by tempol. Tempol alone caused a NO-dependent relaxation of norepinephrine-precontracted aortas that was significantly greater in the aortas of rats on the HS diet than in vessels from rats on the LS diet. These data suggest that a HS diet impairs endothelium-dependent relaxation via reduced NO levels and increased superoxide production.

nitric oxide; endothelium; dietary salt intake; sodium

PREVIOUS STUDIES have demonstrated that elevated dietary salt intake leads to an impaired relaxation of blood vessels to vasodilator stimuli (8, 11). A possible contributor to impaired vascular relaxation to dilator stimuli in animals on a high-salt diet is an impaired function of the endothelium, which normally plays a critical role in regulating vascular tone via the generation of vasodilator and vasoconstrictor substances (13, 22). Impaired endothelium-dependent dilation in vessels of animals on a high-salt diet could occur either because the production of vasodilator substances by the endothelium is impaired or because the vessels liberate vasoconstrictor substances in response to vasoactive stimuli that normally relax the blood vessels.

An important endothelium-derived relaxing factor is nitric oxide (NO), formed by the action of endothelial nitric oxide synthase (NOS) (eNOS). The endothelial cells are also a source of superoxide (), which can contribute to reduced endothelium-dependent dilation (20, 22, 27) and even lead to endothelium-dependent contractions (1, 6). In this respect, eNOS (in addition to xanthine oxidase and NADH/NADPH oxidase) may be an enzymatic source of reactive oxygen species (ROS) in hypertension and other pathophysiological states (1, 12, 22). Excess production could cause a decreased bioavailability of NO, either by formation of peroxynitrite from interactions between NO and (28, 30, 37, 38) or by a process known as NOS uncoupling, where eNOS forms rather than NO.

A variety of evidence suggests that ROS can contribute to impaired endothelial function in several forms of hypertension and that oxidative stress is enhanced in the microvessels of spontaneously hypertensive rats (32) and Dahl salt-sensitive hypertensive rats (33). A recent report by Lenda et al. (22) has suggested that ROS can also contribute to a reduced endothelium-dependent dilation in normotensive rats on a high-salt diet. Despite the potential importance of ROS in contributing to impaired endothelium-dependent vasodilation and reduced NO production during elevated dietary salt intake, the nature and mechanisms of the impaired vascular relaxation with the high-salt diet and the role of enhanced oxidative stress in contributing to salt-induced changes in vascular function are not completely understood.

The purpose of this study was to test the hypothesis that a high-salt diet leads to reduced NO levels, increased generation, and impaired endothelium-dependent vascular relaxation and to investigate the potential role of NOS in contributing to production in the aortas of rats on a high-salt diet. The experiments conducted in the present study were designed to 1) determine whether NO release and vascular relaxation in response to methacholine are impaired in the aorta of rats on a high-salt diet; 2) identify any differences in the generation of in the aortas from rats on high-salt and low-salt diets; and 3) determine the effect of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and the SOD mimetic tempol on NO release, , and vascular relaxation in the aorta from rats on high-salt and low-salt diets.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of thoracic aorta. The experiments were performed on male Sprague-Dawley rats (8–12 wk old). All rats were housed in an animal care facility at the Medical College of Wisconsin (MCW) that is approved by the American Association for the Accreditation of Laboratory Animal Care, and all protocols were approved by the MCW Animal Care and Use Committee.

Before being euthanized, the animals were fed either a high-salt (4% NaCl) or a low-salt (0.4% NaCl) diet for 3 days with water to drink ad libitum. On the day of the experiment, the rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and the thoracic aorta was removed and rinsed with cold physiological salt solution (PSS) having the following composition (in mM): 119 NaCl, 4.7 KCl, 1.17 MgSO4, 1.6 CaCl2, 1.18 NaH2PO4, 24 NaHCO3, 0.026 EDTA, and 5.5 glucose. After removal of any adhering connective tissue, the aorta was cut into several segments. Each segment was opened with fine scissors and pinned onto a piece of rubber with the endothelium facing up, so that the endothelial cells on the inside surface of the aorta were exposed to the solution. After being fixed on the rubber, the segments of the aorta were put into the PSS at 37°C and aerated for 2 h with a gas mixture containing 21% O2-5% CO2-74% N2.

Evaluation of intracellular NO levels with DAF-2DA. NO levels in the vessels were assessed by using 4,5-diaminofluorescein (DAF-2DA). This compound is nonfluorescent but reacts with NO in the presence of oxygen to form the highly fluorescent compound triazolofluorescein (DAF-2T) (18, 34), the intensity of which is proportional to NO levels.

Aortic segments were loaded with 5 µM DAF-DA for 30 min at 37°C in HEPES buffer (pH 7.4). Once loading was finished, the vessels were rinsed with HEPES buffer three times and placed in a chamber containing HEPES buffer maintained at 37°C with a water bath. L-Arginine (100 µM) was put into the chamber during measurements to ensure adequate substrate availability for NOS. The proportional relationship between DAF-2T fluorescence intensity and NO production was demonstrated in vitro by using different concentrations of the NO donor EDTA NONOate (see below and also Fig. 1A).



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  		Fig. 1. Calibration curves for nitric oxide (NO) and superoxide showing the proportional relationships between triazolofluorescein (DAF-2T) fluorescence intensity and NO level (A) and between dihyroethidine (DHE) fluorescence intensity and superoxide production (B) in vitro. Different quantities of Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate) [final concentrations 0 (background, BK), 10–6, 10–5, 10–4, and 10–3 M], xanthine (100 µM), and xanthine oxidase (0, 3, 6, 9, 12, 15 mU/ml) were added to the aorta to produce the changes in fluorescence intensity (n = 3 rats).

 

In some experiments, L-NMMA (100 µM, CalBiochem; San Diego, CA) was used to inhibit NOS activity to verify that the change of fluorescence intensity in the different groups was due to NO release. In other experiments, the membrane-permeable SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-N-oxide (tempol, 50 µM, Sigma Chemical; St. Louis, MO) or the NADH/NADPH oxidase inhibitor apocynin (500 µM, Sigma Chemical) was used to determine the effect of scavenging on NO release and to assess the possible role of NADH/NADPH oxidase as a source of production in the vessels.

Measurements were performed on a Nikon E 600 microscope (Nikon; Tokyo, Japan) by using a x10 Plan Fluo phase objective. The signals were acquired by a Princeton Instruments Micromax Cooled CCD camera (RS Princeton Instruments; Trenton, NJ). To evaluate NO fluorescence, DAF-2T was excited at 490 nm and collected through a 530-nm bandpass emission filter. Fluorescence intensity was analyzed by using MetaMorph version 4.6 software (Universal Imaging; Downingtown, PA), which was run on a Pentium III class computer. NO fluorescence was measured every 5 min for 1 h in the same area of the aortic endothelial surface. Because of the nature of NO release, baseline fluorescence signal exhibited a linear increase. Thus the signal in the initial 30 min was measured to obtain the slope of the line under resting conditions as the control. After the addition of the agonists, the fluorescence intensity changes were evaluated for another 30 min. The ratio of the slopes of the fluorescence intensity changes during the control and treatment conditions was calculated to evaluate the effect of agonists, as described by Yu et al. (41). Because DAF-2DA reacts with NO via an irreversible covalent modification, individual vessels could be studied only once. The NO donor DETA-NONOate (Cayman; Ann Arbor, MI) was used as a positive control in each sample to make sure that a high-salt diet did not affect dye loading and that methacholine treatment did not cause saturation of the dye.

Evaluation of levels with dihydroethidine. To assess the production of superoxide radicals, the endothelial cells on the surface of the aorta were loaded with dihydroethidine (DHE). For these studies, the aortic segments were incubated with DHE (20 µM; Molecular Probes, San Diego, CA) for 30 min in HEPES solution at 37°C. DHE enters the cell and is oxidized by to yield ethidium, which binds to DNA in the cell, resulting in a strong red fluorescence.

Measurements of DHE fluorescence were performed on the same microscope and software employed for the NO measurements by using a 490-nm wavelength for excitation and a 605-nm wavelength for emission. After incubation, aortic segments were washed three times with HEPES buffer and put into a temperature-controlled chamber. In some experiments, PEG-SOD (150 U/ml; Sigma), the SOD mimetic tempol (50 µM, Sigma), or the NOS inhibitor L-NMMA (100 µM, CalBiochem) were used to determine the effects of a scavenger or a NOS inhibitor on the change of fluorescence intensity in each experimental group.

In one series of experiments, we constructed a calibration curve to verify that the intensity of DHE fluorescence is proportioned to the generation of . In these experiments, different concentrations of xanthine oxidase (0, 3, 6, 9, 12, and 15 mU/ml) were added to PSS containing 100 µM xanthine (see Fig. 1B in RESULTS). In other experiments, the mitochondrial superoxide inducer menadione (500 µM) and the SOD inhibitor DETC (1 mM) (Sigma Chemical) were put into the chamber simultaneously as a positive control to demonstrate that the cells were adequately loaded with the dye and that the dye was not saturated after stimulation.

Aortic ring studies. Vascular relaxation was evaluated by measuring the change in active force of aortic rings precontracted with norepinephrine and subsequently treated with methacholine. In these experiments, rats were anesthetized with pentobarbital (50 mg/kg), and the thoracic aorta was removed and cleaned of connective tissue in ice-cold PSS. Four aortic rings (about 3 mm in length) were obtained from each rat. The rings were mounted on two tungsten wires, one connected to a fixed holder and the other to a force displacement transducer (model FT 03, Grass Astro-Med; West Warwick, RI) for continuous measurement of isometric tension. The vessels were immersed in organ baths filled with PSS maintained at 37°C and bubbled with a 95% O2-5% CO2 gas mixture. Data were acquired and analyzed using WINDAQ software (DataQ Instruments; Akron, OH). Aortic rings were initially set between 2 and 3 g of basal tension, washed periodically, and allowed 90 min for equilibration.

After the equilibration period, KCl (60 mM) was added to the chambers three times and washed out with PSS until a reproducible maximal contraction was achieved. After recovery from the KCl-induced contraction, norepinephrine (NE, 0.1 µM) was added to the chamber to precontract the vessel. When the contraction reached a maximal stable level, cumulative concentrations of methacholine (10 nM–100 µM) were added to the chamber. Inhibition experiments were performed by incubating the rings with L-NMMA (100 µM) or tempol (500 µM) before NE addition and for an additional 30 min before the addition of methacholine. To evaluate the contribution of resting levels of to impaired modulation of vascular tone in the absence of methacholine stimulation, aortas were precontracted with NE (0.1 µM) in another series of experiments. After a steady level of tone was reached, tempol (500 µM) was added to the tissue bath in the absence of methacholine, and L-NMMA (100 µM) was then added to the tissue bath to assess the effect of NOS inhibition on tempol-induced relaxation of the vessels.

Reagents. Aliquots of DAF-2DA (5 mM in DMSO), purchased from Calbiochem (San Diego, CA), were stored in an Eppendorf tube at –80°C and subsequently diluted with HEPES buffer to prepare a 5 µM working solution. L-Arginine, methacholine, tempol, and apocynin were purchased from Sigma Chemical. L-Arginine was directly dissolved in the HEPES buffer. Tempol was dissolved in the HEPES buffer to prepare a 100 mM stock solution and was used at a working concentration of 50 or 500 µM. L-NMMA was dissolved in deionized water to make a 100 mM stock solution and stored at –20°C before use at a working concentration of 100 µM. Apocynin was dissolved in DMSO in a 1 M stock solution and used at a working concentration of 500 µM.

Statistics. Data were summarized as means ± SE, One-way ANOVA was employed to identify differences between various groups on a high-salt or low-salt diet. Two-way ANOVA was used to analyze the aortic ring data to evaluate the differences between various treatments and different drug concentrations in vessels from rats on a low-salt and high-salt diet. A Student-Newman-Keuls test was used to identify differences between individual means, post hoc. A probability level of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Concentration dependence of DAT-2T and DHE fluorescence. Figure 1 shows the calibration curves for DAF-2T as a function of increasing NO and for DHE as a function of increasing levels of . DAF-2T fluorescence intensity (490 nm for excitation and 535 nm for emission) showed a steady increase for increasing concentrations of the NO donor DETA-NONOate (Fig. 1A), and DHE fluorescence (490 nm for excitation and 605 for emission) showed a steady increase for increasing concentrations of xanthine oxidase added to a solution containing 100 µM xanthine (Fig. 1B).

Effect of methacholine on NO release. The changes in DAF-2T fluorescence in response to methacholine in the aortas of rats on a low-salt and high-salt diet are shown in Fig. 2. Methacholine significantly increased the slope of the DAF-2T fluorescence change in the aortas of rats on a low-salt diet but did not affect the slope of the DAF-2T fluorescence in the aortas of rats on a high-salt diet. L-NMMA (100 µM) inhibited methacholine-stimulated NO production in the aortas of rats on a low-salt diet but did not affect fluorescence intensity in the aortas of rats on a high-salt diet, reflecting the failure of DAF-2T fluorescence to increase in response to methacholine in vessels of rats on a high-salt diet.



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  		Fig. 2. Effect of methacholine on the slope of DAF-2 fluorescence increase in the aortas of rats on low-salt (LS) diet (A) and high-salt (HS) diet (B) in the presence and absence of NG-monomethyl-L-arginine (L-NMMA). Methacholine caused an increase in DAF-2 fluorescence in the aortas of rats on a LS diet but not in the aortas of rats on a HS diet. L-NMMA (100 µM) completely blocked the enhanced fluorescence intensity in the aortas of rats on a LS diet but had no effect on DAF-2 fluorescence in the aortas of rats on a HS diet. Data are summarized as means ± SE. *Significant difference between control and methacholine treatment within a group (P < 0.05, n = 6 rats).

 

Effect of DETA-NONOate on DAF-2T fluorescence. To confirm that the difference in the change of fluorescence intensity in vessels from rats on a low-salt and high-salt diet resulted from differences in the vessel themselves rather than other factors such as uneven loading of dye, vessel segments were treated with the NO donor DETA-NONOate (1 mM). DETA-NONOate caused a rapid increase in fluorescence intensity in the aortas of rats on either a high-salt or low-salt diet and there was no significant difference in the slope of the fluorescence intensity change in response to DETA-NONOate in the two groups (Fig. 3).



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  		Fig. 3. Change in the slope of DAF-2 fluorescence intensity in response to DETA-NONOate (1 mM) in the aortas from rats on a LS and HS diet. Data are shown as means ± SE. *Significant increase from control value before DETA-NONOate treatment. There was no significant difference in the response of the vessels to DETA-NONOate in the two groups (n = 6 rats).

 

Effect of tempol on NO release in aortas of rats on high-salt and low-salt diet. The effect of the SOD mimetic tempol on NO release in the aortas from rats on a high-salt and low-salt diet is shown in Fig. 4. Under control conditions, methacholine caused a significant increase of NO release in the aortas of rats on a low-salt diet but not in the aortas of rats on a high-salt diet. After treatment with tempol, NO release increased significantly in response to methacholine in the aortas from rats on both a high-salt and low-salt diet. Tempol also caused a significant increase in the slope of DAF-2T fluorescence before methacholine treatment in the aortas of rats on a high-salt diet and low-salt diet.



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  		Fig. 4. Effect of tempol on methacholine-induced NO production in the aortas from rats on a LS diet (A) and HS diet (B). *Significant increase (P < 0.05) in NO production compared with control value before treatment with methacholine (10 µM) or compared with control in the absence of tempol. Data are shown as means ± SE; n = 6 rats. See text for details.

 

Effect of apocynin on NO levels in aortas of rats on high-salt and low-salt diet. To evaluate NADH/NADPH oxidase as a possible source of under resting conditions (acting to reduce resting NO availability), vessels were incubated with the NADH/NADPH oxidase inhibitor apocynin (500 µM) before tempol treatment. In these experiments (Fig. 5), apocynin treatment caused an increase in DAF-2T fluorescence in the aortas of rats on both a low-salt and high-salt diet, indicating that NADPH oxidase contributes to resting production in the aortas of rats on both a low-salt and high-salt diet, and that production reduces NO availability during resting conditions. Addition of tempol to apocynin-treated vessels caused an additional increase in NO levels in the aortas of rats on a low-salt and high-salt diet, suggesting that other sources also contribute to production (and reduced bioavailability of NO) under resting conditions.



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  		Fig. 5. Effect of apocynin (with or without tempol) on NO levels in aortas from rats on a LS and HS diet. Apocynin significantly increased NO release in vessels from rats on HS and LS diet. Addition of tempol to apocynin-treated vessels caused an additional increase in NO levels in aortas from rats on a HS and LS diet. Data are shown as means ± SE. *Significant difference between treated group and control group or between apocynin alone and apocynin + tempol (P < 0.05; n = 6 rats).

 

Effect of methacholine on superoxide levels. Figure 6 shows the effect of methacholine on superoxide levels in the aortas of rats on a high- and low-salt diet. Methacholine caused a significant increase in production in the aortas of rats on a high-salt diet but not in vessels of rats on a low-salt diet. Addition of the SOD mimetic tempol prevented the methacholine-induced increase in levels in the aortas of rats on a high-salt diet. PEG-SOD also inhibited release in the aortas of rats on a high-salt diet (data not shown).



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  		Fig. 6. Effect of methacholine (10 µM) on superoxide production in the aortas from rats on a LS diet (A) and HS diet (B). Methacholine caused a significant increase in superoxide production in vessels from rats on a HS diet but not in aortas from rats on a LS diet. The increase in superoxide production in the aortas of rats on a HS diet was prevented by incubation with tempol. Data are shown as means ± SE. *Significant increase in superoxide production compared with control value before methacholine treatment (P < 0.05; n = 6 rats).

 

Effect of L-NMMA on methacholine-induced superoxide production in aortas from rats on high-salt and low-salt diet. Figure 7 shows the effect of the NOS inhibitor L-NMMA on production during methacholine treatment in the aortic rings from rats on low-salt and high-salt diets. In these experiments, -NMMA inhibited methacholine-induced production in the aortic rings from rats on a high-salt diet but did not affect DHE fluorescence in the presence of methacholine in vessels from rats on a low-salt diet. Inhibition of NOS with L-NMMA also led to a significant increase in resting DHE fluorescence in the aortas of rats on a high-salt diet (P < 0.05), suggesting that some resting NO production was attenuating levels in vessels from animals on a high-salt diet.



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  		Fig. 7. Effect of L-NMMA (100 µM) on methacholine-induced superoxide release in aortas from rats on a LS diet (A) and HS diet (B). Methacholine caused a significant increase in superoxide production in the aortas from rats on a HS diet. The increase in superoxide production in the aortas of rats on a HS diet was inhibited by incubation with L-NMMA. Data are shown as means ± SE for n = 6 rats. *Significant difference from control value before treatment with methacholine. L-NMMA alone also caused a significant increase in DHE fluorescence in the aortas from rats on a HS diet (P < 0.05) but not aortas from rats on a LS diet. See text for details.

 

Effect of dietary salt intake on methacholine-induced relaxation of aortic rings. Figure 8 summarizes methacholine-induced relaxation of the aortic rings from rats on a high-salt and low-salt diet in the presence and absence of L-NMMA. In these experiments, methacholine-induced relaxation of the aortic rings of rats on a high-salt diet was significantly reduced compared with that in vessels from rats on low-salt diet. Aortic relaxation in response to methacholine was completely eliminated by L-NMMA in vessels from animals on a high-salt diet and drastically inhibited in the aortic rings from animals on a low-salt diet, although a small component of relaxation remained at the higher doses of methacholine.



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  		Fig. 8. Dose-dependent relaxation in response to methacholine in the aortic rings from rats on a LS and HS diet. Methacholine-induced relaxations were significantly reduced in the aortic rings from rats on a HS diet compared with those from rats on a LS diet. L-NMMA (100 µM) significantly inhibited methacholine-induced relaxation of the aortic rings from rats on a LS and HS diet. Data are shown as means ± SE (n = 6 rats). +Significant difference between HS and LS diet at a given concentration of methacholine. *Significant difference in the response to methacholine in the presence and absence of L-NMMA.

 

Effect of tempol on methacholine-induced relaxation of aortic rings. The effect of tempol on methacholine-induced relaxation of aortic rings from rats on a low-salt and high-salt diet is summarized in Fig. 9. In these experiments, vascular relaxation in response to methacholine was also reduced in the aortas of rats on the high-salt diet (Fig. 9A). Addition of tempol (500 µM) to the tissue bath significantly enhanced methacholine-induced relaxation of the aortas from rats on both a low-salt diet (Fig. 9B) and high-salt diet (Fig. 9C).



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  		Fig. 9. Effect of tempol (500 µM) on methacholine-induced relaxation of the aortic rings from rats on a LS and HS diet. Methacholine-induced relaxations were significantly reduced in the aortic rings from rats on HS diet compared with those from rats on LS diet (A). Tempol potentiated vascular relaxation in response to methacholine in aortas from rats on either a LS diet (B) or HS diet (C) compared with the response determined in the absence of tempol. Data are shown as means ± SE (n = 6 rats). *Significant difference (P < 0.05) between the response to a given concentration of methacholine in the aortas of rats on a LS diet and HS diet or between the response to a given concentration of methacholine in presence and absence of tempol.

 

Effect of L-NMMA on tempol-induced relaxation of aortic rings from rats on a high-salt and low-salt diet. To evaluate the effect of resting levels of on force generation in aortic rings in the absence of methacholine, tempol (500 µM) was added to the tissue bath after the vessels had been precontracted with NE. After tempol-induced relaxation reached a steady level, L-NMMA (100 µM) was added to the tissue bath to determine whether tempol-induced vascular relaxation was due to enhanced levels of NO following scavenging. As shown in Fig. 10, tempol caused a significant relaxation of vessels from animals on both high-salt and low-salt diet, but the attenuation of contractile force in response to tempol was significantly greater in aortic rings from rats on high-salt diet. Tempol-induced relaxation was completely eliminated by L-NMMA in both groups.



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  		Fig. 10. Effect of tempol (500 µM) on the relaxation of the aortic rings from rats on a LS (n = 5 rats) and HS (n = 6 rats) diet in the presence and absence of L-NMMA. Tempol caused a significantly larger relaxation in the aortic rings from rats on a HS diet compared with controls on a LS diet. Tempol-induced relaxation of the vessels could be completely blocked by L-NMMA in the aortic rings from rats on either HS or LS diet, and successive tempol-induced relaxations were similar in time controls determined in the absence of L-NMMA. Data are shown as means ± SE (n = 6 rats). *Significant differences between the response in the presence and absence of L-NMMA (P < 0.01). Tempol-induced relaxation of the aortic rings from rats on a HS was significantly greater than relaxation of aortic rings from rats on LS diet (P < 0.01).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As noted above, ROS may play a role in mediating impaired vascular reactivity during the development of hypertension and during elevated salt intake (10, 22–24, 32, 33). For example, vascular oxidative stress is increased in spontaneously hypertensive rats (5, 12, 17, 32) and in Dahl salt-sensitive hypertensive rats (33) compared with their respective normotensive controls. Other studies have found that antioxidant therapy or scavengers of ROS attenuate the development of several forms of hypertension (3, 30, 31, 40, 42), supporting a role for oxidative stress in the generation and maintenance of hypertension.

Recent studies in our laboratory have demonstrated that a high-salt diet also causes impaired NO release and loss of endothelium-dependent dilation in response to acetylcholine in cerebral blood vessels (34). The present study demonstrates that aortas from rats on a high-salt diet also show an impaired release of NO and a reduced relaxation in response to cholinergic receptor activation with methacholine. Inhibition of NOS with L-NMMA inhibited the increase in DAF-2T fluorescence in response to methacholine and either eliminated (high-salt group) or drastically inhibited (low-salt group) vascular relaxation in response to methacholine, indicating that the methacholine-induced changes in DAF-2T fluorescence and vascular relaxation that we observed in these experiments were due to NO release. In the case of animals on low-salt diet, the relaxation that remained in the presence of L-NMMA was apparent at higher concentrations of methacholine and was a small fraction of the total relaxation that occurred in response to the agonist. The latter observation is consistent with reports in the literature (29) and suggests that the L-NMMA-resistant component of the response is a minor component of vascular relaxation in response to methacholine. Whereas differences in methacholine-induced vascular relaxation in the aortas of rats on a high-salt and low-salt diet could be due, at least in part, to the loss of this L-NMMA-resistant component of vascular relaxation in animals on a high-salt diet, we believe that this is not the primary mechanism responsible for differences in methacholine-induced vascular relaxation in the two groups, given the observation that the SOD mimetic tempol restores methacholine-induced NO release (Fig. 4) and eliminates the increase in production in response to methacholine (Fig. 7).

In these experiments, we verified the feasibility of assessing NO and levels in vitro with DAF-2DA and DHE by demonstrating that DAF-2T fluorescence and DHE fluorescence increased steadily in the presence of increasing concentrations of the NO donor Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate) and increasing levels of produced by stepwise increases of xanthine oxidase concentration added to a solution containing 100 µM xanthine. Positive controls conducted after measurements in each sample also demonstrated that no dye saturation occurred. Although these two dyes were able to detect increasing levels of NO and , the measurement system employed in the present experiments was not sensitive to reductions in the slope of DAF-2T or DHE fluorescence following inhibition of NOS with L-NMMA or scavenging with tempol. This inability to detect reductions in DAF-2 or DHE fluorescence would explain the apparent discrepancies in the results in which an increase in the DAF-2T signal after treatment with tempol was not associated with a decrease in DHE signal, and increases in DHE fluorescence in the presence of L-NMMA were not associated with a reduction in the slope of DAF-2T fluorescence. This limitation appears to be due to the fact that the fluorescence levels present during resting conditions were in the lower range of detection by the system employed in the present studies. However, DAF-2DA and DHE were clearly able to detect increases in NO or levels in the tissue bath (Fig. 1) and in the vessels during physiological perturbations that would be expected to increased NO or levels. L-NMMA and tempol were also able to inhibit increases in DAF-2 and DHE fluorescence (respectively) under conditions where NO or levels would be expected to increase in the absence of the inhibitor. The effectiveness of L-NMMA in inhibiting methacholine-induced relaxations and increases in DAF-2T fluorescence and in inhibiting the increase in DHE fluorescence in response to methacholine in the aortas of rats on a high-salt diet; the effectiveness of tempol in eliminating the increase in DHE fluorescence with methacholine treatment in vessels from rats on a high-salt diet; and the effectiveness of L-NMMA in blocking the relaxant effect of tempol in NE precontracted aortas in the absence of methacholine stimulation are all consistent with the interpretation that attenuates NO-dependent vascular relaxation in the aorta and that this influence of is greater in animals on a high-salt diet.

The findings of the present study confirm and extend our earlier findings demonstrating impaired relaxation in response to acetylcholine in skeletal muscle arterioles (8), skeletal muscle resistance arteries (39), and cerebral arteries (27) of animals on a high-salt diet. The impaired release of NO and the reduced vascular relaxation in response to methacholine in the aortas from animals on a high-salt diet could result either from a generalized decrease in enzyme function (due to alterations in the enzyme itself or in the availability of essential substrates or cofactors); from increased levels of oxygen free radicals, which would lead to an enhanced breakdown of NO and a subsequent reduction of NO availability (7); or from alterations in signal transduction pathways such as receptor G protein coupling, as previously demonstrated for cAMP-mediated vasodilator responses in animals on a high-salt diet (11, 25).

The present study suggests that increased production likely contributes to the decreased NO release and to impaired vascular relaxation in response to methacholine in the aortas of animals on a high-salt diet, because treatment of the vessels with tempol not only inhibited release in response to methacholine, but also restored methacholine-induced NO release and improved vascular relaxation in the aortas from rats on a high-salt diet. However, even though methacholine-induced increases in NO levels were restored by tempol in the aortas from rats on high-salt diet, methacholine-induced NO release from tempol-treated aortas of rats on a high-salt diet was still less than that from tempol-treated aortic rings from rats on a low-salt diet. As noted above, the impaired NO release in response to methacholine in the presence of tempol could be due to alterations that occur early in the signal transduction pathways, e.g., changes in receptor function or G protein coupling mechanisms. The latter hypothesis is consistent with previous reports demonstrating that GS protein activation in response to cholera toxin is lost and that cAMP-dependent vasodilator responses are impaired proximal to adenylyl cyclase in cerebral and skeletal muscle resistance arteries of rats on a high-salt diet (11, 25). However, the existence of impaired receptor/G protein function in animals on a high-sodium diet remains to be established for the Gq-dependent responses mediated by muscarinic receptor activation in response to cholinergic vasodilators such as methacholine.

Another interesting observation is that tempol led to a significant increase in basal NO release in vessels from animals on either a high-salt or low-salt diet, suggesting that was present under resting conditions in each group. Pretreatment with apocynin also increased NO levels significantly in the aortas from rats on a high-salt and on low-salt diet, indicating that NADH/NADPH oxidase may be involved in production under resting conditions and that basal levels of production lead to reduced NO levels in the vessels under resting conditions. Combined treatment with apocynin and tempol led to a further increase in NO production in the aortas from rats on a high-salt diet and on low-salt diet, suggesting that there may be an additional source of in vessels.

When tempol was added to NE-contracted aortic rings, vascular relaxation occurred in the aortas from animals on either a low-salt or high-salt diet, and tempol-induced vascular relaxation could be eliminated by L-NMMA in both groups. However, tempol-induced vascular relaxation in the absence of methacholine was significantly larger in vessels from animals on a high-salt diet, suggesting that basal levels are elevated in animals on a high-salt diet, and that these elevated levels interfere with the modulation of vascular tone by NO. The latter hypothesis is consistent with studies of arterioles of Dahl salt-sensitive hypertensive rats (36), spontaneously hypertensive rats (35), Zucker obese diabetic rats (10), and normotensive rats on a high-salt diet (22–24) and with the observation that endothelium-dependent relaxation can be restored in those animals by scavenging (10, 22).

Another interesting finding in the present study is that pretreatment of the vessels with L-NMMA prevented the increase in production in response to methacholine in the aortas of rats on a high-salt diet, suggesting that eNOS was the source of stimulated production in these vessels. These findings are consistent with the hypothesis that NOS uncoupling contributes to impaired cholinergic relaxation of blood vessels in normotensive rats on high-salt diet, because enhanced generation (which could be inhibited by L-NMMA) would occur when eNOS activity is stimulated in normotensive animals on a high-salt diet. We also observed that L-NMMA increased the overall level of in the vessels from animals on either a high-salt or low-salt diet. This increase in levels during inhibition of NOS is likely due to reduced degradation of by the combination with NO to form peroxynitrite. Taken together, these data suggest that a likely mechanism for the impaired release of NO by methacholine in the the aortas of rats on a high-salt diet is an enhanced destruction of NO due to an increase in the production of during cholinergic activation of NOS (2).

The reason why NOS changes from NO production to generation during methacholine stimulation in the aortas of rats on a high-salt diet remains unclear. However, some studies suggest that a low concentration of the NOS substrate L-arginine or a deficiency of the essential cofactor tetrahydrobiopterin (BH4) causes eNOS to become uncoupled or partially uncoupled, producing instead of NO, or generating these two radicals simultaneously (4, 14, 38, 42). The latter hypothesis is consistent with the results of studies showing that administration of BH4 prevents the development of endothelial dysfunction in spontaneously hypertensive rats (15). Other studies (21) suggest that BH4 can be a target for oxidative stress in intact arteries, leading to a reduction in BH4 levels via the oxidation of BH4 to dihydrobiopterin. The latter hypothesis is supported by observations that vitamin C stimulates NOS activity in cultured endothelial cells by reducing oxidation of BH4 (16). These observations would suggest that enhanced levels of oxidative stress present from other sources such as NADH/NADPH oxidase could lead to NOS uncoupling and impaired relaxation in response to NO-dependent vasodilator stimuli in animals on a high-salt diet. Finally, other studies suggest that NOS uncoupling could arise from the direct effects of peroxynitrite on the enzyme itself (43) or from inhibition of the association of heat shock protein-90 with the enzyme (26). Regardless of the underlying mechanism(s) of reduced NO release and increased production in vessels from animals on a high-salt diet, the present studies demonstrate that elevation of dietary salt intake leads to an impaired NO release and a reduced endothelium-dependent relaxation in the aorta, which is apparently associated with elevated levels and enhanced oxidative stress.

Whereas many studies have demonstrated that oxidative stress is elevated and that endothelium-dependent vasodilation is impaired in pathophysiological conditions such as hypertension (32, 33) and diabetes (10), the present study demonstrates that impaired endothelium-dependent relaxation, reduced NO release, and increased production also may occur during elevated salt intake in normotensive animals. This impaired control of vascular function during elevated salt intake could be a precursor to the elevated vascular resistance that develops in salt-sensitive forms of hypertension and could also limit the ability of the microvasculature to tolerate circulatory stresses such as hemorrhage, hypoxemia, or exercise, even in the absence of an elevated blood pressure. The present study also demonstrates that the effects of a high-salt diet in producing increased oxidative stress and impaired endothelium-dependent vascular relaxation are extremely rapid, because significant changes were detected after only 3 days on a high-salt diet.


   ACKNOWLEDGMENTS
 
The authors thank Glenn Slocum for advice and technical support and Heather Stefaniak for assistance in the initial studies on the aortic rings. We offer special thanks to Dr. Ai-Ping Zou for helpful advice and criticism during the preparation of the manuscript.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-29587, HL-65289, and HL-72920.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Lombard, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: jlombard{at}mcw.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
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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