In skeletal muscle arterioles of normotensive rats fed a high salt diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced by superoxide anion. Because the impact of dietary salt on resistance vessels in other species is largely unknown, we investigated endothelium-dependent dilation and oxidant activity in spinotrapezius muscle arterioles of C57BL/6J mice fed normal (0.45%, NS) or high salt (7%, HS) diets for 4 wk. Mean arterial pressure in HS mice was not different from that in NS mice, but the magnitude of arteriolar dilation in response to different levels of ACh was 42–57% smaller in HS mice than in NS mice. Inhibition of nitric oxide synthase (NOS) with NG monomethyl l-arginine (l-NMMA) significantly reduced resting diameters and reduced responses to ACh (by 45–63%) in NS mice but not in HS mice. Arteriolar wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction or hydroethidine oxidation, was greater in HS mice than in NS mice. Exposure to the superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) + catalase reduced this oxidant activity to normal and restored normal arteriolar responsiveness to ACh in HS mice but had no effect in NS mice. l-NMMA also restored arteriolar oxidant activity to normal in HS mice. ACh further increased arteriolar oxidant activity in HS mice but not in NS mice, and this effect was prevented with l-NMMA. These data suggest that a high salt diet promotes increased generation of superoxide anion from NOS in the murine skeletal muscle microcirculation, thus impairing endothelium-dependent dilation through reduced NO bioavailability.
- reactive oxygen species
- NOS uncoupling
high dietary salt intake can lead to changes in microvascular function that are unrelated to any change in arterial pressure. For example, studies from our laboratory have demonstrated that in the spinotrapezius muscle of normotensive rats fed a high salt diet, there is a reduction in arteriolar responsiveness to stimuli that normally elicit dilation through endothelial nitric oxide (NO) release (8, 27–29), as well as a loss of NO's influence on resting arteriolar tone (7). These abnormalities are due to local scavenging of NO by superoxide anion (O2−) or one of its immediate metabolites, with increased arteriolar wall NAD(P)H oxidase or xanthine oxidase activity being responsible for this O2− generation under resting conditions (27–29). However, O2− is generated from a different enzyme when arterioles in salt-fed rats are exposed to the endothelium-dependent dilator ACh (28). A plausible candidate for this additional O2− source is nitric oxide synthase (NOS), which can generate O2− if the flow of electrons from the enzyme's heme core is diverted to molecular O2 rather than l-arginine (17, 46). This process, commonly referred to as “NOS uncoupling,” has been found to underlie vascular O2− generation in a variety of other abnormal states, including diabetes (3), hypertension (20), and atherosclerosis (22).
To date, the impact of dietary salt on resistance vessel function has been investigated almost exclusively in the rat. A demonstration of this effect in other species is essential for further establishing the relevance of high salt intake, in general, as a pathological influence on microvascular function. The mouse has become an important species for investigation of the mechanisms underlying normal and abnormal cardiovascular function, largely because of the availability of various murine models with targeted deletion or overexpression of genes relevant to vascular control (25, 39). The use of such models could provide an opportunity to more rigorously define the cellular and molecular changes that are responsible for microvascular dysfunction linked to high salt intake. However, it is first necessary to determine whether high salt intake produces the same microvascular changes in the mouse as we have previously found in the rat. Therefore, the current study was designed to investigate arteriolar responses to endothelium-dependent and endothelium-independent agonists in the spinotrapezius muscle of mice fed normal (0.45%) or high (7%)-salt diets for 4 wk. A second aim of this study was to determine whether reactive oxygen species (ROS) and/or reduced NO bioavailability are responsible for any salt-linked deficit in the endothelium-dependent control of arteriolar tone in the mouse and to explore the possibility that NOS could serve as a source of these ROS.
Ten-week-old inbred male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were placed on a whole-grain diet containing either 0.45% NaCl (normal salt, NS) or 7% NaCl (high salt, HS) (TD88311 and TD92100 diets; Teklad, Madison, WI). As with the Sprague-Dawley rats, we have previously studied (7, 8, 27–29, 32), these mice remain normotensive when placed on a high salt diet (1, 10, 31) (Table 1). The homeostatic mechanisms by which high salt intake leads to increased renal sodium and water excretion in the mouse are similar to those in the rat (2, 10).
Surgical preparation and intravital microscopy.
Mice were studied 4–5 wk after being placed on their respective diets. All surgical and experimental procedures were approved by the West Virginia University Animal Care and Use Committee. Each mouse was anesthetized with sodium thiopental (50 mg/kg ip) and placed on a heating pad to maintain a 37°C core temperature. The trachea was intubated to ensure a patent airway, and in some mice, the right carotid artery was cannulated to measure arterial pressure. Using an approach similar to that used for rats (8), we exteriorized the right spinotrapezius muscle for microscopic observation, leaving its innervation and all feed vessels intact. Throughout this surgery and the subsequent experimental period, the muscle was continuously superfused with an electrolyte solution (in mM), 119 NaCl, 25 NaHCO3, 6 KCl, and 3.6 CaCl2, warmed to 35°C and equilibrated with 95% N2-5% CO2 (pH = 7.35–7.40). Superfusate flow rate was maintained at 4–6 ml/min to minimize equilibration with atmospheric O2 (9).
The mouse was transferred to the stage of an Olympus BHMJ microscope (Hyde Park, NY) fitted with a color CCD video camera (Dage-MTI, Michigan City, IN). Images were displayed on a high-resolution video monitor and stored on magnetic tape for later analysis. Observations were made with an Olympus ×20 water immersion objective (final video image magnification = ×1460), and arteriolar inner diameters were measured off-line during videotape replay with a video caliper (Cardiovascular Research Institute, Texas A&M University). As in the rat, the mouse spinotrapezius muscle features a proximal network of anastomosing “arcade” arterioles that extends throughout the muscle (4). We chose to study these arterioles because in studies on rat spinotrapezius muscle, we have consistently found that the functional changes associated with high salt intake are most evident in this portion of the arteriolar network (7, 8, 27–29, 32, 36–38).
Experimental protocol 1: arteriolar responses to ACh and sodium nitroprusside.
To determine the effect of high salt intake on endothelium-dependent dilation, we evaluated arteriolar responsiveness to iontophoretically applied ACh (Sigma Chemical, St. Louis, MO) in each dietary group. A micropipette, beveled to an outer tip diameter of 2–3 μm, was filled with 0.025 M ACh in distilled water and positioned with its tip in light contact with the arteriolar wall. A retaining current of 70–100 nA was used to prevent passive diffusion of ACh from the micropipette tip during the control period, and net ejection currents of 5, 20, and 80 nA (delivered in random order) were used for ACh application. For each level of ACh, the vessel was continuously observed during a 1-min control period, a 2-min application period, and a recovery period of at least 2 min. To determine the contribution of NO release to these endothelium-dependent responses, the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA, Sigma) was then infused into the superfusate delivery line to produce an l-NMMA concentration of 10−4 M in the muscle bathing solution. After a 10-min equilibration period, arteriolar responses to all levels of ACh were reassessed, with l-NMMA superfusion continued throughout to ensure maximal NOS inhibition.
In other mice fed the NS and HS diets, we evaluated the possible contribution of ROS to any diet-related differences in endothelium-dependent arteriolar responses. In these animals, arteriolar diameter changes in response to ACh were evaluated before and then during exposure to 2, 2,6,6-tetramethylpiperidine-N-oxyl (TEMPO; 1 × 10−4 M in superfusate; Sigma) + catalase (Cat; 50 U/ml in superfusate; Sigma). TEMPO belongs to a family of a nitroxide spin labels that act as membrane-permeable SOD mimics (15), and we have previously verified in rat spinotrapezius muscle that these superfusate concentrations of TEMPO and Cat are sufficient to effectively scavenge ROS in the immediate vicinity of the arteriolar wall (29).
In other mice from each dietary group, the intrinsic responsiveness of arteriolar smooth muscle to NO was evaluated by iontophoretically applying the NO donor sodium nitroprusside (SNP; Sigma) to the arteriolar wall. Beveled micropipettes were filled with 0.05 M SNP in distilled water, retaining currents of up to 100 nA were used to prevent passive diffusion, and net ejection currents of 5, 10, and 20 nA were used to apply SNP (1-min control, 2-min application, minimum 2-min recovery).
At the end of all experiments, adenosine (ADO, Sigma) was added to the superfusate at a concentration of 10−4 M, and passive arteriolar diameters were measured so that all dilations could be expressed as “percent maximum response” (see Data and statistical analysis).
Experimental protocol 2: in vivo measurement of microvascular wall oxidant activity.
Steady-state oxidant activity in the arteriolar wall was assessed by the tetranitroblue tetrazolium (TNBT) reduction assay and by the hydroethidine (HE) oxidation assay, adapted for use in vivo (27–29, 43).
TNBT reduction was used as a general index of microvascular oxidant activity. The muscle was continuously superfused for 60 min with a solution containing 2% TNBT or 2% TNBT + TEMPO and Cat at the concentrations indicated above, then fixed with 10% formalin and removed. After slow dehydration by sequential immersion in solutions with progressively higher ethanol content, the muscle was cleared with methyl salicylate so that microvessels could be more easily visualized with brightfield microscopy. Images of arcade arterioles were then captured, digitized, and analyzed with MetaMorph imaging software (Universal Imaging, Downingtown, PA) to assess the deposition of formazan, a dark TNBT reduction product that indicates oxidant activity. Using a 5 × 5 μm photometric window, measurements of average tissue light intensity were made at a series of sites along each vessel wall, and in a corresponding series of extravascular regions immediately adjacent to those sites. This allowed for the normalization of each wall measurement to that of the immediate extravascular environment, thus controlling for any site-to-site or muscle-to-muscle variations in background intensity. To assess arteriolar formazan levels, the average pixel intensity measurements were used to calculate arteriolar wall light absorption (A): A = -ln (Iw/Io), where Iw is the vessel wall intensity and Io is the intensity for the adjacent extravascular region. We have previously verified that the amount of formazan that forms in arteriolar walls during TNBT exposure is proportional to the level of oxidant activity and that the calculated light absorption is linearly related to the amount of formazan present (27).
For the HE oxidation assay, HE was added to the muscle superfusate at a concentration of 1 × 10−3 M, and the muscle was continuously exposed to this HE-containing superfusate for 30 min in complete darkness to prevent HE photo-destruction. Because it is not oxidized by H2O2, HOCl, ONOO−, or O2, HE is considered to be relatively specific for the detection of O2− (5, 6, 34). HE easily permeates cell membranes and, when oxidized by O2−, is converted to fluorescent ethidium bromide that intercalates into nuclear DNA (5, 34). In theory, any differences among vessels in the extent of cellular HE loading could also lead to differences in ethidium bromide fluorescence that would be erroneously interpreted as differences in O2− activity. To circumvent this problem, we calculated the ratio of oxidized-to-unoxidized substrate in the arteriolar wall from paired fluorescence intensity measurements at the peak emission wavelengths for ethidium bromide and HE, respectively. Tissue autofluorescence at each of these wavelengths was measured before HE exposure and then subtracted from signals measured after HE exposure to determine specific ethidium bromide and HE fluorescence intensities.
In some preparations, the effect of ROS scavengers on O2− levels was assessed by also adding TEMPO + Cat, at the concentrations indicated above, to the superfusate. In other preparations, the role of NOS in O2− production was assessed by adding either 10−4 M l-NMMA, 1 × 10−6 M ACh, or ACh + l-NMMA to the HE-containing superfusate. After the 30-min exposure period, the muscle was rinsed with normal superfusate for 15 min to remove extracellular HE, and then briefly (1–2 s) illuminated with a mercury lamp, using appropriate excitation and emission filters for detection of ethidium bromide fluorescence (480- to 550-nm bandpass, 590-nm barrier), and HE fluorescence (330- to 385-nm bandpass, 420-nm barrier). Fluorescence images were acquired, stored, and analyzed with Metamorph 6.01.
Data and statistical analysis.
Arteriolar diameter (D, μm) was sampled at 5-s intervals during control and ACh or SNP application periods. Resting vascular tone (T) was calculated for each vessel as follows: T = [(Dpass − Dc)/Dpass] × 100, where Dpass is passive diameter under ADO, and Dc is the diameter measured during the control period. A tone of 100% represents complete vessel closure, whereas 0% represents the passive state. The magnitude of dilation to ACh or SNP was expressed as the percentage of the maximum response for that vessel: %maximum response = [(Dss - Dc)/(Dpass − Dc)] × 100, where Dpass is passive diameter. All data are reported as means ± SE. One-way repeated measures ANOVA was used to determine the effect of a treatment within a dietary group. Two-way repeated measures ANOVA was used to determine the effects of diet, treatment, and diet-treatment interactions on each variable. For all ANOVA procedures, the Student-Newman-Keuls method for post hoc analysis was used to isolate pairwise differences among groups. Significance was assessed at the 95% confidence level (P < 0.05) for all tests.
The general characteristics of mice used in this study are shown in Table 1. At the time of study, body weight and mean arterial pressure of mice fed high salt diets were not significantly different from those of mice fed the normal diet. Table 2 displays the characteristics of all arterioles studied by in vivo microscopy in protocol 1. Under control conditions, there was no significant difference between dietary groups in either resting or passive arteriolar diameter. However, the calculated resting tone of these vessels was slightly lower in HS mice than in NS mice.
Iontophoretically applied ACh consistently caused arteriolar dilation in both dietary groups, but for each level of ACh, the magnitude of this dilation was significantly lower in HS mice (20 ± 4, 24 ± 3, and 36 ± 4% of maximum dilation for 5, 20 and 80 nA ACh, respectively) than in NS mice (34 ± 6, 55 ± 4, and 78 ± 4% of maximum dilation for 5, 20, and 80 nA ACh, respectively) (Fig. 1). Exposure to l-NMMA significantly reduced resting arteriolar diameters and increased resting arteriolar tone in NS mice but had no effect on these variables in HS mice (Table 2). l-NMMA also significantly reduced the magnitude of arteriolar dilation to all levels of ACh (by 45–63%) in NS mice, but not in HS mice (Fig. 2). Iontophoretically-applied SNP also caused arteriolar dilation in both dietary groups, but in contrast to the responses to ACh, there were no significant differences between groups in the magnitude of these dilations (Fig. 3).
Figure 4 summarizes arteriolar responses to ACh in each dietary group before and then during exposure to TEMPO + Cat. Before addition of TEMPO + Cat to the superfusate, ACh delivered at 5, 20, and 80 nA caused arteriolar dilations of 26 ± 7, 47 ± 4, and 73 ± 5% of maximum in NS mice, but dilations of only 22 ± 4, 28 ± 3, and 34 ± 4% of maximum in HS mice. These differences between groups were significant for 20 and 80 nA ACh. In the presence of TEMPO + Cat, arteriolar responses to 20 and 80 nA ACh were significantly increased in HS mice, so that there were no longer any significant differences between groups in responses to ACh (dilations of 22 ± 6, 50 ± 4, and 70 ± 4% of maximum in NS vs. dilations of 30 ± 5, 51 ± 6, and 62 ± 6% of maximum in HS).
In muscles exposed to TNBT, calculated arteriolar wall light absorption in HS mice (0.55 ± 0.03 units) was significantly greater than that in NS mice (0.38 ± 0.03 units) (Fig. 5). Exposure to TEMPO + Cat significantly reduced this value in HS mice to a level that was not different from that in NS mice under the same conditions (0.36 ± 0.03 units in HS vs. 0.41 ± 0.03 units in NS).
After exposure of muscles to HE, control arteriolar wall ethidium bromide fluorescence, normalized to HE fluorescence, was significantly greater in HS mice (ratio = 1.57 ± 0.10) than in NS mice (1.10 ± 0.03) (Fig. 6). In the presence of TEMPO + Cat, this fluorescence ratio was significantly reduced in HS mice (to 1.04 ± 0.03) but not in NS mice (1.02 ± 0.05), and there was no longer a difference between dietary groups. Exposure to l-NMMA, ACh, or ACh + l-NMMA had no effect on normalized ethidium bromide fluorescence in NS mice (ratios of 1.14 ± 0.05, 1.12 ± 0.08, and 1.1 ± 0.08, respectively). In contrast, l-NMMA had the same effect as TEMPO + Cat in HS rats (reducing the fluorescence ratio to 1.02 ± 0.04), and ACh significantly increased normalized ethidium bromide fluorescence to a value greater than control (2.28 ± 0.09). This effect of ACh in HS mice was completely prevented by l-NMMA (1.06 ± 0.03).
The major findings of this study are as follows: 1) Arteriolar responses to the endothelium-dependent dilator ACh are smaller in mice fed a HS diet than in those fed a NS diet, and NOS inhibition significantly attenuates these responses in mice fed a NS diet but not in those fed a HS diet. 2) Exposure of the vascular bed to ROS scavengers restores normal arteriolar responsiveness to ACh in mice fed a HS diet but has no effect in mice fed a normal diet. 3) Arterioles exposed to TNBT or HE in mice fed a HS diet show greater formazan staining or ethidium bromide fluorescence, respectively, than those in mice fed a NS diet, but not if the arterioles are simultaneously exposed to either ROS scavengers or a NOS inhibitor. 4) ACh increases ethidium bromide fluorescence in arterioles of mice fed a HS diet, but not in those fed NS diet, and this effect is prevented by NOS inhibition. These findings suggest that ingestion of a HS diet leads to increased O2− generation in mouse skeletal muscle arterioles and that this O2− is responsible for decreased arteriolar responsiveness to endothelium-dependent dilators. Furthermore, this O2− appears to be generated exclusively by the NOS enzyme.
Effect of dietary salt in rats vs. mice.
Our laboratory has previously documented that in rats fed a 7% salt diet, microvascular NO activity in the spinotrapezius muscle is lost as a result of ROS-mediated breakdown of NO within the arteriolar wall (29). Under resting, steady-state conditions, this ROS accumulation is due to reduced Cu/Zn SOD activity in combination with increased NAD(P)H oxidase and xanthine oxidase activity (27, 28), implicating O2− as the molecule responsible for the decrease in microvascular NO. However, although treatment with the O2− scavenger TEMPO in combination with catalase restored the normal contribution of NO to ACh-induced dilation in rats fed a HS diet (29), inhibition of NAD(P)H oxidase and xanthine oxidase did not (28), indicating that another source of O2− arises during endothelium-dependent dilation.
In the current study on spinotrapezius muscle arterioles in the mouse, we found that NOS inhibition reduced resting arteriolar diameters in animals fed NS diet but not in those fed HS diet (Table 2), suggesting that the tonic influence of NO on arteriolar tone is lost in salt-fed mice. Furthermore, arteriolar responses to ACh, which were reduced ∼50% by l-NMMA in mice fed a NS diet, were smaller and insensitive to l-NMMA in mice fed HS diet (Figs. 1 and 2). This is consistent with our previous findings in salt-fed rats (7, 8) and further confirms that there is a selective loss of arteriolar NO activity in the salt-fed mice as well. Our finding of unaltered arteriolar responses to SNP in mice fed a HS diet (Fig. 3) indicates that a deficit in vascular smooth muscle responsiveness to NO does not contribute to this effect. Instead, the current study strongly suggests that as in the rat, the salt-dependent loss of arteriolar NO activity in the mouse is due to rapid inactivation of NO by O2−. This conclusion is based on our findings that steady-state arteriolar wall oxidant activity, as judged by TNBT reduction and HE oxidation, is elevated in mice fed a HS diet (Figs. 5 and 6) and that treatment with TEMPO + catalase for as little as 30 min returns arteriolar wall oxidant activity to normal and restores normal arteriolar responsiveness to ACh (Fig. 4). Because HE oxidation is a fairly specific indicator of O2− activity (5, 6, 34), it appears that O2− is the particular reactive molecule that is responsible for scavenging arteriolar NO in salt-fed mice.
Although we found a trend toward smaller passive arteriolar diameters in mice fed a high salt diet, this difference between groups was not statistically significant (Table 2). In earlier studies on normotensive rats, we sometimes found that passive arteriolar diameters were significantly reduced after 4 wk on this same HS diet (36–38), which is consistent with other findings of hypotrophic vascular wall remodeling in salt-fed rats (26), possibly in response to the reduction in circulating ANG II that normally occurs with high salt intake (18). This remodeling process may also occur in mice, but it may require a longer period of high salt intake than the one used here.
NOS as a source of arteriolar O2− in mice fed a HS diet.
Because our previous studies suggested that O2− from a source other than NAD(P)H oxidase or xanthine oxidase could be responsible for the blunting of endothelium-dependent dilation in arterioles of salt-fed rats (28), we investigated whether NOS could serve as this O2− source in the current study on mice. Studies from other laboratories have provided evidence that uncoupled NOS can be responsible for vascular O2− production and reduced endothelial NO levels in diabetes (3), hypertension (20), and atherosclerosis (22), as well as for the blunted endothelium-dependent dilation of cerebral arterioles associated with acute nicotine exposure or chronic alcohol consumption (13, 42). Our current findings indicate that NOS uncoupling could also be the source of microvascular O2− in animals ingesting a high salt diet. We found that the NOS inhibitor l-NMMA was just as effective as TEMPO + catalase in reversing increased arteriolar O2− levels (as judged by ethidium bromide fluorescence) in arterioles of mice fed a HS diet (Fig. 6). Furthermore, exposure to ACh increased arteriolar O2− to even higher levels in mice fed a HS diet, but not in those fed a normal diet, and this effect was completely abolished in the presence of l-NMMA. To our knowledge, this is the first study to demonstrate that HS intake can lead to microvascular O2− generation through NOS uncoupling.
Despite the general similarity of these current findings in the mouse with our previous findings in the rat, there may be some important differences in the mechanism(s) of salt-induced superoxide production between the two species. Whereas accumulation of ROS in the arteriolar wall under resting, steady-state conditions is due to increased activity of NAD(P)H oxidase and xanthine oxidase in the rat (28), this appears to be due exclusively to NOS uncoupling in the mouse (Fig. 6). This raises the possibility that elevated salt intake produces a greater degree of NOS uncoupling in mice than in rats.
Future studies will be needed to identify the mechanism(s) through which HS intake can lead to uncoupling of arteriolar NOS in the mouse and to determine whether this same process occurs in the rat. A reduction in BH4 availability is the most widely reported cause of NOS uncoupling in other abnormal conditions (3, 11, 20, 42, 46), but other possible mechanisms include reduced intracellular pH (16), oxidation of the zinc-thiolate complex that stabilizes the NOS dimer (49), reduced l-arginine availability (17), and increased levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) (44). The last two of these possibilities may be the least likely in salt-fed animals, because ingestion of a HS diet leads to either no change or an increase in l-arginine synthesis and plasma l-arginine levels, with no change in l-arginine metabolism (23, 47) and does not trigger increased ADMA levels unless hypertension develops (33, 41).
The scavenging of NO by O2− is not the only mechanism through which arteriolar responsiveness to endothelium-dependent dilators can be reduced in animals fed a high salt diet. In the rat, ingestion of a high salt diet for only 3 days has been found to eliminate ACh-induced increases in cerebral artery NO production and the attendant dilation of these vessels, but this is due to disruption of the ACh signaling pathway proximal to NOS activation, rather than to ROS formation in the vascular wall (45). Ingestion of a HS diet for 3 days also does not lead to increased resting O2− levels in rat thoracic aorta (48). The discrepancy between these findings and those of our current and previous studies (27–29, 32) may be due to fundamental differences between arteries and arterioles in the cellular or biochemical pathways that are affected by HS intake, or to a difference in the length of time that animals were fed a HS diet (3 days vs. 4 wk).
In mice fed a NS diet, l-NMMA, at a concentration that maximally inhibits arteriolar NO production in spinotrapezius muscle (30), blocked approximately half of the ACh-induced dilation (Fig. 2). This is consistent with other reports that l-arginine analogs do not completely block arteriolar responses to ACh in mouse, rat, hamster, and rabbit striated muscle (12, 21, 30, 40). A portion of the endogenous l-arginine pool in these vessels may be inaccessible to l-NMMA (35), but there is also strong evidence that other endothelial factors, such as vasodilator prostaglandins and endothelium-derived hyperpolarizing factors, can also contribute importantly to ACh-induced arteriolar dilation in skeletal muscle (12, 21, 30). Some of the enzymes that produce these other endothelial relaxing factors, most notably cyclooxygenase and cytochrome P-450 epoxygenases, can also produce O2− when activated (14, 19). However, our finding in HS mice that l-NMMA completely abolished the ACh-induced increase in arteriolar O2− (Fig. 6) argues against a significant production of O2− by any of these other enzymes under the conditions of our study.
In summary, the current study demonstrates that a HS diet leads to increased oxidant activity, decreased NO availability, and reduced endothelium-dependent responses in skeletal muscle arterioles of the mouse and also suggests that NOS-derived O2− plays an important role in this process. These findings open up the possibility of using various transgenic mouse models to gain further insight into the cellular and molecular mechanisms that underlie the altered arteriolar control linked to high salt intake. The current findings are consistent with our earlier findings in the rat, and suggest that this effect of dietary salt may be common to a number of species. Organizations such as the American Heart Association, the National Academy of Sciences, the National Heart, Lung and Blood Institute, and the World Health Organization recommend lowering sodium intake for better health (24). Whether the current observations are germane to human cardiovascular function awaits the demonstration of a similar effect in human patients.
This investigation was supported by National Heart, Lung, and Blood Institute Grants HL-44012 and HL-67562.
The authors would like to acknowledge the expert technical assistance of Kimberly Wix in this study.
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- Copyright © 2007 the American Physiological Society