INTRODUCTION

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THE SMALL RESISTANCE ARTERIES that lie immediately proximal to the microcirculation are responsible for a large pressure drop within the arterial system and are important regulators of vascular resistance. Therefore any structural and functional alterations of these vessels could affect both vascular resistance and the regulation of blood flow in peripheral vascular beds. Previous studies by Liu et al. (13) demonstrated that the relaxation of skeletal muscle resistance arteries in response to a variety of vasodilator stimuli, including reduced PO₂, the stable prostacyclin analog iloprost, and ACh is impaired in rats with volume-expanded hypertension caused by renal mass reduction with chronic (4-8 wk) exposure to a high-salt diet. However, those studies also indicated that vessels from sham-operated normotensive controls on a high-salt diet also exhibit a decreased sensitivity to reduced PO₂, ACh, and iloprost, relative to those of sham-operated controls on a low-salt diet. Vascular reactivity in salt-induced models of experimental hypertension has been studied extensively, but the effect of elevated salt intake alone on vascular reactivity in normotensive animals is not well characterized.

In addition to their effects on vasodilator responses, hypertension and a high-salt diet also lead to a reduction of microvessel density in the cremaster muscle and to changes in the ultrastructure of microvessels in reduced renal mass (RRM) hypertensive rats and in normotensive sham-operated rats on a high-salt diet (7-9, 14). This reduction of cremasteric microvessel density in rats fed a chronic high-salt diet can be prevented by an infusion of ANG II (10). An important and surprising observation regarding the effect of RRM hypertension and high-salt diet in normotensive animals is that changes in microvessel density and ultrastructure can occur quite rapidly. For example, Hansen-Smith et al. (9) demonstrated that microvascular rarefaction and profound ultrastructural alterations occur in microvessels of RRM hypertensive rats and normotensive animals after only 3 days on a high-salt diet. These rapid ultrastructural changes in vessels of animals on a high-salt diet (9) raise the question of whether short-term elevations in dietary salt intake in normotensive animals will also lead to an impaired relaxation of resistance arteries in response to vasodilators and, if vasodilator reactivity is altered, could the impaired responses be related to suppression of ANG II by the high-salt diet?

The present study tested two major hypotheses. The first hypothesis was that both chronic (4 wk) and short-term (3 days) exposures of normotensive rats to elevated salt intake impair the relaxation of skeletal muscle resistance arteries in response to vasodilator stimuli. The second hypothesis was that normal vasodilator responses would be restored by preventing the ANG II suppression that occurs in rats on a high-salt diet. The specific goals of these experiments were to determine: 1) whether an impaired reactivity of skeletal muscle resistance arteries to vasodilator stimuli will occur in normotensive animals on high-salt diet; 2) whether any reduction in the ability of resistance vessels of animals on a high-salt diet to relax in response to vasodilator stimuli occurs as quickly and dramatically as the alterations in microvessel density and structure that occur in rats after short-term exposure to a high-salt diet (9); and 3) whether impaired vasodilator responses in animals on a high-salt diet are caused by suppression of ANG II.

	MATERIALS AND METHODS

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Experimental animals. Male Sprague-Dawley rats (Harlan, Madison, WI) weighing 250-400 g at the time of the experiment were used for these studies. Rats were fed either a high-salt (4% NaCl) or low-salt (0.4% NaCl) diet (Dyets, Bethlehem, PA) with tap water to drink ad libitum. Rats were maintained on the diet for either 3 days (short term) or 4-8 wk (chronic) before studies of the isolated vessels. An additional group of instrumented rats was fed a high-salt diet for a period of 1 wk and received an intravenous infusion of a low dose (5 ng · kg¹ · min¹) of ANG II for 3 days before isolated vessel studies to prevent the ANG II suppression that occurs in response to a high-salt diet (10).

Instrumented animal preparation. Rats used for the ANG II infusion were anesthetized with an injection containing ketamine HCl (78.0 mg/kg ip, Ketaject, Phoenix Pharmaceutical, St. Joseph, MO) and acepromazine maleate (2.2 mg/kg, Fermenta Animal Health, Kansas City, MO). Catheters were placed in the femoral vein under sterile conditions to allow infusion of ANG II. Chronic indwelling catheters were made from a 3.5-cm piece of single lumen vinyl tubing [inner diameter (ID) 0.50 mm and outer diameter (OD) 0.80 mm, Critchley Electrical Products, Auburn, Australia] connected to a 1-cm 22-gauge pin (Small Parts, Miami Lakes, FL) and 24 in. of microbore tubing (ID 0.020 in. and OD 0.060 in., Tygon). The catheter was inserted into the vessel, advanced into the inferior vena cava via the femoral vein, secured in the vessel with a 3-0 suture (Ethicon, Somerville, NJ), and anchored to the abdominal muscle in the groin area with surgical nylon suture (Braunamid). The catheters were filled with heparin, tunneled subcutaneously, and exteriorized at the back of the neck. A flexible spring (Exacto Spring, Grafton, WI) was held in place by a leather jacket that wraps the upper torso of the rat to protect the catheters. The spring was connected to a swivel outside of the cage, which allowed the rat free movement within the cage. On completion of surgery, all areas of incision were thoroughly cleaned with a 2% H₂O₂ solution, and animals were given an intramuscular injection of penicillin G (300,000 U/kg, Phoenix Pharmaceutical) to prevent infection. After recovery for a minimum of 5 days, the rats were placed on a high-salt diet for 1 wk, and ANG II was infused intravenously at a dose of 5 ng · kg¹ · min¹ for the final 3 days before the experiment.

General procedures. On the day of the isolated vessel experiment, the rat was anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg; Abbott, North Chicago, IL), and the carotid artery was cannulated with polyethylene tubing (PE-50, Clay Adams, Parsippany, NJ) to determine mean arterial blood pressure. The small muscular branch of the femoral artery supplying the gracilis muscle (diameter, 100-200 µm) was carefully removed, as previously described (4, 5, 13), and placed in warmed physiological salt solution (PSS) bubbled with 21% O₂-5% CO₂-74% N₂. The PSS used in these experiments had the following constituents (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.

Response to reduced PO₂. After the initial control period in PSS equilibrated with 21% O₂, the response to reduced PO₂ was determined in gracilis arteries from each group of animals. PO₂ reduction was achieved by simultaneous perfusion and superfusion of the artery for 20 min with PSS equilibrated with 0% O₂-5% CO₂-95% N₂, as previously described (4). Under these conditions, control values for PO₂ during 21% O₂ perfusion/superfusion are ~140 Torr, whereas equilibration of the PSS reservoirs with 0% O₂ reduces both the luminal and extraluminal PO₂ to 35-40 Torr (4, 5). After the period of PO₂ reduction, the perfusate and superfusate were reequilibrated with 21% O₂ for a minimum of 20 min, and the recovery of the vessel from hypoxia was verified by measuring vessel diameter at the end of the recovery period in 21% O₂.

Response to vasodilator agents and Ca²⁺-free solution. In addition to determining the response of the vessels to reduced PO₂, we assessed the response of the arteries from each group of rats to the following vasodilator agents: 1) the endothelium-dependent vasodilator ACh (10⁶ M); 2) the stable prostacyclin analog iloprost (10¹¹ g/ml); 3) the NO donor sodium nitroprusside (10⁶ M); and 4) forskolin (10⁵ M), a direct activator of adenylyl cyclase. ACh, sodium nitroprusside, and forskolin were purchased from Sigma Chemical (St. Louis, MO), and iloprost was a gift from Berlex (Wayne, NJ). The concentrations of the vasoactive agents used in these experiments were selected on the basis of prior experiments conducted by Liu et al. (13). To administer the drugs, superfusion was briefly interrupted in the bath, and an appropriate amount of drug was added to the PSS to achieve the final desired concentration in the vessel chamber. All measurements were made with the vessel fully pressurized by clamping the outflow pipette.

Statistical analysis. Dilator responses were determined as a percentage of maximum dilation in Ca²⁺-free PSS ([diameter change with drug/diameter change in Ca²⁺-free PSS] × 100). All data are summarized as mean percentage of maximum dilation ± SE for each group of animals, except for the dilation in response to Ca²⁺-free PSS, which is expressed as percent increase from control diameter. Differences between the means of individual experimental groups were determined by ANOVA with a subsequent Newman-Keuls test, or by an unpaired Student's t-test, when two means were compared. P < 0.05 was considered to be statistically significant.

	RESULTS

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Mean arterial pressure, body weight, and vessel diameter. Mean arterial pressures, body weights, and resting diameters of the vessels isolated from the various groups of animals used in this study are summarized in Table 1. There were no significant differences in mean arterial pressure between any of the experimental groups. Body weights of animals on the chronic high- and low-salt diets were significantly greater than those of animals on the short-term high- and low-salt diets, but dietary salt content had no effect on body weight within an experimental group. Diameters of arteries isolated from rats on short-term, high-salt and short-term, low-salt diets were significantly less than those of the other experimental groups.

Maximum dilation of resistance arteries in Ca²⁺-free solution. Figure 1 summarizes the diameter increase that occurs during maximum relaxation of the arteries in Ca²⁺-free solution in the various experimental groups. The maximum relaxation of arteries isolated from rats maintained on a chronic high-salt diet was significantly less than that of vessels isolated from their corresponding low-salt controls (Fig. 1A). In the animals maintained on short-term dietary regimens (Fig. 1B), there were no significant differences in the maximal relaxation of the arteries in response to Ca²⁺-free solution in any of the experimental groups.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Dilation of skeletal muscle resistance arteries during maximal relaxation produced by equilibrating the vessels in Ca2+-free solution. Vessels were obtained from rats maintained on chronic high-salt (HS) diet (n = 15) and chronic low-salt (LS) diet (n = 12) (A), and on short-term HS (n = 10) diet, short-term LS (n = 10) diet, and short-term HS diet receiving intravenous ANG II infusion (HS/ANG II) (n = 5) (B). Data are means % increase (±SE) from control diameter measured during equilibration of the arteries in physiological salt solution (PSS) before equilibration in Ca2+-free solution. * Significant difference (P < 0.05) from respective LS control.

Response to reduced PO₂. Figure 2 summarizes the response of skeletal muscle resistance arteries to reduced PO₂ in rats maintained on short-term and chronic high-salt and low-salt diets. Arteries from rats maintained on either the chronic or the short-term low-salt diet exhibited a significant increase in diameter in response to reduced PO₂, as previously reported for skeletal muscle (4) and cerebral (5) resistance arteries. However, arteries of rats maintained on the high-salt diet for 4-8 wk (Fig. 2A) and for 3 days (Fig. 2B) did not dilate in response to the reduction in perfusate and superfusate PO₂. Maintenance of plasma ANG II levels via intravenous infusion restored the dilator response to hypoxia in resistance arteries of rats fed a high-salt diet for 3 days (Fig. 2B).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Response of skeletal muscle resistance arteries to simultaneous reduction of perfusate and superfusate O2 concentration from 21 to 0% in vessels from normotensive rats maintained on chronic HS diet (n = 15) and chronic LS diet (n = 12) (A) and on short-term HS diet (n = 10), short-term LS diet (n = 10), and short-term HS diet receiving intravenous ANG II (HS/ANG II) (n = 5) (B). Change in diameter in response to 0% O2 is expressed as % maximum dilation occurring in Ca2+-free PSS (means ± SE). * Significant difference (P < 0.05) from respective LS control.

Response to vasodilator drugs. Figures 3 and 4 summarize the response of the arteries to ACh and iloprost in the various experimental groups. In these experiments, vessels from animals on a low-salt diet for 3 days or for 4-8 wk exhibited a pronounced vasodilation in response to both of these agents. In contrast, the dilator responses to both ACh (Fig. 3) and iloprost (Fig. 4) were either absent or drastically reduced in arteries from animals maintained on chronic or short-term, high-salt diets. Infusion of ANG II restored the relaxation of the vessels in response to ACh and iloprost in rats fed a short-term, high-salt diet. Figures 5 and 6 show the response of skeletal muscle resistance arteries of animals on high- and low-salt diets to sodium nitroprusside and forskolin. In contrast to the responses to ACh and iloprost, neither chronic nor short-term exposure to a high-salt diet affected vascular relaxation in response to nitroprusside (Fig. 5) or forskolin (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Response of skeletal muscle resistance arteries to ACh (106 M) in rats maintained on chronic HS diet (n = 9) and chronic LS diet (n = 9) (A) and on short-term HS diet (n = 10), short-term LS diet (n = 10), and short-term HS diet receiving intravenous ANG II (HS/ANG II) (n = 5) (B). Change in diameter in response to ACh is expressed as % maximum dilation occurring in Ca2+-free PSS (means ± SE). * Significant difference (P < 0.05) from respective LS control.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Response of skeletal muscle resistance arteries to iloprost (1011 g/ml) in rats maintained on chronic HS diet (n = 12) and chronic LS diet (n = 15) (A) and on short-term HS (n = 10) diet, short-term LS (n = 10) diet, and short-term HS diet receiving intravenous ANG II (HS/ANG II) (n = 5) (B). Change in diameter in response to iloprost is expressed as % maximum dilation occurring in Ca2+-free PSS (means ± SE). * Significant difference (P < 0.05) from respective LS control.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Response of skeletal muscle resistance arteries to sodium nitroprusside (106 M) in rats maintained on chronic HS diet (n = 9) and chronic LS diet (n = 9) (A) and on short-term HS diet (n = 9), short-term LS diet (n = 10), and short-term HS diet receiving intravenous ANG II (HS/ANG II) (n = 5) (B). Change in diameter in response to sodium nitroprusside is expressed as % maximum dilation occurring in Ca2+-free PSS (means ± SE). There was no significant difference between groups in either chronic or the short-term studies.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Response of skeletal muscle resistance arteries to forskolin (105 M) in rats maintained on chronic HS diet (n = 15) and chronic LS diet (n = 12) (A) and on short-term HS diet (n = 9), short-term LS diet (n = 10), and short-term HS diet receiving intravenous ANG II (HS/ANG II) (n = 5) (B). Change in diameter in response to forskolin is expressed as % maximum dilation occurring in Ca2+-free PSS (means ± SE). There was no significant difference between groups in either chronic or short-term studies.

	DISCUSSION

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Although changes in the sensitivity of blood vessels in response to vasoactive agonists have been extensively studied in hypertension, there have been very few studies of the effect of high-salt diet alone on vascular reactivity, and some of these have been confounded by changes in arterial blood pressure. In one study, Soltis et al. (16) demonstrated that administration of a high-salt diet to Sprague-Dawley rats for 4 wk after weaning was associated with a reduced dilation of the thoracic aorta in response to ACh and an enhanced constriction in response to norepinephrine. However, maintenance of the rats on the high-salt diet after weaning increased their blood pressure, and the authors noted that it was uncertain whether the high-salt diet caused direct alterations in vascular structure and function or whether the observed changes were the result of the elevated pressure. In another study, Obiefuna et al. (15) reported that there was no change in ACh-induced relaxation of aortic rings of Sprague-Dawley rats on a high-salt diet, but there was a significant blunting of the vasodilator response to histamine. Although blood pressures were not determined in that study, the interpretation of those experiments may have also been complicated by an elevation of blood pressure, because the authors had reported that arterial pressure of animals on the high-salt diet was elevated in earlier studies utilizing similar dietary protocols. Finally, Boegehold (2) reported that high-salt diet led to a reduced relaxation of arcade arterioles in response to ACh in the spinotrapezius muscle of the Dahl salt-resistant (Dahl R) genetic strain of rat compared with Dahl R rats maintained on a low-salt diet. This appeared to be caused by an impaired ability of the endothelium to release nitric oxide, because there was no difference in the response of the vessels to the NO donor sodium nitroprusside in that study.

In the present study, chronic exposure to a high-salt diet completely eliminated the dilation of isolated skeletal muscle resistance arteries in response to hypoxia (Fig. 2), ACh (Fig. 3), and iloprost (Fig. 4), providing further evidence in support of the hypothesis that chronic elevations in dietary salt intake may impair vascular relaxation mechanisms independent of an elevated blood pressure. In contrast, chronic exposure to a high-salt diet did not affect dilator responses that are mediated via direct activation of second messengers (cGMP and cAMP) operating further down the signal transduction cascades activated by ACh and iloprost (Figs. 5 and 6). As discussed below, these observations indicate that the impaired vasodilator function that we observed following exposure to a chronic high-salt diet is most likely caused by alterations that occur early in the signal transduction pathway, e.g., changes in receptor function or G protein coupling mechanisms, rather than alterations in second messenger function.

In the present study, the maximal relaxation of the arteries in response to Ca²⁺-free PSS was also significantly reduced in gracilis arteries of rats on a chronic high-salt diet relative to the arteries of the low-salt rats (Fig. 1). The latter observation indicates that structural changes occur in resistance arteries during chronic elevations in dietary salt intake, restricting the ability of the vessels to increase their diameter when the smooth muscle cells are fully relaxed. However, the impaired responses to hypoxia, ACh, and iloprost that we observed in arteries of animals on the high-salt diet for 4-8 wk were not caused by structural narrowing of the vessel, because individual vessels of animals on a chronic high-salt diet either failed to dilate in response to these stimuli or exhibited diameter changes that were much less than those occurring in response to Ca²⁺-free PSS. Although the mechanism for the reduction in the maximum diameter of the vessels in animals on the high-salt diet was not determined in the present study, it is likely that ANG II suppression in response to the high-salt diet contributes to structural alterations in the vessel, because Wang and Prewitt (18) reported that ANG-converting enzyme inhibition with captopril leads to a reduction in the passive lumen diameter of arterioles in the rat cremaster muscle.

One of the major goals of the present study was to determine whether vasodilator responses are also altered in response to short-term elevations in dietary salt intake. Hansen-Smith et al. (9) demonstrated a significant reduction in microvessel density in the cremaster muscle and profound ultrastructural changes in microvessels after only 3 days exposure to a high-salt diet. The ultrastructural changes that occurred during short-term elevations in dietary salt intake in that study involved both the endothelium and the vascular smooth muscle cells. The drastic effect of high-salt diet on microvessel structure in the study of Hansen-Smith et al. (9) naturally raises the question of whether short-term elevations in dietary salt intake can also result in functional changes in the resistance arteries, such as alterations in their response to vasoactive stimuli.

In the present study, vessels of animals on a high-salt diet for 3 days failed to dilate in response to hypoxia (Fig. 2), ACh (Fig. 3), and iloprost (Fig. 4), demonstrating that short-term elevations in dietary salt intake also lead to an impaired relaxation of skeletal muscle resistance arteries in response to these vasodilator stimuli. Similar to chronic high-salt diet, the responses of the vessels to direct activation of second messengers were unchanged after short-term exposure to high-salt diet (Figs. 5 and 6). There was also no difference in the maximal relaxation occurring in response to Ca²⁺-free PSS in arteries from rats on short-term, low-salt diet, short-term, high-salt diet, and short-term, high-salt diet with ANG II infusion (Fig. 1), demonstrating that structural narrowing of the vessel does not occur in response to short-term elevations of dietary salt intake.

Hernandez and co-workers (10) demonstrated that the loss of microvessel density in the cremaster muscle of rats on a chronic high-salt diet could be prevented by intravenous infusion of low-doses of ANG II, implicating the angiogenic effects of this hormone in maintaining normal microvessel density. Our observation that intravenous infusion of ANG II preserves the dilator response to hypoxia, ACh, and iloprost in rats maintained on a short-term, high-salt diet suggests that ANG II suppression is responsible for the impaired relaxation in response to vasodilator stimuli in resistance arteries of animals on a high-salt diet. A further implication of this finding is that ANG II is important in maintaining normal functional responses of the resistance vessels, such as the response of the vessels to vasodilator stimuli. The latter interpretation is consistent with the observation that chronic captopril treatment also leads to impaired vasodilator responses in rat skeletal muscle arterioles (6) and preliminary findings suggesting that the protective effect of ANG II on vasodilator responses in resistance arteries is mediated via its interaction with the AT₁-receptor subtype (19).

The mechanism by which ANG II suppression contributes to the reduced response to vasodilator stimuli in resistance arteries of animals on a high-salt diet remains to be determined. However, it appears that the failure of the vessels to dilate in response to hypoxia, iloprost, and ACh is caused by an intrinsic change in the mechanisms responsible for the relaxation of vascular smooth muscle in response to these stimuli. In an earlier study, Stekiel and co-workers (17) demonstrated that in situ arterioles and venules of reduced renal mass hypertensive rats on a high-salt diet exhibited an impaired hyperpolarization of their vascular smooth muscle cells in response to -adrenergic stimulation with isoproterenol compared with those of their normotensive controls. Similarities in the second messenger pathways of isoproterenol and iloprost include the activation of a G protein-mediated pathway working through adenylyl cyclase. There are several studies (1, 3, 11, 12) suggesting that changes in G protein-subunit populations may occur in hypertensive states, which could lead to alterations in vascular responses; and it is possible that similar changes also may occur in the high-salt animals in the present study. Another possible mechanism for the loss of normal vasodilator responses in vessels of animals on the high-salt diet is an impaired coupling between the G protein and the second messenger systems. The latter interpretation would be consistent with the results of Stekiel et al. (17), who showed that vascular smooth muscle hyperpolarization in response to direct activation of the G_s protein with cholera toxin was impaired in RRM hypertensive rats, while the electrophysiological response to direct activation of adenylyl cyclase with forskolin was unaffected.

An elevated salt intake is associated with an increased risk for the development of hypertension and other cardiovascular diseases in some individuals. As noted above, evidence is accumulating that suggests that an elevated salt intake alone may affect vascular structure and function. The novel aspects of the current study are the demonstration that the relaxation of skeletal muscle resistance arteries in response to several different vasodilator stimuli is profoundly impaired by even a short-term (3 days) exposure to high-salt diet, and that ANG II has a protective effect to maintain normal vasodilator responses in animals on a high-salt diet. These rapid changes in the ability of resistance arteries to relax in response to dilator stimuli indicate that a short-term elevation in dietary salt intake can lead to fundamental alterations in vessel function, in addition to the rapid structural alterations and reduced vessel density that Hansen-Smith et al. (9) have reported previously. Although additional studies are needed to determine the exact mechanism of the impaired relaxation of resistance arteries in response to vasodilator stimuli in animals on a high-salt diet, the observation that high-salt diet leads to rapid and drastic changes in vascular responses in normotensive animals may be pivotal in identifying factors that may predispose an individual to salt-sensitive forms of hypertension. The demonstration of salt-induced decreases in the reactivity of resistance arteries to vasodilator stimuli in normotensive animals may also reveal previously undescribed defects in the ability of normotensive individuals on a high-salt diet to regulate blood flow and to respond to circulatory stress such as hypoxia, hemorrhage, and exercise.